The 20S proteasome is a valuable target for the treatment of a number of diseases including cancer, neurodegenerative disease, and parasitic infection. In an effort to discover novel inhibitors of the 20S proteasome, many reseaarchers have looked to natural products as potential leads for drug discovery. The following review discusses the efforts made in the field to isolate and identify natural products as inhibitors of the proteasome. In addition, we describe some of the modifications made to natural products in order to discover more potent and selective inhibitors for potential disease treatment. The 20S proteasome is a valuable target for the treatment of a number of diseases including cancer, neurodegenerative disease, and parasitic infection. Ubiquit4 The 20S core particle is composed of four stacked heptameric rings in an α1–7β1–7β1–7α1–7 arrangement.5 The outer α-rings are responsible for recognition of the regulatory particles, and the inner β-rings house the catalytic activity of the protease. The β-rings each consist of seven unique subunits, and three of these subunits  are responsible for the proteolytic activity of the proteasome. Binding of the 19S caps to the CP facilitates gate-opening, which in turn allows for the proteolytic degradation of polyubiquitinylated proteins. The 19S caps are responsible not only for this gate-opening,6 but also the recognition of polyubiquitinylated proteins, unravelling of these into linear peptides, and feeding the peptides into the catalytic chamber.7 Once fed into the chamber, unravelled proteins are cleaved into oligopeptides by the catalytic sites, which all proteolytically cleave the peptide chains with the aid of an N-terminal threonine residue (Thr1). Although all three catalytic sites use a nucleophilic threonine to carry out their activity, slight differences between their substrate channels allow for preference towards cleavage of specific peptide residues. The proteasome exhibits caspase-like , trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities which are carried out by its β1, β2, and β5 subunits, respectively.8–10 The first 20S proteasome structure determined was that of the archaebacterium Thermoplasma acidophilum in 1995,5 and many eukaryotic proteasomes have since been characterized. Structural similarity between eukaryotic proteasomes is highly conserved, which has allowed for the use of models such as the yeast 20S proteasome11 and mammalian 20S proteasome12,13 for identification of molecules which exhibit modulatory activity against the proteasome. These models have been especially useful considering that while their X-ray crystal structures were solved throughout the 1990s and early 2000s, which has facilitated inhibitor design greatly, the X-ray crystal structure of the human 20S proteasome was only recently solved in 2015.14 Two other core particles have also been identified as the immunoproteasome15 and the thymoproteasome.16 The differences of selectivity between these are governed by slight differences in the topology within their substrate channels.17The 26S proteasome is comprised of one or two 19S regulatory particles (RP) which associate with the 700 kDA barrel-like 20S core particle (CP).18–20 Additionally, the proteasome of parasitic species has recently been targeted in the treatment of parasitic diseases.21 Inhibition of the proteasome has been implicated as a veritable strategy for the treatment of certain cancers, given that inhibition leads to ER stress and apoptosis.22 The invention of the proteasome-inhibitor Velcade® (bortezomib) is perhaps the best representation of how targeting the ubiquitin-proteasome pathway can lead to the treatment of some cancers. This dipeptide boronate was approved by the FDA in 2003 as a treatment for multiple myeloma, and its mechanism of action is through direct inhibition of the ChT-L activity of the proteasome.23 Activation of the proteasome has also been considered for the treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's disease.24–26Due to its integral role in maintaining cell homeostasis, modulation of the activity of the 20S proteasome has been considered as a potential way to treat several diseases including cancer, autoimmune disease, and