Alzheimer’s disease (AD) is multifactorial, progressive and the most predominant cause of cognitive impairment and dementia worldwide. The current “one-drug, one-target” approach provides only symptomatic relief to the condition but is unable to cure the disease completely. The conventional single-target therapeutic approach might not always induce the desired effect due to the multifactorial nature of AD. Hence, multitarget strategies have been proposed to simultaneously knock out multiple targets involved in the development of AD. Herein, we provide an overview of the various strategies, followed by the multitarget-directed ligand (MTDL) development, rationale designs and efficient examples. Furthermore, the effects of the linkers and substitutional functional groups on MTDLs against various targets of AD and their modes of action are also discussed. For a long time, the paradigm of “one-drug, one-target, one disease” has had countless successes for the pharmaceutical industry in the 20th century . Many prIn 2021, the World Health Organization (WHO) data projected that 55 million people were affected with dementia worldwide, and this figure is expected to escalate to 78 million in 2030 and 139 million in 2050 . CurrentTo overcome the drawbacks of the existing drugs, a “one-drug, multi-target” paradigm has been developed for AD candidates. Increasing amounts of evidence indicate that the multi-target approach may improve therapeutic efficacy and safety ,12,13,14The cholinergic hypothesis concerns major neurotransmitters and cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)), which are extracellular enzymes present abundantly in the central nervous system and at peripheral junctions. The reactions between the choline acetyltransferase (ChAT) enzyme and acetyl-coenzyme A (Ac-CoA) yield acetylcholine (ACh). The nicotinic receptors and muscarinic receptors play a key role in cholinergic transmission and, among other important functions, are responsible for learning and memory generation in the brain. Thus, the maintenance of ACh levels in the brain is essential for proper brain function. AChE is a serine hydrolase enzyme that decomposes neurotransmitter ACh at the neuronal synapses into acetic acid and choline. The choline is recycled and reused by the presynaptic neuron. In AD patients, the brain-elevated activity of AChE is observed, which reduces the required Ach levels in the brain, impairs synaptic functions and eventually produces toxicity in neuronal cells, leading to brain damage, as well as memory loss and dementia . It is wAmyloid-β-peptide (Aβ) is the connective agent in AD pathology. Neurofibril tangle formations, vascular damage and dementia follow as a result of Aβ deposition in the brain. This was postulated by Hardy and Higgins in 1992. This hypothesis has dominated the understanding of AD for more than two decades. It states that the pathogenic procedure begins with abnormal processing of the amyloid precursor protein (APP) . The pro•OH) or the superoxide anion (Reactive oxygen species (ROS) are the main cause of oxidative stress in cells. Generally, ROS are highly reactive and unstable chemical species because they have one or more unpaired valence shell electron. These species are derived from oxygen molecules such as the hydroxyl radical  and redox-inactive (Zn) metals are involved in oxidative stress. Normally, the bio-essential metals Fe, Cu and Zn are present in the neocortex and play several critical roles in the brain. They are cofactors for numerous enzymes with important catalytic activities  and serve as active participants in metal-dependent neurotransmission. Redox-active metals act as catalysts to generate free radicals in addition to the Fenton reaction. The generated free radicals increase the oxidative stress of the brain cells. In addition to that, redox-active metals promote amyloid fibril formation in the brain by the interaction with the amino acid residues. A therapeutic approach would consist of the use of small molecules  to deplete the excess Cu, Zn and Fe . Other hA recent strategy to achieve better AD drugs is to simultaneously tackle several AD-related pathological features . TherefoThe basic rational design of MTDLs is to take two or more chemical entities with biologically known targets  and combine them into a single chemical entity . In the 1) has a postulated dual mode of action: in addition to inhibiting AChE, it has a facilitating effect on the nicotinic receptor-mediated transmission via allosteric modulation of the α-subunit of nicotinic receptors. In particular, 1 can enhance synaptic N-methyl-D-aspartate receptor (NMDAR) activity by activating nicotinic receptors located on presynaptic glutamatergic neurons. On the other hand, Memantine (2) is the only clinically available NMDAR antagonist and is prescribed to treat moderate-to-severe dementia. Moreover, 1 has a memory-enhancing activity, and it can also utilize the complement of the effect of 2 in AD [1 and 2) linked by flexible spacers , micromolar-binding affinity for NMDAR (Ki = 4.6 µM) and NMDAR-containing 2B subunit (NR2B) . It was observed that the five to six methylenes units were the minimal distance to contact both active sites  of AChE; when the spacer length of 3 was reduced to four carbons, the inhibition efficacy decreased significantly (IC50 = 695.9 nM). Moreover, the linker length did not affect the binding affinity against NMDAR. In addition to that, 3 was able to inhibit NMDA-induced neurotoxicity with sub-nanomolar potency and exhibited remarkable neuroprotective activity  compared to marketed drugs 1 (IC50 = 747 nM) and 2 (IC50 = 718 nM) [The linking strategy is most common for constructing MTDLs against AD. The selection of linkers, substituted functional groups, position of tailoring on the two distinct pharmacophores (should not join a ligand at the position with a steric effect) and length of the linkers should maintain the activity of the MTDLs. Most importantly, linkers themselves may sometimes interact with the target residues . Overall 2 in AD . Based o spacers A. Intere 718 nM) .4) in combination with Trolox (5) by coupling reactions for various AD targets  was the optimum length for hAChE, and the activity was even 125-fold better than that of 4 and 5. On the other hand, the unit of four carbon-length linkers showed slightly more activity against hBuChE. Further, the linkers themselves interacted hydrophobically with the surrounding hydrophobic amino acid residues , which was identified by molecular modeling. The flexibility and length of the linker played a major role in making compound 6 to reach both the catalytic site and peripheral anionic site of hAChE. In addition to that, the antioxidant activity of compound 6  was reported to be due to the structural feature of the Trolox moiety, as 5 is a standard antioxidant. The authors suggested that compound 6 could be a suitable candidate for mild to severe forms of AD [Eugenie et al. synthesized MTDLs based on the standard drug tacrine ( targets B. Compoums of AD .9 was developed by fusing the benzofuran ring from Moracin M (7) (Phosphodiesterase 4D (PDE4D) and Aβ inhibitor) and the hydroxyquinoline ring from clioquinol (8) using Witting regents. The synthesized compound 9 exhibited significant inhibitory potency towards PDE4D (IC50 = 0.32 μM), antioxidant activity and self- and metal-induced Aβ aggregation properties (3) played a vital role in its biological activity, particularly Aβ aggregation inhibition (67.5%), which was much higher than Moracin M (43.1%) [In the fusion strategy, two distinct pharmacophores fused chemically using various reaction conditions. The active functional groups should be maintained to retain the activity against various targets, and the developed MTDLs are smaller than the linked pharmacophores and provide better access to the pharmacokinetic profile. By adopting this strategy, compound operties A. Notabl (43.1%) .10) and donepezil was (11) [N2 nucleophilic substitution with excellent yield (47–97%). The most active compound, 12, was identified by the initial biological characterization, and it is worth noting that all the fused pharmacophore series were shown to be weak inhibitors of hAChE, which did not exceed 21% in the 100-µM range; in the case of hBChE, all the compounds showed a significant inhibitory profile . The substituted functional groups  and the position of substitution  on the benzyl moiety significantly influenced the inhibition profile of hBChE. For example, when the position of the methoxy group moved from position 2 to 3 or 4, the inhibitory efficacy (IC50 = 47.2 µM) was eight times lower than the substitution of position 2 . On the other hand, the substitutional functional group and position of the substitution did not influence the Aβ42 self-aggregate inhibition (38.1–59.4%). Compared to 12a, compound 12b showed potent inhibitory activity for the Aβ42 amyloid (65%), and the value was closer to the known antiaggregating agent curcumin (73.7%). Other serious MTDLs were synthesized via the fusing strategy (13 was found to possess rapid H2O2 scavenging activity and glutathione peroxidase-like (GPx-like) activity. The type of substitution group at the oxine moiety played a major role in GPx-like activity, e.g., the chlorine substituted showed much better GPx-like activity as compared with the iodine substitution. Along with this, 13 could be able to form complexes with biometals such as Cu, Zn and Fe with the stoichiometry of a 1:2 Cu2+:ligand molar ratio. According to the results of the in vitro blood–brain barrier model assay, 13 was able to penetrate the CNS, and the toxicity tests in mice showed that 13 had no acute toxicity at doses of up to 2000 mg kg−1 [A series of fused MTDLs with the combination of clioquinol (was (11)  preparedstrategy C, and th mg kg−1 .14, which showed balanced ChE inhibition  , SKF-64346 (Aβ aggregation inhibitor) and deferiprone  by using the merging strategy. The synthetic conditions involved multiple steps; the initial intermediates were achieved by a microwave apparatus with 155 °C heating for around 10–20 min. The final compounds were synthesized with a good yield (93–99%) in the presence of the pd/C catalyst. Most of the compounds exhibited antagonism toward the H3 receptor, Aβ aggregation inhibition, metal ion chelation and radical scavenging activity. Especially, the most promising compound, 15, displayed nanomolar IC50 values in H3 receptor antagonism (IC50 = 0.32 nM) with high selectivity, efficient capability to interrupt the formation of Aβ1–42 fibrils (IC50 = 2.8 µM), good copper and iron-chelating properties and more potent 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) radical cation (ABTS•+) scavenging activity. Significantly, 15 did not show any significant toxicity in human glioma U251 cells at a concentration of 20 µM. In vivo data provided the BBB penetration ability of 15. Overall, 15 was able to hit four important AD targets [Due to the low molecular weight and efficient pharmacokinetic profile, the merged MTDLs have received much attention in AD drug design. A series of 3,4-dihydropyrimidin-2(1H) derivatives  and tacrine (ChEs inhibitor) were merged via the Friedlander-type reaction by Bartolini et al. They investigated the detailed ChE inhibition and calcium intake blockade profile. All the compounds showed moderate-to-good ChEs inhibition, and the most important compound was ty (30%) A. It was16, showed a nanomolar inhibition (IC50 = 66 nM) profile on GSK-3β, and the activity was improved significantly while increasing the length of the linker from two to five carbon units. Ellman’s test showed that 16 inhibited hAChE in the nanomolar range (6.4 nM). It was interesting to note that the substitution on the tacrine moiety (6-Cl-tacrine or 7-MeO-tacrine) increases the inhibitory activity of hAChE more compared to BuChE, suggesting that such a modification could help to design highly selective hAChE inhibitors. The binding mechanism of 16 with hAChE was identified by molecular docking data, which revealed that 16 occupied both the catalytic active site (CAS) and peripheral anionic site (PAS). Endocyclic nitrogen was involved in the hydrogen bond interaction with the mainchain carbonyl oxygen of His447 ; thus, the catalytic triad was disrupted. The authors found that the pyridothiazole moiety was located in the PAS of the hAChE-binding groove and formed π–π stacking contacts with the side chain of Trp286. In addition to that, the cognition-improving potency of 16 was assessed by the Morris water maze test, and the results indicated that 16 showed better cognitive improvement and less hepatotoxicity than tacrine [17 was synthesized by reacting intermediate (E)-ethyl3-amino)propyl)carbamoyl)phenyl)acrylate with NH2OH in the presence of KOH in MeOH. All the tested compounds showed better ChEs inhibitory activity than the standard (tacrine) in the nanomolar range. Compound 17 was found to show the most potent inhibition for AChE (IC50 = 0.12 nM); along with the superior selectivity of ChEs, the tacrine unit could be responsible for the inhibition of ChEs. When the spacer between N and hydroxamate is longer and the substitution of chlorine at the sixth position in tacrine benefits the inhibition of AChE, the inhibitory activity of BuChE remains comparable. Interestingly, when the connecting amide bond between N and hydroxamate was changed to a sulfonamide bond, the inhibition on AChE was decreased while the inhibition on BuChE was increased, and a similar pattern was observed on HDAC inhibition, such as exchanging the amide (IC50 = 0.23 nM) to sulphonamide (IC50 = 6810 nM) to reduce the inhibitory activity. Significantly, compound 17 was proposed as a mixed-type inhibitor, since it could bind CAS and PAS, which is evident from the kinetic data and molecular modeling studies [Numerous MTDLs for AD have been reported in the literature, and a few efficient MTDLs are discussed in this part . Sun et  tacrine . Few tac studies .6 antagonism and antioxidant properties [18, a systematic synthetic protocol was followed. In brief, the key intermediate of 4-(1-benzyl-1H-indol-4-yl)piperazin-1-hydrogen chloride was alkylated in acetonitrile reflux with ω-bromoalkylphthalimides and K2CO3 as a base (yield = 73%). From these MTDLs, compound 18 exhibited dual activity against selected targets: 5-HT6 receptor (Ki = 41.8 nM) and BuChE (IC50 = 5.07 μM). The authors noted that the length of the alkyl linkers affected the affinity of the compounds for the 5-HT6 receptors. For example, the elongation and extension of the linkers drastically reduced the affinity, which was reflected in the results of the radioligand-binding assay. The same results were observed for the BuChE inhibitory profile as well; few longer-length linker compounds displayed modest activity or appeared inactive at the screened concentration. The favorable antioxidant property of 18 was screened by a FRAP assay, and 18 exceeded the activity of ascorbic acid at the corresponding concentration; the indole moiety could be the reason for this, as was previously confirmed in numerous studies. The authors suggested that compound 18 might be a promising scaffold for starting further modifications toward the development of MTDLs against AD [Wieckowska et al. synthesized 12 MTDLs based on indole scaffold and investigated their biological activities against selected AD targets, including BuChE, serotonin HToperties . To isolainst AD .19a, IC50 = 35nM), the carbamate  substitution at the fourth position of the phenyl ring significantly improved the PDF9A inhibitory activity. A similar trend was followed by the hybrids in ChE inhibition; of note is that 19b exhibited better selectivity against BuChE (IC50 = 3.3 mM) than AChE [19 were developed by Huang et al., but in those analogs, pyrazolopyrimidinone was retained and the benzyl piperidine moiety (donepezil) fused via various linkers (20a showed better inhibitory activity against AChE (IC50 = 0.298 μM) than the one-carbon units linker hybrid (IC50 = 0.340 μM). The inhibition activity was slightly increased, while the -N atom was replaced by an O atom  and drastically increased with the CH2 molecule . Of significance, the favorable linker length between piperidine and pyrazolopyrimidinone to inhibit PDE9A was four atoms ; the activity was decreased when the linker length increased to five or decreased to three atoms. The substitution in the linker showed minor effects on the PDE9A inhibitory activity [19a,b) did not show any significant inhibition against AChE, while the donepezil scaffold-installed compounds (20a–c) showed significant inhibition against AChE.Pyrazolopyrimidinone is a nitrogen-containing heterocyclic compound that acts as a building block for several pharmaceutically relevant compounds. Pyrazolopyrimidinones showed excellent biological activity against various diseases, such as cancer, obesity and cystic fibrosis . In addihan AChE . The nan linkers B. The leactivity . Taken c21, in which the piperidine fragment of donepezil was replaced by the piperazine moiety and studied the inhibitory activities against AChE, BACE-1 and the Aβ protein . The results of the study clearly showed the influence of the linker length; the increased length resulted in a decreased activity against AChE. On the other hand, the substitution of electron-donating and -withdrawing groups such as -CH3, -F, etc. plays a vital role in the hybrid performance. For example, methyl substitution on the phenyl ring improved the activity over that of the -F substitution. All the compounds showed good BACE-1 and Aβ inhibitions. The authors also employed molecular modeling studies to reveal the mode of interaction with target receptors. It is interesting to observe that the phenyl ring of piperazine interacts with the Phe20 residue via π–π stacking and carbonyl oxygen, and the amino group of piperazine forms hydrogen bonds with Lys16, which are significant for effective Aβ inhibition. The mixed type of interaction was identified by kinetic data. The neuroprotective activity and the BBB penetration abilities suggested that these hybrids deserve further in vivo studies [22a, IC50= 0.46 µM and 22b, IC50= 16.74 µM and the selectivity of BuChE were very poor. The hydroxyl group of the feruloyl moiety confirmed the antioxidant activity . Regiochemistry plays a major role in the antioxidant activity of the compounds [23a–e) was synthesized by a fusion strategy, and their multitarget abilities were studied by Wang et al. , carboxamide  and alkyl (2 carbon spacer) linkers. All the compounds exhibited micromolar to sub-micromolar inhibition of the ChEs . Of note, the hybrids showed more potency toward BuChE compared to AChE. The substitutions of electronic properties and the position of the substitution on the phenyl ring influenced their inhibitory activity. While the two-position substituted methyl hybrids  showed better inhibition efficacy than the 2- or 3-Cl or -NO2 substitutions, which indicates that the electron-donating group and the position of the substitution have appeared more favorable in ChE inhibitions. However, increasing the linker length (23e) provided the strongest inhibition of ChEs . The anti-Aβ aggregation, antioxidant and BBB abilities showed potential multitarget molecules for the treatment of AD [Donepezil is a rapidly reversible inhibitor of AChE approved for the treatment of AD, and it is the first and only AChE inhibitor approved for the treatment of severe AD. Donepezil derivatives and hybrids target various AD-related targets, such as BuChE, Aβ cascade and metal ions ,63,64,65 protein A. The hy studies . To findompounds . Anotherg et al. C. These nt of AD .24a and 24b) with electron-donating -(CH3) and electron-withdrawing (-F) substitutions on the phenyl ring and introduced the alkyl linker between the Trolox and donepezil scaffolds (24a exhibited more selectivity in BuChE (IC50 = 5.49 µM) over AChE (IC50 = 19.5 µM). While the linker length was increased to the two-carbon spacer 24b, the AChE inhibitory potency was much greater than 24a . In the case of MAO selectivity, the linker length did not influence the activity, and the most interesting factor was that the -F-substituted (electron-withdrawing group) hybrid showed better potential to inhibit AChE than the -CH3 (electron-donating group) group-substituted hybrids. Additionally, the antioxidant ability of 24b was ABTS = 1.79 Tr and ORAC = 1.6 Tr, which was relatively close to the standard Trolox. The good neuroprotectivity, less cytotoxicity and BBB penetration abilities showed the value of the hybrids for AD [As the aforementioned hybrids significantly improved the AChE inhibitory activity in the presence of methyl (electron-donating group), a similar trend did not follow in all the cases. For example, Kong et al. developed a few donepezil–Trolox hybrids  but had slightly decreased anti-AChE activity. The introduction of the methoxy group on the benzofuran moiety improved the potency . The varying results can be ascribed to the various positions of the benzofuran unit at the entrance to the AChE gorge, depending on the structure of the quinoline fragment. Interestingly, the introduction of an amino group in the spacer chain drastically increases the efficacy of the AChE inhibition , and it was 493 times more potent than tacrine. This could be explained by the fact that the amino group may involve π−cation interactions with the PAS, greatly enhancing the anti-AChE activity. On the other hand, with the introduction of another benzofuran moiety on the spacer, the activity decreased significantly  but showed better activity on BuChE . This indicates that the bulky group would not fit well into the AChE gorge, and as previously reported, the volume of the upper portion of the catalytic hBChE gorge is approximately 340 Å larger than the corresponding portion of the hAChE gorge, thus showing good selectivity in BuChE. Moreover, 25c reduced the hAChE-induced amyloid fibrillization by 58% at a 100-mM concentration, which was comparable to bis(7)-tacrine (68%), and tacrine failed to inhibit AChE-induced Aβ1–40 aggregation in similar experimental conditions. In contrast, 25d showed better BACE-1 inhibition than other tested hybrids (IC50 = 0.19 µM) [Tacrine belongs to acridine compounds and is a potent noncompetitive reversible AChE inhibitor. Tacrine was the first medication licensed by the FDA for the treatment of AD. The compound was withdrawn from the market due to the severe hepatotoxic side effects. Numerous tacrine derivatives and hybrids showed excellent inhibitory activity for ChEs ,72,73,740.19 µM) .26a showed an IC50 value for AChE of 30.9 nM. It is worth noting that, by decreasing the linker length from 1 to 0 carbon spacers and -Cl atom substitution on tacrine, the activity was improved significantly . After the introduction of the -OH group on the linker, the activity  was lower compared to 26b, and the activity further decreased while the -Cl substituted tacrine . These activity changes were explained by the molecular modeling data; the substituted chlorine atom fit well into the hydrophobic pocket of the AChE hybrids, where it was able to fit into the space formed by Trp432, Met436 and Ile439. The lengthening of the linker may keep away hydroxyphenyl benzimidazole from PAS. The installation of the -OH group on the linker may contribute to the improvement in enzyme inhibition via the H-bond with Asp72. In addition to that, the study confirmed that the hybrids were mixed-type inhibitors. The Aβ inhibition efficacy and the metal chelation abilities have added advantages to the hybrids [A novel series of tacrine–hydroxyphenyl benzimidazole hybrid molecules was synthesized and evaluated for their multitarget potency by Santos et al. The rationale for the design of the hybrids was as follows: the tacrine moiety was expected to provide AChE inhibition, and the hydroxyphenyl benzimidazole moiety should ensure Aβ inhibition and metal chelation; these two scaffolds were coupled with aliphatic linkers B. During hybrids .27a–d) was synthesized, and their ChEs and Aβ inhibition abilities were evaluated  showed better inhibitory activity than the dimethoxy-substituted  and hydroxy-substituted  hybrids, and a selectivity for AChE over BuChE was observed. The most potent inhibitor for AChE was 27b, and its potency was 13 times stronger than the reference compound tacrine. However, a similar trend was not followed in Aβ inhibition . Despite that, the same electron-rich aromatic ring was afforded (27b), and the IC50 value for AChE of compound 27d was