neurodegeneration.27 which had been shown to inhibit several types of proteases including serine and cysteine proteases. These peptide aldehydes act as reversible covalent inhibitors of the 20S proteasome, forming a hemiacetal with the nucleophilic threonine residue. Design of more selective inhibitors stemmed from alteration of the electrophilic warhead, leading to the eventual inclusion of boronic acids, vinyl sulfones,28 and α-ketoaldehydes29 within the scaffolds of synthetic inhibitors of the 20S proteasome. The interaction between covalent inhibitors and the catalytic sites is demonstrated in 30 Noncovalent inhibitors typically capitalize upon purely hydrophobic and hydrogen-bonding interactions to confer inhibition.Following the discovery of the 26S proteasome complex in the mid-1990s, researchers focused upon the development of novel inhibitors of the 20S proteasome. Most compounds which have been designed as inhibitors of the 20S proteasome are built upon peptide-based scaffolds. Peptide-based scaffolds contain optimized amino acid residues to mimic substrates of the 20S proteasome and improve recognition by the different catalytic subunits. The residues also participate in hydrogen-bonding and hydrophobic interactions to optimize interaction with the substrate channel. Specific design of the peptide chain has allowed for the generation of subsite-specific inhibitors of the proteasome. The design of such inhibitors is based upon the inherent preference of the C-L, T-L and ChT-L activities towards acidic, basic, and hydrophobic residues, respectively. Additionally, these compounds typically contain an electrophilic warhead at their C-terminus which interacts directly with the N-terminal threonine hydroxyl residue of proteolytic sites to block their catalytic activity. The first synthetic proteasome inhibitors were peptide aldehydes31 These methods have been especially helpful in regards to identification of natural product inhibitors, as some may be utilized with crude mixtures before isolation. Examples of the use of these methods are included in the review. Elucidation of the mechanisms of action by natural products through the methods of X-ray crystallography and computational docking allow for the design of more potent and selective inhibitors based upon the natural product scaffold. The focus of this review is to discuss several classes of natural products which have been identified for their inhibitory activity towards the 20S proteasome, their mechanism of action, and the efforts taken to optimize these scaffolds for potency and selectivity towards human 20S proteasome inhibition.Nature is an attractive source for the identification of novel proteasome inhibitors: the inherent stereochemical complexity of many natural products may allow researchers insight into unique interactions relative to the established clinically available inhibitors. A variety of methods exist for mining nature for identification of novel proteasome inhibitors including the use of fluorogenic peptide assay, site-specific probes, pathway-specific accumulation assay and NMR spectroscopic assay.Kitasatospora sp. MK993-dF2, a strain originally discovered in a soil sample from Kami-gun, Japan.32,33 After extensive spectroscopic analysis, the structures of the tyropeptins A and B were determined as isovaleryl-l-tyrosyl-l-valyl-dl-tyrosinal and n-butyryl-l-tyrosyl-l-leucyl-dl-tyrosinal, respectively. For confirmation of their structures, the total syntheses of the natural products were completed and compared with the isolated samples. The peptide backbone and reactive aldehyde moiety had already been established as a promising proteasome inhibiting scaffold,34 and therefore the potential biological activities of tyropeptins A and B were of interest to researchers. Tyropeptin A (1) competitively inhibits the ChT-L and T-L activities of the 20S proteasome with IC50 values of 0.20 μM and 2.9 μM, respectively. Tyropeptin B (2) is a weaker competitive inhibitor of the 20S proteasome, with IC50 values for the ChT-L and T-L sites of 0.39 μM and 7.8 μM, respectively. Tyropeptin A also exhibited cytotoxicity against HeLa S3 human cervical cells and HL-60 promyelocytic leukemia cells with IC50 values of 33.2 μM and 8.6 