decreased to 16.88 nM. A possible reason for this result could be that the additional vinyl group enlarged the conjugated regions in the same substituents of an aromatic ring, which meant that the electron density of the phenyl group was averaged and thus led to an electron-deficient effect. A Lineweaver–Burk plot and molecular modeling study proved that 27b targeted both the CAS and PAS of ChEs [Another series of electron-rich tacrine–multialkoxybenzene hybrids  showed a weak ratAChE inhibitory activity, and the activity was slightly improved with cyclic amine  substitution and showed better inhibition potency with aliphatic  substitution. The substituted aliphatic CH2 of 28c interacted with key residue Trp86 via σ–π interactions, the carbonyl group interacted with the key Phe295 via intramolecular hydrogen bonding and the hydroxyl group (Apigenin) interacted with important residue Ser293 through intramolecular hydrogen bonding, which was identified by molecular modeling studies. Importantly, this carbamate moiety did not show any significant activity in antioxidant and Aβ inhibition. The parent apigenin scaffold plays a major role in antioxidant activity, particularly in the hydroxy group . Furthermore, the ideal neuroprotectivity, hepatoprotective activity and good BBB penetration abilities showed the potential of the hybrids against AD [Rivastigmine is a carbamate derivative that is structurally related to physostigmine, which acts as a cholinergic agent for the treatment of mild-to-moderate dementia of the Alzheimer’s type. It is a reversible inhibitor that inhibits both cholinesterases (AChE and BuChE). Liu et al. synthesized a novel series of apigenin–rivastigmine hybrids and assayed their activity in vitro against AD targets A. In ChEainst AD .29a) exhibited the strongest inhibition of BuChE with an IC50 of 6.2 µM. On the other hand, hybrids 29b (IC50 = 0.34 mM) and 29c (IC50 = 0.57 µM), containing the N,N-diethylcarbamate moiety, showed the most potent inhibition for AChE. It can be reasonably concluded that the N,N-diethylamine and N-ethyl-N-methylamine moieties fit better inside the gorge of the CAS in AChE. In general, when the N,N-diethylamine and N-ethyl-N-methylamine moieties were replaced by N,N-diisopropylamine and morpholine groups, the activity was decreased significantly. In addition to this, hybrid 29c showed better antioxidant activity and caused less cytotoxicity. Overall, the aliphatic-substituted carbamates were the better choice than the aromatic or cyclic amine substitution [Other derivatives of rivastigmine-based MTDLs were synthesized by Deng and his coworkers B. The autitution .30a) installation at position 6 of the coumarin core led to improved potency on human AChE inhibition (IC50 = 11.7 nM) over the marketed drug donepezil (IC50 = 23.1 nM) and to a significant decrease in the self-aggregation of Aβ42 (~60%), whereas the replacement of the diethylamino group with a phenylpiperidine group (30b) decreased the hAChE inhibitory activity (IC50 = 4.1 µM) significantly and did not induce any activity on hBuChE and the self-aggregation of Aβ42. In addition, 30a was able to block the neurotoxic effects induced by Aβ42 oligomers, thereby showing neuroprotective behavior, which made it a potential disease-modifying agent [31a) with three carbon unit lengths between tacrine and piperazine showed better inhibitory activity toward AChE  than compound 31b with a two-carbon unit linker . However, this linker effect was not reflected in BuChE inhibition; the most potent compound was 31b (IC50 = 16.12 nM). Most importantly, the bulky group substitution (phenyl) on coumarin reduced the ChE inhibition activity. On the other hand, the linker length did not show any significant profile on the MAO inhibition. The simultaneous substitution positions 3 and 4 at the coumarin moiety with the methyl group afforded the most potent MAO-B inhibitor, compound 31c , the inhibitory activity of which was about 10-fold more potent than that of compound 31a . The hybrids also exhibited low toxicity to SH-SY5Y cells, and in vitro studies confirmed their ability to penetrate the BBB [In recent years, coumarin derivatives have received a lot of attention because of their biological profiles related to AD . Coumaring agent . Kong et the BBB .32) through molecular docking, molecular dynamics (MD) simulation and binding-free energy calculations. The rational design of the MTDL adapts the combination of tacrine (AChE inhibitor) and modified dehydrozingerone (myeloid differentiation 2 protein inhibitor (MD2), which is necessary for TLR4 signaling), and the two chemical entities were linked with different lengths of linkers. The calculated binding energy of the designed MTDL  was much better than the standard drug tacrine  in both enzymes. The results of the MD simulation confirmed that the designed ligand acts as a suitable inhibitor for AChE and MD2 proteins. Therefore, it can be applied as an effective antioxidant that can also target two other agents of AD appropriately [33 . With the help of this assessment, the authors synthesized the compounds and performed biological experiments [34 was a more effective AChE inhibitor as compared to the fluoro derivative, while the reverse trend was observed in MAO-B inhibitory activity. Though the potential binding orientations of the MTDLs to AChE and MAO-B were identified by molecular modeling, the experimental data did not stand in agreement with the computational data [35) and performed molecular docking studies against MAO-A/B and ChE  and epidermal growth factor receptor (EGFR) tyrosine kinases ,90. Anot


Heating to 1200 °C or higher resulted in the formation of a single spinel phase regardless of whether the powders were mechanically activated or not. Neutron diffraction analysis was used to determine cation site occupancy and revealed that mechanical activation resulted in a lower degree of cation site inversion compared to the nonactivated materials, which indicated that the powders were closer to thermodynamic equilibrium. This is the first study to characterize the effects of mechanical activation on crystallographic site occupancy in magnesium aluminate spinel using neutron diffraction.The synthesis and crystallographic site occupancy were investigated for MgAl It is the prototype for the spinel structure and is attractive due to its high melting point, corrosion resistance, mechanical properties, and low cost Z4, where i is the inversion parameter, with tetrahedral site occupancy given in parentheses and octahedral site occupancy given in square brackets [2O4 can range from 0.1 to 0.6, but powders synthesized by solid-state reaction typically have an inversion parameter that is around 0.25 [In naturally occurring MgAl lattice . Similarbrackets . Site inund 0.25 . Site inund 0.25  and Al [und 0.25 . In contund 0.25 .Mechanical activation is a high-energy ball milling process that induces physical and chemical changes in materials ,15,16. M2O4 spinel (MAS).The aim of the present study was to determine the influence of mechanical activation on site occupancy in the MgAlMagnesium oxide powder  was heated in air at 10 °C/min and calcined at 1000 °C for 4 h to decompose any hydroxide or carbonate species. The calcined MgO powder was then mixed with alumina powder  by dry ball milling for 24 h with ½” alumina medium using a medium-to-powder mass ratio of 1:1. After mixing, the precursor powder was ground using a pestle and mortar and sieved to 80 mesh. Approximately 12 g of the mixed powder was mechanically activated (MA) using high-energy ball milling  with 5 mm alumina spherical medium and an alumina mill jar. The medium-to-powder mass ratio was approximately 3:1. The powder was milled for 60 min using a cycle of 30 min on, 15 min off, and then the final 30 min to minimize powder heating. After milling, the powder was passed through an 80-mesh sieve with minimal grinding.Powders that were either as-mixed or mixed plus mechanically activated were loaded into alumina crucibles and reacted at temperatures from 1200 °C to 1500 °C for 2 h in stagnant air . The heating rate was 10 °C/min, and the furnace was allowed to cool at its natural rate after the isothermal hold. Powders were denoted as either mixed (XD) or mechanically activated (MA) along with the calcination temperature .Reacted powders were lightly ground using a mortar and pestle and sieved to −200 mesh for X-ray diffraction . Phase analysis of XRD data was performed by Rietveld refinement . Phases were modeled using the appropriate ICSD data. Powder morphologies were examined using scanning electron microscopy . Powders were coated with a conductive Au/Pd layer before SEM.12 C. The central wavelength was 1.5 Å, which was used to assess lattice spacing from 0.485 Å to 13 Å. The total number of refined diffraction peaks was 133 per sample.Neutron diffraction patterns were collected on POWGEN at ORNL through the mail-in program . The aveiso) on a specific site were constrained to be equal for Al and Mg. The specific site was constrained to be fully occupied . Both parameters are heavily correlated with Mg and Al occupancy. Furthermore, site occupancy could not be refined without these constraints or the restrains in the next paragraph.Rietveld refinement was performed using GSAS-II . As a stThe chemical composition target was 8 Mg and 16 Al per unit cell with a restrain weight factor of 150. If the initial assumption for the weight factor were too high, the refinement would stay at the initial configuration without changing the site occupancy. If the initial assumption for the weight factor were too low, then the chemical composition would deviate to become Mg-deficient and Al-rich. The weight factor was manually adjusted to restrict the nominal Mg content per unit cell to 8.00 ± 0.05 atoms for all refinements. The procedure described herein enabled optimization of all fitting parameters in the first step and then site occupancy in the second step. This refinement procedure was reproducible in contrast to sequential fitting methods that resulted in different site occupation with each iteration.2O4. Analysis of the XRD patterns (2O4 (PDF: 01-074-1133) without any detectable residual MgO or Al2O3, or other impurity phases [2O4.Heating the mixed or mechanically activated precursor powders to 1200 °C, 1300 °C, 1400 °C, and 1500 °C produced crystalline MgAlpatterns  for XD ay phases . All of 2O4.As with the XRD results, the neutron diffraction patterns of both the XD and the MA materials indexed to the spinel structure. 