μM, respectively. Derivatives of tyropeptin A were designed by Momose et al. using computational methods.35,36 At the time, the crystal structure of the human 20S proteasome had not yet been solved; however, the crystal structure of the bovine 20S proteasome had been determined and was therefore used as a model.12,13 Tyropeptin A was believed to undergo nucleophilic attack by the Thr1Oγ residue of the active site to form a hemiacetal, and its side chains interact with the subpockets to form an antiparallel β-sheet. Introduction of bulky aromatic groups at the N-terminus enhanced inhibitory activity towards the ChT-L site of the proteasome. Optimization led to TP-110 (3), an N-terminal naphthyl derivative with methylated hydroxy positions (IC50: 0.027 μM). TP-110 exhibited improved growth inhibitory activity against cancer cells, likely due to increased hydrophobicity of the molecule to improve cell permeability. In subsequent in vitro studies, TP-110 also repressed the chymotrypsin-like activity of the proteasome in human prostate cancer PC-3 cells, inhibiting the proteasome activity by 56% with 0.16 μM.37 Furthermore, TP-110 induces apoptosis in the prostate cancer cell line. Substitution of the formyl group in TP-110 with boronates resulted in enhanced inhibitory potency towards the ChT-L site.38 Optimization of this scaffold led to boronate 4, which bore an N-terminal 3,6-dichloro-2-pyridyl substituent and exhibited comparable inhibitory potency towards the chymotrypsin-like activity of the proteasome (IC50: 0.053 μM) to that of bortezomib.39 Analog 4 also displayed good cytotoxic activity towards the RPMI8226 multiple myeloma cells, and was chosen for further in vitro and solid tumor studies.40Several families of peptide aldehyde natural products have been identified for their inhibitory activity towards the 20S proteasome . The fir5) was originally isolated from Penicillium fellutanium, a fungus which is found in the gastrointestinal tract of the marine fish Apogon endekataenia.41 Fellutamide B demonstrated potent cytotoxicity against murine leukemia P388 (IC50: 0.18 μM) and L1210 (IC50: 1.26 μM) cells, as well as human epidermoid carcinoma KB cells (IC50: 1.26 μM) in vitro, though the mechanism of cytotoxicity was not established. The total synthesis of fellutamide B was first achieved by the Crews group.42 Since then, other groups have also set out to achieve total syntheses of not only fellutamide B, but other members of the fellutamide family as well.43–45 Fellutamide B potently inhibits the chymotrypsin-like site (IC50: 9.4 ± 2.5 nM), but the trypsin-like and caspase-like sites to a lesser extent .46In vitro studies revealed the ability of the natural product to inhibit the proteasome within cells, as treatment of L–M mouse fibroblasts resulted in accumulation of ubiquitinated proteins.Fellutamide B (47 The Kelleher group utilized the method of Proteomic Investigation of Secondary Metabolism (PrISM) to screen Streptomyces species for non-ribosomal peptide synthases (NRPS) responsible for secondary metabolite synthesis. Using the PrISM approach, the group was able to identify a novel NRPS gene cluster from Streptomyces sp. NRRL F-6652. With the help of bioinformatics analysis and metabolomics analysis, six flavopeptins were discovered as the products of this gene cluster. Flavopeptins I (6) and II (7) were synthesized using solid phase peptide synthesis (SPPS), along with the N-terminal amine derivative 8. Due to the similarity of the flavopeptins to other established inhibitors, the natural products were tested for their inhibitory activity towards the ChT-L and C-L sites of the 20S proteasome. Flavopeptins I and II are low micromolar inhibitors of both sites, and the terminal amine derivative 8 exhibits submicromolar IC50 values for both sites. The flavopeptins were also tested for their cytotoxicity against the multiple myeloma cell lines MM.M1S and FR4, as well as the histiocytic lymphoma cell line, U-937. Flavopeptin I displayed values of IC50: ∼35 and 13 μM for the MM cell lines and the lymphoma line, respectively. The N-terminal amine derivative 8 displayed no cytotoxic activity. The lack of activity is believed to be due to lower cell permeability and stability as compared to