2O3, and it was not included in the refinement. In i = 0.13, indicating that 13% of the Mg atoms on octahedral sites were replaced by Al atoms. This was the lowest inversion parameter of the XD materials.The fractions of 16c and 8b sites occupied by Mg and Al were refined with isotropic displacement parameters for each powder. All 133 diffraction peaks that were observed for each specimen were used in each of the refinements. An impurity phase indicated by two small peaks was detected by NPD that was not detected by XRD. This impurity phase is likely residual MgO and/or AlThe Mg1 and Al2 columns correspond to the site occupancy of the 16c or octahedral position in the spinel structure. As stated above, the refinements were set such that each site was fully occupied, i.e., Al1 + Mg2 = 1 and Al2 + Mg1 = 1. The last column labeled “Mg” is the number of Mg atoms per unit cell after refinement, which was used as a final check of the validity of the fit. The weight factor was manually adjusted to keep this deviation ±0.05 from the nominal composition of eight Mg atoms per unit cell. The Rietveld refinement produced results with similarities with previous work on site occupancy and mechanical activation, as discussed in greater detail below.i = 0.149, significantly lower than the XD1200C sample. This trend of decreasing inversion value as calcination temperature increased continued, with XD1400C and XD1500C having inversion parameter values of 0.132 and 0.130, respectively. All of the mixed powders had inversion parameters of at least 0.13, with inversion decreasing as calcination temperature increased. The decrease in inversion parameter with increasing calcination temperature is thought to be due to the higher mobility of the cations at higher temperatures allowing the cations to shift to their equilibrium positions.The site occupancy data from i = 0.075 for MA1300C and 0.074 for MA1400C. In contrast, increasing the calcining temperature to 1500 °C for MA1500C led to an increase in the inversion parameter to 0.097. As with the XD powders, the decrease in inversion parameter when calcination temperature increased from 1200 °C to 1400 °C was thought to be due to same mechanism that led to the decrease in inversion with increasing calcination temperature in the XD powders, i.e., increased cation mobility with increasing temperature. The increase in inversion parameter for MA1500C is thought to be due to a competing mechanism that increases cation disorder. While increasing temperature increases mobility and allows cations to move to their equilibrium positions, higher temperatures may also promote disorder through the increase in entropy and site disorder with increasing temperature.The degree of inversion of the mechanically activated powders did not show a clear trend with temperature. The inversion parameter was about 0.124 for MA1200C. The MA1300C and MA1400C samples each had lower values of cation site disorder with values of Regardless of temperature, the inversion parameters for the MA materials were all lower than the lowest inversion value for the XD samples. Mechanical activation is known to produce defects in powder particles that result in a higher energy state compared to the nonactivated powders. Our hypothesis is that this higher-energy starting state allowed the MA powders to get closer to equilibrium than the XD powders for the same heat treatment temperature. These results generally agree with a previous study by Obradovic et al., who used Raman spectroscopy to characterize cation inversion in mechanically activated spinel powders . Whereas2O4 powders were studied to determine the effect of temperature and mechanical activation on the cation site occupancy. Heating the various powders to at least 1200 °C produced single-phase MgAl2O4 spinel powders, with or without mechanical activation, which was confirmed by both XRD and neutron diffraction analysis. The mechanically activated MgAl2O4 powders had lower degrees of inversion than the lowest inversion parameter for mixed powders. The relationship between temperature and the degree of inversion indicated that competing mechanisms affect cation site occupancy. The lower inversion in MA powders compared to XD powders was attributed to the higher-energy state of the MA powders prior to calcination that provided a higher driving force to approach equilibrium  at the same calcining temperature. In addition, higher calcining temperatures appear to increase atom mobility and allow for lower degrees of inversion, but higher calcining temperatures can also result in more thermally activated site disorder. More research into how mechanical activation and temperature affect cation site disorder is needed to determine the mechanisms at play here.The crystallographic site occupancies of stoichiometric MgAl


Escherichia coli or by purification as natural proteins to study possible associations with different phenotypes of cat allergy  by analyzing molecular IgE recognition profiles in a representative cohort of clinically well-characterized adult cat allergic subjects (n = 84). IgE levels specific to each of the allergen molecules and to natural cat allergen extract were quantified by ImmunoCAP measurements. Cumulative IgE levels specific to the cat allergen molecules correlated significantly with IgE levels specific to the cat allergen extract, indicating that the panel of allergen molecules resembled IgE epitopes of the natural allergen source. rFel d 1 represented the major cat allergen, which was recognized by 97.2% of cat allergic patients; however, rFel d 3, rFel d 4, and rFel d 7 each showed IgE reactivity in more than 50% of cat allergic patients, indicating the importance of additional allergens in cat allergy. Patients with cat-related skin symptoms showed a trend toward higher IgE levels and/or frequencies of sensitization to each of the tested allergen molecules compared with patients suffering only from rhinitis or asthma, while there were no such differences between patients with rhinitis and asthma. The IgE levels specific to allergen molecules, the IgE levels specific to cat allergen extract, and the IgE levels specific to rFel d 1 were significantly higher in patients with four different symptoms compared with patients with 1–2 symptoms. This difference was more pronounced for the sum of IgE levels specific to the allergen molecules and to cat extract than for IgE levels specific for rFel d 1 alone. Our study indicates that, in addition to rFel d 1, rFel d 3, rFel d 4, and rFel d 7 must be considered as important cat allergens. Furthermore, the cumulative sum of IgE levels specific to cat allergen molecules seems to be a biomarker for identifying patients with complex phenotypes of cat allergy. These findings are important for the diagnosis of IgE sensitization to cats and for the design of allergen-specific immunotherapies for the treatment and prevention of cat allergy.Cat allergy is a major trigger factor for respiratory reactions (asthma and rhinitis) in patients with immunoglobulin E (IgE) sensitization. In this study, we used a comprehensive panel of purified cat allergen molecules  that were obtained by recombinant expression in  P allergy . Accordi allergy ,17,18,19 allergy . In addi allergy ; Fel d 3 allergy ; Fel d 4 allergy ,24; and  allergy .4 antibodies that block the binding of IgE to Fel d 1 in allergic patients [Without any doubt, Fel d 1 is the most important cat allergen, showing high IgE reactivity in the majority of cat-sensitized patients and high allergenic activity . It is apatients ,32. The However, to understand the possible relevance of IgE sensitization to cat allergen molecules other than Fel d 1, it is important to study, as a first step, the IgE sensitization and IgE levels to the complete panel of cat allergen molecules in a representative cohort of clinically well-characterized cat allergic patients. So far, limited data regarding the IgE reactivity of each allergen molecule are available from studies characterizing these allergens, while the full panel has not been studied. Only IgE reactivity to a limited number of cat allergen molecules has been studied for adults ,7 and evSera were collected from patients with clinical symptoms of cat allergy living in Moscow, the Moscow region, and Yekaterinburg. A/L, respectively  were performed in the two groups of patients, those with and without skin test results . We founectively .A/L for cat allergen molecules were detected in 75 out of the 84 patients with cat-related allergic symptoms  of the patients, and the mean level was 16.9 kUA/L (n = 22), with a mean level of 2.7 kUA/L. rFel d 3-specific IgE was found in 50.6% of patients (n = 36), with a mean level of 2.18 kUA/L. rFel d 4-specific IgE was found in 52% of patients (n = 38), with a mean level of 5.69 kUA/L. nFel d 6-specific IgE was found in 32.8% of patients (n = 24), with a mean IgE level of 0.82 kUA/L. rFel d 7-specific IgE was found in 54.7% of patients (n = 40), with a mean level of 4.53 kUA/L. rFel d 8-specific IgE was found in 42.4% of patients (n = 32), with a mean level of 0.37 kUA/L . Accordingly, they may be considered as major allergens according to the definition in . After rA/L for the sum of allergen molecules, cat allergen-specific IgE reactivity was detected in 75 out of 84 patients with cat-related allergic symptoms. Thus, two more patients were detected with allergen molecules than with the cat allergen extract  were significantly correlated with IgE levels specific to cat allergen extract, indicating that the seven allergen molecules can be used to replace the cat allergen extract for measuring allergen-specific IgE levels . Specifi extract . However extract . In five extract .Next, we were interested in understanding if molecular allergen-specific IgE recognition profiles and cumulative allergen-specific IgE levels are associated with different phenotypes of cat allergy as defined by the number of different cat-related allergic symptoms. We found that 21 patients suffered from 1–2 symptoms of cat allergy, 39 patients suffered from 3 different symptoms of cat allergy, and 13 patients suffered from 4 different symptoms of cat allergy . Figure When the sum of the IgE levels specific to allergen molecules, the IgE levels specific to the cat allergen extract, and the IgE levels specific to rFel d 1 were compared, it turned out that the allergen-specific IgE levels were significantly higher in patients with four symptoms compared with patients with 1–2 symptoms B. IntereA/L may cause a clinically relevant sensitization. Lastly, it is possible that, in certain patients, allergen-specific IgE antibodies are mainly bound to effector cells via IgE receptors and, thus, cannot be detected by IgE serology. This explanation is not unlikely because all of the patients who were skin-tested without allergen-specific IgE in serum showed positive skin reactions. Support for this explanation comes from a study that showed that treatment of allergic subjects with the anti-IgE antibody omalizumab, which prevents IgE binding to IgE receptors, could indeed visualize new IgE specificities in the sera of treated patients [To the best of our knowledge, our study is the first to determine IgE reactivity profiles and IgE levels specific to the complete panel of purified cat allergen molecules  as well as cross-reactive carbohydrate epitopes, as represented by nFel d 6, in a clinically well-characterized group of adult cat-allergic patients. Several relevant results were obtained. First, we found that the panel of tested allergen molecules seemed to resemble the majority of IgE epitopes present in natural cat allergen extracts. This aspect was studied by quantifying the cumulative sum of cat allergen molecule-specific IgE concentrations and by comparing it with allergen extract-specific IgE levels using a test system, allowing for their quantification, i.e., the ImmunoCAP system . We founpatients . At presThe determinations of the molecular IgE reactivity profiles were performed in a representative cohort of clinically well-characterized patients for whom cat-related symptoms of allergy were carefully recorded by case history, so that they could be definitively attributed to cat exposure. The fact that we did not perform provocation studies by exposing patients under controlled conditions to cats to confirm the symptoms of rhinitis, asthma, conjunctivitis, and dermatitis may be considered as a limitation of our study, but this could only have been performed in a large prospective study. Regardless of this limitation, our study demonstrated unambiguously that, in addition to the major cat allergen, rFel d 1, which was recognized by 97% of the cat allergic patients, other cat allergen molecules are important. rFel d 3, rFel d 4, and rFel d 7 were recognized by the IgE antibodies of more than 50% of the study population; hence, they must be considered as major or mid-tier allergens according to the frequency of IgE recognition. It is understood that measuring allergen-specific IgE reactivity alone is not sufficient to demonstrate the clinical relevance of additional allergen molecules in cat allergic patients; however, this finding highlights that it will be important to investigate in depth the contribution of allergens other than Fel d 1 to symptoms of cat allergy by provocation testing or at least by studying their allergenic activity in surrogate assays for allergic inflammation using freshly collected blood samples for basophil activation testing. Our study does, however, provide indirect evidence that allergen molecules other than Fel d 1 are clinically important because we found that the significance of the correlation of IgE levels specific to the sum of allergen molecules with complex phenotypes of cat allergy was higher than that of Fel d 1-specific IgE levels. Furthermore, we noted a trend toward higher IgE levels for allergens other than rFel d 1 in patients with symptoms of cat-related dermatitis.Our study, thus, indicates that, in addition to rFel d 1, rFel d 3, rFel d 4, and rFel d 7 must be considered as important cat allergens. Accordingly, it will be important to consider additional cat allergens for allergen-specific treatment by AIT or passive immunization therapy for cat allergy. Furthermore, our study demonstrates the importance of molecular IgE diagnosis for cat allergy and indicates that the cumulative sum of cat allergen-specific IgE could be a biomarker for identifying patients with complex phenotypes of cat allergy. Our findings are, thus, important for the molecular diagnosis of IgE sensitization to cat and the design of specific molecular immunotherapies for treatment and prevention of cat allergy.As a first step, all patients completed the standardized ISAAC questionnaire for obtaining first-line evidence for or against an allergic phenotype . SubjectEscherichia coli and purified as previously described [E. coli expression coding for Fel d 3, Fel d 4, Fel d 7, and Fel d 8 with 3′ DNA coding for a C-terminal hexahistidine tail, were synthesized and inserted into the NdeI/EcoRI sites of plasmid pET27b . The DNA sequences and their correct insertion into the plasmid were confirmed by automatic sequencing of both DNA strands (ATG). E. coli BL21 DE3 transformed with the plasmids expressing Fel d 3, Fel d 4, Fel d 7, and Fel d 8 were grown at 37 °C in an incubator in LB medium containing kanamycin (34 μg/mL) at 37 °C until they reached a cell density (OD600 nm) of 0.3–0.6. The expressions of rFel d 3 or rFel d 7 proteins were induced by the addition of 1 mM isopropyl-β-thiogalactopyranoside for 3 h at 37 °C. The cells were harvested by centrifugation at 10,000× g for 10 min and frozen. The pellets from a 400 mL culture were opened with three cycles of freezing and thawing, resuspended in 40 mL of 20 mM Tris-HCl (pH 7) and 10 mM imidazole, and then incubated overnight at 4 °C with stirring. After centrifugation, the pellet was discarded and the supernatants were subjected to nickel affinity purification under native conditions, as described in the Qiagen purification protocol .Recombinant Fel d 1 was expressed in escribed . Purifieg for 10 min and frozen. The pellets from a 400 mL culture were resuspended in 40 mL of 8 M urea, 10 mM Tris-HCl (pH 8), and 100 mM NaH2PO4; further homogenized with an Ultra-Turrax  on ice; and then incubated overnight at 4 °C under constant stirring. The supernatant was subjected to nickel affinity chromatography under denaturing conditions (Qiagen). Fractions containing pure protein were united and refolded by extensive stepwise dialysis against 2 mM HEPES (pH 8) in the case of Fel d 4 and against 150 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl (pH 8.5) in the case of Fel d 8; they were stored in PBS as soluble proteins at −20 °C until use. The purity of the recombinant proteins and natural allergens was analyzed by SDS-PAGE  was used according to the instructions by the manufacturer. Streptavidin ImmunoCAPs  were used to prepare ImmunoCAPs containing rFel d 1, nFel d 2, rFel d 3, rFel d 4, nFel d 6, rFel d 7, and rFel d 8. Cat allergen extract-specific IgE levels were determined using the commercial ImmunoCAP e1 (Thermo Fisher Scientific/Phadia). For preparation of the molecular ImmunoCAPs allergens with streptavidin o212 ImmunoCAPs, each allergen was dialyzed extensively against 0.1 M NaHCOescribed ,49,50.A/L were considered positive [Allergen-specific IgE levels greater than or equal to 0.1 kUpositive ,51.SPSS Statistics version 15.0 and Graphpad Prism version 6.0 were used for statistical calculations. Significant differences were calculated using the Mann–Whitney U test.Our study showed that the panel of cat allergens consisting of rFel d 1, nFel d 2, rFel d 3, rFel d 4, nFel d 6, rFel d 7, and rFel d 8 resembled the IgE epitopes of the allergen source. The sum of cumulative IgE levels specific to allergen molecules was best correlated with different phenotypes, whereby higher levels indicated more symptoms. Our study also indicated that allergens in addition to rFel d 1 are important and need to be considered for molecular diagnosis and the development of molecular forms of allergen-specific treatment.


Increases in antibiotic usage and antimicrobial resistance occurrence have caused a dramatic reduction in the effectiveness of many frontline antimicrobial treatments. Topoisomerase inhibitors including fluoroquinolones are broad-spectrum antibiotics used to treat a range of infections, which stabilise a topoisomerase-DNA cleavage complex via intercalation of the bound DNA. However, these are subject to bacterial resistance, predominantly in the form of single-nucleotide polymorphisms in the active site. Significant research has been undertaken searching for novel bioactive molecules capable of inhibiting bacterial topoisomerases at sites distal to the fluoroquinolone binding site. Notably, researchers have undertaken searches for anti-infective agents that can inhibit topoisomerases through alternate mechanisms. This review summarises work looking at the inhibition of topoisomerases predominantly through non-intercalating agents, including those acting at a novel allosteric site, ATPase domain inhibitors, and those offering unique binding modes and mechanisms of action. Infectious disease contributes significantly to worldwide mortality rates, with over 10 million deaths attributed to this class of disease in 2019 . As suchResistant infections are increasing due to a multitude of reasons, including overprescribing antibiotics, their use in agriculture, and poor public understanding of the causes and implications of antibiotic resistance ,5,6. CurBroad-spectrum antibiotic activity can be achieved through the inhibition of essential proteins and enzymes found to be relatively well conserved across bacteria. For example, topoisomerases are found in all prokaryotes and eukaryotes, and some viruses . TopoisoEscherichia coli contains topoisomerase I and topoisomerase III (type I), both of which are dimeric and reduce negative supercoiling in DNA .,33.5, 32bacteria ,35. Workbacteria , whilst teria 6, ,38. Advateria 6, .In addition to NBTIs and spiropyrimidinetriones, significant work has been produced studying inhibition of alternative binding sites of topoisomerases, including that of the second catalytic domain within DNA gyrase and topo IV, the ATPase domain found in GyrB/ParE, as well as a novel allosteric site identified in the vicinity of the GyrA active site. Through the targeting of distal sites within topoisomerases, it may be possible to reduce the current usage of FQs, thereby extending their lifespan and giving potential for combined-therapeutic treatments targeting DNA gyrase and topo IV. This review aims to summarise the research to date primarily focussing on non-intercalating inhibitors targeting alternative sites in bacterial topoisomerases.7 to 8  and Gram-negative bacteria . Efflux of both compounds was proposed, with inhibition in efflux pump mutants of E. coli, P. aeruginosa, and A. baumannii improving efficacy by up to 64-fold.Researchers at GSK identified an allosteric site in DNA gyrase in 2017, providing a potential route to novel alternatives to FQs. A high-throughput screen by Chan et al. identified a thiophene-bearing compound, which was subsequently shown to bind to DNA gyrase in a previously unidentified pocket, dubbed the allosteric site, at the interface between GyrA and GyrB in the vicinity of the active site of the enzyme  41]. Op. Op41]. 7 to 8  improved8 was found be unaffected by mutations to key residues in GyrA, ParC, and ParE, with potent inhibition observed in FQ-resistant strains. However, one alternate mutation in GyrB-Glu793  was identified which reduced inhibition caused by 8. Further studies confirmed that the mechanism of action was different to that of FQs, as single- and double-strand DNA cleavage complexes were formed in equal quantities, as opposed to the entirely double-strand cleavage caused by the actions of ciprofloxacin. Although compound 8 showed promise due to its novel mode of action, this particular series was discontinued due to in vivo toxicity issues  and 7PQM [(S)-enantiomer]), as well as P. aeruginosa (PDB ID 7PTG [(S)-enantiomer]).Hu et al. developed a and ParE . This leParE indol-8-amine core , derived and 24b  to creat25 . It also makes distinct interactions with GyrB compared to novobiocin, leading to differences in resistance patterns. For example, novobiocin forms a key bonding interaction with Arg141, but site-directed mutagenesis proved that interaction with this residue is not a requirement for 26 to maximise its potency.However, subsequent work by Henderson et al. determined compound ricycles . MoreoveE. coli GyrB ATPase sub-domain, highlighting the high hit-rate a fragment-based approach can yield , with moderate potency against topo IV (IC50 E. coli = 113 μM) [29, Kibdelosporangium sp. (MA7385), which also shows ATPase inhibitory activity, whilst acting with a different mode of inhibition to novobiocin [89 (S. aureus numbering) to form a bonding interaction, which is key to many other DNA gyrase ATPase inhibitors. The natural product displays a U-shaped conformation, which shares no common features to the way in which novobiocin binds to the same region, and hence its ATPase inhibition differs from that of novobiocin [23a and 23b, highlighting its versatility across scaffolds [50 = 60 nM E. coli), though this does not translate to potency in Gram-negative bacteria (MIC >64 μg mL−1 of E. coli) [Several natural products have been previously identified, which branch away from the modes of inhibition outlined above. The first, a polythioamide produced by mide 28, , was fou 113 μM) ,69. The vobiocin . Kibdelovobiocin . Kibdelocaffolds ,72. KibdE. coli) .30, Streptomyces antibioticus Tü 6040, has further been the subject of much research. This bifunctional molecule features both an aminocoumarin head and polyketide, and studies have found that it binds in two sites on DNA gyrase [The natural product simocyclinone D8 30, , derivedA gyrase . The priA gyrase . HoweverE. coli in in vitro testing (IC50 = 0.41 μM E. coli DNA gyrase) [X-ray crystallography shows that the polyketide head does indeed occupy a space adjacent to ciprofloxacin, and hence likely intercalates DNA, but the site is nonetheless distinct. In fact, the dominant mechanism of action of simocyclinone D8 is to prevent cleavage complex formation, in stark contrast to FQs which allow formation and then stabilise the cleavage complex. Although simocyclinone D8 does also bind to a site on GyrB, this is the minor binding site, and whilst it cooperatively functions with binding on GyrA, the GyrB binding is calculated to be approximately 1000-fold weaker ,76. It f gyrase) .The natural products reported in this section have all been characterised as bacterial topoisomerase inhibitors via macromolecular biosynthesis and supercoiling DNA gel-based assays with ciprofloxacin and novobiocin as controls. To our knowledge, their potential off-target effects have not been characterised with respect to structural features such as Michael acceptors and redox-active aromatics. Therefore, follow-up characterisation is warranted to rule out chemical promiscuity or pan-assay interference before pursuing further as bacterial topoisomerase inhibitors .Although improvement upon and development of new FQs will prove fruitful in the generation of new antibiotics, other approaches are required to prevent the antibiotic pipeline from drying up. Topoisomerases are a known target, which have been validated in both Gram-positive and Gram-negative bacteria, making them an excellent drug target. This review has summarised key research into topoisomerase inhibitors with different modes of action to FQs, particularly those which target a recently discovered allosteric site in the GyrA/ParC subunit, and GyrB/ParE inhibitors which bind to the ATPase site. Moreover, a discussion of compounds with novel modes of action highlighted that other DNA-intercalating compounds exist which do not proceed via the FQ mechanism of action, nor that of NBTIs. The research highlighted above shows that the scope for novel topoisomerase inhibitors is broad, and future research may produce new life-saving antibiotics, with some research mentioned already in clinical trials.