flavopeptins I and II.The flavopeptin class of peptide aldehydes was discovered with the help of proteomics.Polyangium brachysporum sp. nov. K481–B101.48–51 Glidobactins A–C are cytotoxic against melanotic melanoma B16 cells as well as human colon cancer HCT-116 cells, and exhibit potent in vivo antitumoral activity towards P388 leukemia in mice.48 Further modification of the glidobactin A structure by Oka et al. led to the preparation of several derivatives, which were also evaluated for their antitumoral activity.51 Glidobactin A (9) was later identified as a nanomolar inhibitor of the ChT-L site of the 20S proteasome, with a Ki value of 49 ± 5.4 nM.52 The compound also inhibited T-L activity of the 20S proteasome, albeit at considerably higher concentrations . The mechanism of inhibition by syrbactins was determined through elucidation of the X-ray crystal structure of the 20S proteasome in complex with glidobactin A and syringolin A. The α,β-unsaturated moiety of glidobactin A undergoes a nucleophilic Michael-type 1,4-addition by the Thr1Oγ of the chymotrypsin-like and trypsin-like sites of the proteasome. The lipophilic alkyl chain of glidobactin A contributes to its inhibitory activity and served as inspiration in the development of novel syrbactin proteasome inhibitors; the activity of these analogs is discussed in a later section.Several syrbactin natural products have also been identified for their proteasome inhibition . Syrbact10) was recently identified as an inhibitor of the human constitutive and immunoproteasomes through a competitive metabolite profiling technique by the Böttcher group.53 Glidobactin C exhibited in vitro inhibitory activity towards the ChT-L and T-L sites of the human constitutive proteasome . The compound also inhibits the human immunoproteasome . The two additional methylene groups of 10 relative to 9 are thought to improve its ability to penetrate the cell membrane, resulting in greater activity in cell assays.Glidobactin C  was identified as a proteasome inhibitor in later studies: Stein et al. developed an NMR-based proteasome assay to identify natural product inhibitors of the yeast 20S proteasome.56 Cepafungin I potently inhibits the ChT-L activity of the proteasome (IC50: 4 nM), five times more potent than the known inhibitor glidobactin A (IC50: 19 nM). Cepafungin I also inhibits the T-L activity of the proteasome with an IC50 value of 24 nM. The improved inhibitory activity of cepafungin I as compared to glidobactin A is believed to be due to the increased stabilization of the fatty acyl tail through van der Waals interactions.The cepafungins were observed as products of the bacteria 12) is another natural product of the syrbactin family which has recently gained attention as a potential proteasome inhibitor. A deoxy derivative of glidobactin A, luminmycin A contains the hallmark 12-membered macrolactam ring responsible for the inhibitory activity of the syrbactin family towards the human 20S proteasome. Luminmycin A was identified as a metabolite of a silenced gene cluster plu1881–plu1887 of Photorhabdus luminescens, and the associated gene cluster was utilized to produce luminmycin A using the method of heterologous expression.57 The natural product was also observed as a metabolite of crickets following their infection with Photorhabdus asymbiotica.58 Luminmycin A and other natural products belonging to the luminmycin class were successfully isolated following heterologous expression of the same gene cluster, using E. coli as host.59,60 Recently, luminmycin A was successfully synthesized in the laboratory by Servatius et al.61 Luminmycin A inhibits the constitutive proteasome (CP) as well as the immunoproteasome (IP), (ChT-L IC50: 0.039 ± 0.002 μM (CP), 0.016 ± 0.006 μM (IP); T-L 0.026 ± 0.008 μM (CP), 0.017 ± 0.0016 μM (IP)).53 The natural product also exhibits cytotoxic activity against human carcinoma HCT-116 cells (IC50: 91.8 nM).Luminmycin A  inhibits the ChT-L and C-L activities of the 20S proteasome . The macrolactam binds covalently to the 20S proteasome by direct interaction with Thr1Oγ within the active sites.52,67 The α,β-unsaturated carboxamide of syringolin A is rendered especially electrophilic due to the ring strain of the macrocycle