The influence of thread profile on the fixation stability of bone screws remains unclear. This study aimed to compare the fixation stability of screws with different thread profiles under several loading conditions.Bone screws that differed in thread profile  only were made of stainless steel. Their fixation stabilities were evaluated individually by the axial pullout test and lateral migration test, besides, they were also evaluated in pairs together with a dynamic compression plate and a locking plate in polyurethane foam blocks under cyclic craniocaudal and torsional loadings.The triangle-threaded and square-threaded screws had the highest pullout forces and lateral migration resistance. When being applied to a dynamic compression plate, higher forces and more cycles were required for both triangle- and square-threaded screws to reach the same displacement under cyclic craniocaudal loading. On the other hand, the triangle-threaded screws required a higher torque and more cycles to reach the same angular displacement under cyclic torsional loading. When being applied to a locking plate, the square-threaded screws needed higher load, torque, and more cycles to reach the same displacement under both cyclic craniocaudal and torsion loadings.The triangle-threaded screws had superior pullout strength, while square-threaded screws demonstrated the highest lateral migration resistance. Moreover, dynamic compression plate fixation with triangle- and square-threaded screws achieved more favorable fixation stability under craniocaudal loading, while triangle-threaded screws demonstrated superior fixation stability under torsional loading. Locking plate fixation with a square-threaded screw achieved better fixation stability under both loading types. Bone screw, a powerful mechanical device with versatile functions, has been frequently applied in orthopedic surgeries . It can Even with good clinical outcomes, the screw failure rate reported clinically is high, especially among patients with poor bone quality –7. The iImproving the fixation strength of bone screws is a feasible way to reduce the failure rate . Screw tThe thread design of a bone screw can vary in pitch, depth, and shape. The relationships between the mechanical properties of bone screws and the modifications of thread pitch and depth have been well documented . The ratLocking screws and compression screws of 3 different thread designs  were manufactured from the 316 LVM stainless steel. All the screws were self-tapping screws with a thread pitch of 2.0 mm, major and minor diameters of 4.5 mm and 3.2 mm, respectively, screw shaft length of 38 mm, and had the same cutting flute design. The 4.5-mm screws were used for compatibility with the standard instrumentation for the 4.5-mm AO cortical screws by relying on the identical drill bits . All the screws were used once in the following tests.LP and DCP were specially designed for the above locking and compression screws and manufactured from the 316 LVM stainless steel  that adhered to ASTM F-1839-08 were used [Solid rigid polyurethane foam blocks with a density of 0.32 g/cmere used . The denere used –25.Thereafter, individual screw axial pullout and lateral migration tests, and multi-screws stability of plate fixation test, which were based on our previously published work , were coIn brief, polyurethane foam blocks were cut into small pieces  Fig.  a. ThereThe polyurethane foam blocks were cut into small pieces   and later a loading force was applied onto the upper surface of the foam block using an MTS Mini Bionix system Fig.  c and d.The force-displacement curves in the axial pullout test are presented in Fig. Figure In the DCP/compression screw constructs subject to craniocaudal loading, compared wiht buttress-threaded screws, both the squared- and triangle-threaded screws required more cycles and stronger force to achieve an identical displacement. For triangle- and square-threaded screws, the cycle number and load needed to obtain an identical displacement were similar, with no significant difference Fig.  a and b.In the DCP/compression screw constructs subject to torsional loading, compared with buttress- and square-threaded screws, a greater number of cycles and increased torque were needed for triangle-threaded screws to obtain an identical torsion angle. For buttress- and square-threaded screws, similar cycle number and torque were needed to achieve an identical displacement, and the difference was not significant Fig.  c and d.In the LP/locking screw constructs subject to craniocaudal loading, a greater number of cycles and an increased load were required for the square-threaded locking screws to obtain the same displacement as those of buttress- and triangle-threaded screws. There were significant differences in the cycle number and load to achieve 1–5 mm displacements between screws of the two thread types. Buttress- and triangle-threaded screws required similar cycle number and force for obtaining an identical displacement, and there was no significant difference (Fig. In the LP/locking screw constructs subject to torsional loading, a greater number of cycles and a higher torque were needed for the square-threaded screws to acquire an identical torsion angle to those of buttress- and triangle-threaded screws. Differences in the cycle number and torque needed to obtain the torsion at 2/4/6/8/10 degrees were significant between the two thread types. The buttress- and triangle-threaded screws had similar requirements on cycle number and torque to obtain an identical displacement, with no significant difference Fig.  c and d.The individual screw tests revealed that screws with triangle threads were superior in axial pullout strength, while those with square thread were superior in lateral migration resistance, and those with buttress thread were inferior in both axial pullout strength and lateral migration resistance. The buttress thread was proposed by Robert Danis in the 1940s as the standard thread for bone screws, which did not perform well in the axial pullout test. The triangle thread, which was the standard thread of the industrial screw for connection, however, had the best axial pullout strength. In this study, the pullout strength of the triangle-threaded screw was 31.3% higher than that of the buttress-threaded screw. This was consistent with the finding by Kim et al. in a pedicle screw-related biomechanical test that the strangle thread had the highest pullout strength compared with buttress thread and square thread . This inLater, this study compared the screws applied to a DCP in pairs under cyclic craniocaudal loading, as a result, the square-threaded screws had comparable fixation stability to triangle-threaded screws, and both of them had better fixation stabilities than buttress-threaded screws. Under the cyclic torsional loading, screws with triangle threads had better fixation stability than those with buttress and square threads, and those with buttress thread achieved comparable fixation stability to those with square thread. These results indicate that when standard buttress-threaded screws are used as the compression screws to repair fractures in conjunction with a DCP, they have the worst fixation stability under both craniocaudal and torsional loadings. The triangle thread, however, displayed better performance. This suggests that a compression screw product should have a triangle thread design, rather than the standard buttress thread design, so as to improve its fixation strength.For the locking screws applied to a LP in pairs, screws with square thread had better fixation stability than those with buttress and triangle threads, and those with buttress thread attained comparable fixation stability to those with triangle thread under both craniocaudal and torsional loadings. These results reveal that when being used as the locking screws to repair fractures in conjunction with a LP, the standard buttress thread and triangle thread demonstrate the worst fixation stability under both craniocaudal and torsional loadings. The square thread, however, had better performance. This demonstrates that a locking screw product should have a square thread design, rather than the standard buttress or triangle thread design, for the sake of improving its fixation strength. However, the area of the square thread profile is twice of that of buttress and triangle threads, which suggests that more bones will be damaged and removed, and a higher insertion torque will be required when the screw is inserted. This may limit its application in bone screw design.Thread is an important structure for bone screws when designing devices that will be subject to multiple loading conditions. Different thread shapes allow for modifications in the contact surface area and shear force development at the screw-bone interface, thereby affecting the fixation stability of the device. Previously, the fixation stability of bone screws has been consistently evaluated by a pure axial pullout test –37. It iThe buttress thread, triangle thread, and square thread tested in this study were all from the industrial screw thread designs. None of them demonstrated superior performance in all the individual screw biomechanical tests and multi-screws biomechanical tests in our study. Moreover, the influences of these three different thread designs on the insertion torque of the bone screw remain unclear. Screw threads designed specifically for human-related physiological loading with modest insertion torque should be developed, instead of directly relying on the existing thread designs from the industrial screws.Several limitations of the present study should be considered. First, synthetic bone models are a consistent medium for mechanical testing, which do not perfectly simulate normal human bone. However, the polyurethane foam is microstructurally similar to human bone and exhibits similar mechanical properties while avoiding the inconsistencies induced by interindividual variations in human bones. Second, since the bone samples used in this study were synthetic cancellous bones, which were unable to represent the clinical environment in which the cortical bone was the main factor of bone screw stability. Thus, more research is needed to evaluate how the three different thread types perform in a bone analog with cortical shell. Third, the present work utilized torsional loading and cyclic craniocaudal loading to simulate physiological loading, while these two loading types did not comprehensively mimic the extremely complicated arm or leg loading conditions in human body during daily activities. Nonetheless, the loadings utilized in the present work mostly simulated our daily activities. At last, the present biomechanical research analyzed the simulated bone under normal density condition as an example. In the future, more studies should be conducted to test bone densities that represent osteoporosis and osteopenia to strengthen our conclusion. The conclusions of this study were drawn taking these limitations into account.In summary, the triangle-threaded screws had superior pullout strength, while square-threaded screws demonstrated the highest lateral migration resistance. The DCP fixation stability under craniocaudal loading was not different between triangle- and square-threaded screws, while both of them had superior fixation stabilities to buttress-threaded screws. Under torsional loading, DCP fixation with triangle-threaded screws achieved superior fixation stability. LPs with square-threaded screws required a larger force and more cycles to reach the same amount of displacement under both craniocaudal loading and torsional loading. The findings of this study may have limited clinical significance, but they will provide new insight for surgeons and bone-implant device companies and will eventually benefit clinical practice.


S)-hydroxycholesterol (24S-OHC)—has been shown to cause cytotoxicity when esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the endoplasmic reticulum (ER). Here, we show that the accumulation of 24S-OHC esters caused phosphorylation of eukaryotic translation initiator factor 2α (eIF2α), dissociation of polysomes, and formation of stress granules (SG), resulting in robust downregulation of global protein de novo synthesis in human neuroblastoma SH-SY5Y cells. We also found that integrated stress response (ISR) activation through PERK and GCN2 activation induced by 24S-OHC treatment caused eIF2α phosphorylation. 24S-OHC-inducible SG formation and cell death were suppressed by inhibition of ISR. These results show that ACAT1-mediated 24S-OHC esterification induced ISR and formation of SG, which play crucial roles in 24S-OHC-inducible protein synthesis inhibition and unconventional cell death.Perturbation of proteostasis triggers the adaptive responses that contribute to the homeostatic pro-survival response, whereas disruption of proteostasis can ultimately lead to cell death. Brain-specific oxysterol—i.e., 24( Cellular protein homeostasis, which is also known as proteostasis, is essential for living cells to maintain their normal cellular function . ProteosThe UPR, which is activated by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER), is mainly an adaptive response that encompasses ISR activation , 6. The Upon phosphorylation of eIF2α, when translation is initiated in the absence of a ternary complex consisting of eIF2, GTP, and initiator methionyl-tRNA, a stalled 48S pre-initiation complex results . This prS)-Hydroxycholesterol (24S-OHC) is an enzymatically formed oxysterol that is catalyzed by the brain-specific cholesterol 24-hydroxylase (CYP46A1). As 24S-OHC is able to effectively pass through the blood–brain barrier, it plays a vital role for regulating brain cholesterol homeostasis [de novo synthesis. Although ER dysfunction contributes to the 24S-OHC-induced cell death [24 condition; moreover, these effects could be suppressed by cotreatment with the ACAT inhibitor F12511 Fig. . In contervation , we obseBecause phosphorylation of eIF2α causes the dissociation of polysomes and the accumulation of monosomes, leading to a decrease in translation initiation , we analAs phosphorylation of eIF2α and reduction of de novo protein synthesis are tightly linked to formation of SGs , 9, we nWe next wondered whether disease-associated proteins might also be recruited to SGs in response to 24S-OHC treatment. We therefore examined the localization of TAR DNA-binding protein 43 (TDP-43)—an amyotrophic lateral sclerosis (ALS)-associated protein—because TDP-43 is known to be recruited to SGs in response to neuronal injury . As expede novo synthesis.We next carried out a time-course study to ascertain the effect of 24S-OHC treatment on SG formation and de novo protein synthesis. An immunocytochemical analysis showed that SGs appeared at 1.5 h and became more prominent with time Fig. . In corrWe then sought to examine the possible role of PERK–eIF2a pathway activation in 24S-OHC-inducible SG formation and cell death. Whereas in our previous report  we had sWe further observed that GSK2606414 inhibited SG formation in cells treated with 24S-OHC or thapsigargin Fig.  without Whereas 24S-OHC-inducible PERK phosphorylation was almost completely blocked by treatment with GSK2606414, a small amount of phosphorylated eIF2α was still observed Fig. , lane 4 As both PERK and GCN2 were found to be activated in cells treated with 24S-OHC, we took this to indicate possible activation of the ISR. To examine the involvement of ISR signaling, we investigated the effects of the inhibitor ISRIB, ISRIB being an activator of the eIF2 guanine nucleotide exchange factor ; the resISR is influential in maintaining or restoring proteostasis in response to various physiological and pathological conditions , 6. ISR We previously reported that ER dysfunction caused by accumulation of 24S-OHC esters and the accompanying decrease in ER chaperones induces PERK–UPR activation . ActivatAs we also observed that the global blockade of protein synthesis was significantly restored by ISRIB, indicating that a part of this translational downregulation is likely to be a downstream consequence of eIF2α phosphorylation. We postulate that, because an inhibitor of PERK, GCN2, and ISR cannot suppress ER membrane disruption, the inhibitory effects of GSK2606414, GCN2iB, and ISRIB on translational attenuation and cell death were partial. As we reported previously, RIDD might partially account for the observed decrease in newly synthesized proteins . Furtherde novo protein synthesis. Defects in SG dynamics have been linked to various degenerative disorders, such as ALS and AD [As SG formation is caused by eIF2α phosphorylation, various stressors that activate the ISR signaling pathway are involved in the triggering of SG formation –11. ThisS and AD , 45. We S and AD , it woulS and AD –22, induS and AD . It is aS and AD . It woulRegulated cell death (RCD) is a controlled cellular process in which a cell activates its own molecular autodestruction machinery . RCD is 24S-OHC  was dissHuman neuroblastoma SH-SY5Y cell line was purchased from the European Collection of Cell Cultures  and routinely maintained as described previously . The culde novo global protein synthesis, cells were exposed to 10 μg/ml puromycin for 15 min before lysis. Whole-cell lysates were immunoblotted with an antibody specific for puromycin to detect levels of puromycin-labeled proteins.Preparation of whole-cell extracts and immunoblotting were performed as described previously , 53. For2, and 0.1% NP-40) containing a protease inhibitor cocktail, 100 μg/ml CHX and 40 U/ml RNase inhibitor  at 4 °C for 30 min. The lysate was centrifuged for 10 min at 13,000 ×g. The supernatant (500 μl) was loaded onto a 10–50% sucrose gradient in polysome lysis buffer. The gradient was subjected to centrifugation in a Beckman SW55Ti rotor at 100,000 xg at 4 °C for 1 h, following which fifty 100-μl fractions were collected from the top. Absorbance was measured at 260 nm for each fraction.Cells were treated with 100 μg/ml CHX for 10 min prior to being harvested. Cells washed with ice-cold PBS containing 100 μg/ml CHX were lysed in 550 μl of polysome lysis buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 30 mM MgClImmunofluorescence staining was performed as previously described . Cover sP < 0.05 was employed.Unless otherwise indicated, data shown are the mean ± SD of at least three independent experiments. Statistical significance was calculated by analyzing variance using ANOVA and the Tukey–Kramer multiple comparisons test. A significance threshold of Supplemental figure


Moreover, we found that the peptide could significantly inhibit the proliferation and migration of Ang II-stimulated VSMCs. Further transcriptomic analysis revealed that differentially expressed genes (DEGs) were enriched in the cardiovascular disease-related pathways, and that the peptide may have the ability to regulate vascular remodeling. Our findings indicate the potential anti-hypertensive effects of FPQYLQYPY, as well-implicate its role in regulating vascular dysfunction.Hypertension is a major risk factor leading to cardiovascular disease, and is frequently treated with angiotensin I-converting enzyme (ACE) inhibitory peptides. The objective of this study was to separate and identify an ACE-inhibitory peptide from goat milk casein hydrolysates, and to evaluate its potential for improving angiotensin II (Ang II)-mediated adverse effects on vascular smooth muscle cells (VSMCs). A novel ACE-inhibitory peptide with the highest activity from the goat milk casein hydrolysates as determined by four steps of RP-HPLC was purified and identified as Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr (FPQYLQYPY). The results of inhibitory kinetics studies indicated that the peptide was a non-competitive inhibitor against ACE. Gastrointestinal digest  Hypertension is a major preventable risk factor for cardiovascular disease and all-cause mortality globally. The prevalence of hypertension is increasing worldwide due to unhealthy diet and lack of exercise , and in in vitro (Angiotensin I-Converting enzyme (ACE) is a key enzyme in the regulation of blood pressure and is the subject of most research . It can in vitro .The current standard care for the treatment of hypertension includes drugs which are mostly synthetic ACE-inhibitors, such as captopril and enalapril, to name a few. But, regardless of their anti-hypertensive effects, synthetic ACE-inhibitors are accompanied by undesirable side effects, including headache, cough, dizziness, fatigue, and diarrhea . TherefoIt has been shown that bioactive peptides from milk proteins have potential health benefits for humans and are used in health-promoting food as well as drug applications . Based oMilk protein is one of the good sources of ACE-inhibitory peptides, and various ACE-inhibitory peptides have been purified and identified from it using enzymatic hydrolysis or fermentation . The benin vitro activity of ACE-inhibitory peptides in goat milk remains insufficient  was provided by Ausnutria (Netherlands) and contained 63% protein and 22% lactose.−1) was purchased from China Pangbo Biological Engineering Co., Ltd. . Angiotensin Converting Enzyme from rabbit lung, hippuryl-L-histidyl-L-leucine (HHL), and α-amylase (109.8 U mg−1) were purchased from Sigma-Aldrich (USA). The Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), 0.25% trypsin-EDTA, penicillin-streptomycin solution, and Fetal bovine serum (FBS) were purchased from Gibco Life Technologies . Angiotensin II (Ang II) and the Cell Counting Kit-8 were purchased from MedChemExpress .Trypsin (10–20% activity protein) was purchased from Novozymes . Pepsin (10000 NFU mgThe hydrolysis process parameters were according to the reported method with some modifications . The speACE inhibitory activity was measured using an RP-HPLC method with Hippuryl-His-Leu (HHL) as a substrate, which generated hippuric acid (HA) under enzymatic hydrolysis . The samACE inhibitory activity (%) was confirmed by monitoring the formation of HA at 228 nm as detected by RP-HPLC on a C18 column . The mobile phase was 25% acetonitrile and 75% water with 0.01% trifluoroacetic acid with a flow rate of 1 mL/min. The inhibition activity was calculated using the following equation:0 = the HA peak area of control with HCL before pre-incubated.Where, A = the HA peak area of the control; B = the HA peak area of the sample; AThe inhibitory activity of the hydrolysates or collected fractions was expressed as percentage of ACE inhibition at a given protein concentration .−1 for 66 min, and 90% B in the next 5 min. Second, the active fraction was further purified by a RP Shim-pack PRC-ODS(K) column , and the elution was performed with 25–60% solvent B for 70 min. Third, the active fraction peptide was further purified by a RP ECOSIL C18 column , and the elution was performed with 30–43% solvent B for 60 min. Last, the fraction solution with the highest ACE-inhibitory activity was further separated using an analytical HPLC equipped with a C18 column , and the elution was performed with 25 to 30% solvent B for 60 min. The peak of highest activity was further analyzed using an analytical RP-HPLC equipped with the same C18 column, and the elution was performed with 10–60% solvent B for 40 min, 90% B for the next 10 min, and 10% B in the last 10 min.The hydrolysate was subjected to a four-step separation process. First, the hydrolysate was separated by a reverse-phase (RP) C18 column . Elution was performed with mobile phase A (0.1% trifluoroacetic acid in water) and mobile phase B (0.1% trifluoroacetic acid in acetonitrile) with a gradient of 10–48% B at a rate of 10 mL minThe absorption peaks of every step were fractionated, collected and lyophilized in order to measure the ACE-inhibitory activity. The eluted samples were monitored at a dual wavelength of 214 and 280 nm.The molecular mass of the peptide with the highest ACE-inhibitory activity was determined using an ABI 4800 MALDI-TOF-MS/MS . The N-terminal sequence of the purified peptides was identified by Protein Sequencer using the Edman degradation procedure .The peptide (FPQYLQYPY) was synthesized by Synpeptide Co., Ltd . The purity of the synthesized peptide was 98%, as evaluated by HPLC and the molecular mass was determined by LC/ESI–MS.To clarify the inhibitory mechanism of the most potent purified peptide on ACE, different concentrations of the ACE inhibitory peptide were added to each reaction mixture, according to a previously reported method . BrieflyIn vitro digestion of the potent purified ACE inhibitory peptide was carried out according to the reported method, with some adjustments (−1) was incubated with 2% (w/w) α-amylase, pepsin, or trypsin at 37°C.ustments , 23. Pep−1 and incubated with α-amylase for 5 min. Then the pH was decreased to pH 2 with the addition of 1 M HCl. Subsequently, the gastric digestion was initiated by adding pepsin at an enzyme-substrate ratio of 2:100 (w/w). The reaction was stopped after 2 h by raising the pH to 8.0 with 1 M NaOH, and trypsin was added to the reactor to continue gastrointestinal digestion for 4 h. Last, the reaction mixture was heated for 15 min in boiling water to inactivate the enzyme.In successive digestion tests, the peptide was first dissolved in buffer solution (pH 6.8) to 2 mg mLThe rat aortic vascular smooth muscle cell line A7r5, was purchased from Cell bank of Chinese Academy of Sciences . A7r5 cells between passage 8 and 20 were grown in DMEM supplemented with 10% FBS and antibiotics until they reached confluence. For experiments, the confluent cells were placed in a quiescing medium (DMEM + 1% FBS + antibiotics)  for 24 h2 incubator at 37°C for 24 h. Then, the medium of each well was replaced by 100 μL of fresh medium with different concentrations of FPQYLQYPY . After further incubation for 24 h, 10 μL CCK-8 solution was added to each well. Then, the plate was incubated for another 2 h. The absorbance of samples at a wavelength of 450 nm was measured using a microplate reader. The cell survival rate (%) was determined by the percentage of living cells in the test wells compared to the control wells.The cytotoxic effects of FPQYLQYPY in cell culture were evaluated using the Cell Counting Kit-8. First, A7r5 cells were seeded at 1 × 104 cells per well into 96-well plates  and cultured in a 5% CO3 cells per well into 96-well plates and grown in DMEM with 10% of FBS for 24 h, then the medium was replaced by quiescing medium overnight. The cells were co-treated with different concentrations of FPQYLQYPY  and Ang II (0.1 μM) for 24 h. The absorbance of samples at a wavelength of 450 nm was measured using a microplate reader. The cell survival rate (%) was determined by the percentage of living cells in the test wells compared to the control wells.Cell proliferation was measured by the Cell Counting Kit-8. A7r5 cells were seeded at 5 × 105 cells per well, and grown in complete medium until the cells covered the bottom of the plate, then the medium was replaced by quiescing medium for overnight.Migration of A7r5 cells was evaluated using the wound healing assay . Three l−1) and Ang II (1 μM) was added, then the cells were cultured in an incubator at 37°C and 5% CO2 for 24 h. The degree of cell migration was observed with inverted microscope and photographs were taken at 0 and 24 h. The migration distance was analyzed by ImageJ software.Afterwards, a sterile 200 μL spear head was used to quickly delimit at an average equal distance in the vertical direction according to the marking line, and washed with PBS to remove the floating cells. Quiescing medium with FPQYLQYPY  (A7r5 cells were collected and suspended in DMEM (1% FBS), and then the density was adjusted to 2 × 10NY, USA) , and at A7r5 cells were incubated with FPQYLQYPY and Ang II for 24 h. Then, total RNA was extracted using the TRIzol reagent , according to the manufacturer's protocol. After the quality test, the libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit  following the manufacturer's instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. .® PCR System 9700 . Then, Real-time PCR was performed using the LightCycler® 480 II Real-time PCR Instrument . The relative expression level was calculated using the 2ΔCt−Δ.Total RNA was extracted from A7r5 cells using TRIzol reagent, according to the manufacturer's specifications. The information of primer sequences is shown in post-hoc test using SPSS (version 22). P < 0.05 were considered statistically significant.All data are expressed as means ± standard deviation (SD). Statistical significance among multiple experimental groups was analyzed by ANOVA coupled with Tukey's Before separating the enzymatic hydrolysate, the ACE-inhibitory activity of goat milk casein and its enzymatic hydrolysate was first compared. It was found that the casein had no ACE-inhibitory ability, but the product after trypsin enzymolysis of casein had certain activity , indicatIn this study, the ACE-inhibitory activity of the hydrolysate was determined before the separation process. The hydrolysate was divided into six fractions by preparative reversed-phase C18 column, and fraction Y3 showed a highest inhibition which was notably higher than the hydrolysate . Then, YIn order to obtain the exact sequence of the purified peptide, MALDI-TOF-MS/MS combined with Protein Sequencer (PPSQ) was used. According to MALDI-TOF-MS/MS analysis, the molecular mass of ACE inhibitory peptide F3 was 1218.552 Da . As showm and Vmax for the reaction at different concentrations of the peptide were determined according to Lineweaver–Burk plots. As Km and Vm are important parameters of enzymatic reactions, the inhibition types of ACE-inhibitory peptides can be determined by them. There are two common inhibition types of ACE-inhibitory peptides, competitive inhibition and non-competitive inhibition. As shown in To clarify the type of inhibition, the peptide was co-incubated with various substrate (HHL) concentrations and an ACE solution, and the double reciprocal velocity-substrate is shown in in vitro. FPQYLQYPY was progressively incubated with various digestive enzymes, including α-amylase, pepsin, and trypsin. Then, the ACE inhibitory activity was assessed, and we observed that peptide digestion by α-amylase and pepsin promoted ACE inhibitory activity slightly when compared with the original (with inactivation of enzymes at every stage). However, peptide digestion by trypsin after 4 h reduced the ACE inhibitory activity a bit . And this test results provided a reference for the selection of the peptide concentration in the subsequent experiments.The cytotoxicity test result  showed tThe proliferation of cultured A7r5 cells was determined by CCK-8 assay. As expected, stimulation with Ang II for 24 h significantly increased the proliferation of VSMCs . Treatme−1 could restore the distance of the wound healing area to the level of the control group . However, after 48 h treatment with FPQYLQYPY, the decrease in the level of cell migration was noteworthy  were detected between the FPQYLQYPY treated group and the Ang II-treated group, and 797 genes between the Ang II-treated group and the control group.Transcriptome sequencing was performed using RNA from A7r5 cells. The typically changed genes were identified and shown in the heatmaps and volcano maps . The resAfter obtaining the differentially expressed genes, GO enrichment analysis was performed in order to describe their functions, covered biological processes, cellular components, and molecular functions. There were 212 significantly upregulated and 117 significantly downregulated genes in the comparison of the FPQYLQYPY and Ang II-treated groups. The top 30 GO-terms of significantly differential genes were mainly involved in stress fiber, actin cytoskeleton organization, positive regulation of cell migration, wound healing, and actin binding. These enriched GO terms may be related to vascular remodeling. Furthermore, the top 30 GO-terms of DEGs in the Ang II-treated group and the control group are shown in Pathway analysis using KEGG can further appreciate the function of genes and their interactions. Differential genes were enriched in cardiovascular disease-related pathways between the FPQYLQYPY-treated group and the Ang II-treated group, and between the Ang II-treated group and the control group. The top 20 enriched pathways in the FPQYLQYPY-treated group and the Ang II-treated group are presented in Overall, the analysis of pathways showed that the ACE inhibitory peptide may have a regulatory effect on Ang II -induced changes in cardiovascular-related pathways at the cellular level.Based on the GO and pathway results, we selected 9 genes among all identified DEGs for verification by RT-PCR. As shown in in vitro using gastrointestinal enzymes . In previous studies, the effective ACE inhibitory peptides released from goat whey protein and casein by pepsin hydrolysis, PEQSLACQCL of β-lactoglobulin (f 113-122), QSLVYPFTGPI of β-casein (residue 56-66), and ARHPHLSFM of κ-casein (f 96-106), were first introduced by Ibrahim et al. , which was derived from α-casein of goat milk . Based oIn vitro gastrointestinal enzyme simulation can simply describe the changes of a peptide during oral administration  and insulin-like growth factor 1 receptor (Igf-1R) of the growth factor family, and these genes tend to affect cell proliferation and migration, they are likely to cause fibrotic effects on vascular tissue or angiogenesis , 51. At in vitro.The pathways in which these DEGs are involved suggest that the peptide had the ability to modulate cellular dysfunction caused by Ang II on VSMCs. And the regulatory effects of this peptide were manifested in vascular remodeling, cardiac remodeling, and cardiomyopathy. These results correspond to the inhibitory ability of the peptide on excessive proliferation and migration of the Ang II-stimulated VSMCs shown in experiments. Simultaneously, our findings illustrated other antihypertensive mechanisms of this peptide in vitro gastrointestinal digest, and the ACE-inhibitory model of the peptide was confirmed as non-competitive. The anti-hypertension of FPQYLQYPY was evaluated by Ang II-stimulated VSMCs in vitro. This peptide could inhibit the excessive proliferation and migration of VSMCs. Besides, transcriptome sequencing analysis revealed that DEGs were enriched in cardiovascular disease-related pathways and implicated the regulatory effects of the peptide on vascular remodeling. The results of this study show that, in addition to inhibiting ACE, this peptide also regulates vascular dysfunction, which may broaden the application of the ACE-inhibitory peptides as functional food ingredients. However, the precise antihypertensive mechanism of this peptide remain unclear, and more studies are needed in the future to fully understand this process.In conclusion, a novel ACE-inhibitory peptide was separated and purified from goat milk casein hydrolysates, and the amino acid sequence was identified as FPQYLQYPY. The bioactivity of the hydrolysate persisted against The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI; PRJNA809493ZQ: data curation, formal analysis, investigation, writing—review and editing, and validation. JW, LP, XP, YG, and SC: funding acquisition, resources, and validation. ZH and QL: data curation, formal analysis, and investigation. KF: investigation and writing—review and editing. MS: methodology, writing—reviewing and editing, and investigation. YCh: investigation, writing—review and editing, and supervision. YCa: project administration, funding acquisition, writing—reviewing and editing, investigation, supervision, and resources. GL: project administration, funding acquisition, writing—reviewing and editing, investigation, and supervision. All authors contributed to the article and approved the submitted version.The authors express their gratitude to the Guangdong Province Engineering Research Center for Bioactive Natural Products (2016B090920093), Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods (2018B030322010), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019ZT08N291), the Project funded by China Postdoctoral Science Foundation (2020M672651), and the Innovation Platform and Talent Plan of Hunan Engineering and Technology Research Center for Nutrition and Health Products (2019TP2066).JW, LP, XP, YG, and SC were employed by Ausnutria Dairy (China) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.