resulting from the presence of two trans alkenes; upon 1,4-Michael addition of Thr1Oγ, this ring strain is relieved. In comparison, syringolin B (14), which lacks one of the trans alkenes, is a significantly less potent inhibitor of the 20S proteasome (Ki: 7.7 ± 2.3 μM (ChT-L); 107.8 ± 39.2 μM (T-L).The syringolin family of natural products were identified as secondary metabolites of the pathogen 15, which exhibits inhibitory activity towards all three catalytic sites of the proteasome (Ki: 8.65 ± 1.33 nM (ChT-L); 79.6 ± 29.3 nM (T-L); and 943 ± 100 nM (C-L)).68 TIR-199 (16) contains a slightly longer lipophilic tail than SylA-LIP and inhibits the ChT-L (Ki: 18 nM) and T-L (Ki: 194 nM) sites of the human proteasome in in vitro enzymatic assays.69 TIR-199 also inhibits the proteasome in multiple myeloma cell line MM1.RL and neuroblastoma cell line MYCN2 and has demonstrated exciting cytotoxic activity against bortezomib-resistant cell lines (MM1.S BzR and U266 BzR), indicating it can overcome bortezomib resistance in vivo.70 Clerc et al. further investigated the effects of substitution at the N-terminus through the synthesis of a syringolin A–glidobactin A hybrid 17.71 The inhibitory activity and subsite selectivity of the analogue was tested using competitive activity-based protein profiling (ABPP) in HEK cell lysates and living cells, compared to known syrbactin inhibitors. Further in vitro studies revealed that 17 inhibits all three catalytic sites of the 20S proteasome (Ki: 12.5 ± 1.5 nM (ChT-L), 136.9 ± 12.4 nM (T-L), 3.7 ± 1.2 μM (Casp-L)).72 Cell culture-based experiments from the same study revealed that the analogue carries out inhibition of the proteasome in several cancer cell lines. Introduction of hydrophobic sidechains allowed the group to target the S3 subpocket of the β5 subunit for inhibition. Compound 18, which contained a benzyl substituent and lipophilic side chain, displayed strong inhibitory activity against the ChT-L site of the proteasome (Ki′: 0.12 nM) and cytotoxicity towards human RPMI8226 multiple myeloma cells (IC50: 2.2 nM).66 Further optimization of 18 has focused on improving cytotoxicity.73Additional derivatization of the syringolin A scaffold has been pursued by several groups . Alterat74 The N-terminal amide of analogue 18 is believed to participate in a hydrogen-bonding interaction with Asp114 (β6), similarly to its parent syringolin A. The group hypothesized that removal of the N-terminal amide group and replacement with a longer alkyl chain may allow for a switching in the hydrogen-bonding interactions within the β5 subunit and allow for a novel interaction between the substrate and Ala49. The hypothesis was verified through the synthesis of analogue 19, which displayed nanomolar inhibitory activity towards the ChT-L site of the proteasome (IC50: 107 nM). Additionally, analogue 19 exhibits nanomolar growth inhibitory activity against several cancer cell lines.Simplification of the syringolin A scaffold has also been a focus of research by the Ichikawa group. One report focused on analogues that exhibited proteasome inhibition and cytotoxicity against cancer cells by targeting a different intermolecular H-bonding interaction.20) and its analogue 21, which both exhibited inhibition towards the ChT-L site .75 Compound 21 also exhibited potent cytotoxicity against OPM-2 human myeloma cancer cells (IC50: 6.7 nM) and bortezomib-resistant OPM-2 cells (IC50: 60 nM), displaying greater cytotoxicity as compared to bortezomib (IC50: 146 nM). Expansion of the syringolin A ring by Ibarra-Rivera et al. led to oxa-SylA-LIP (22), which displays potent cytotoxicity against the MYCN-2 cells (IC50: 0.4 μM) and also inhibits the proteasome in cell-based studies.76,77Alteration of the macrocycle structure was also explored. Researchers hypothesized that a more accessible alkene isomer with a similar three-dimensional structure might be also be able to carry out inhibitory activity. The group synthesized isosyringolin A (23) was developed as a direct comparison to SylA-LIP (15).76 Totaro et al. designed more potent syringolin B analogues with greater inhibition towards the β5 subunit based upon the synthesis of syringolin B by Pirrung et al.65,78 Introduction of aromatic groups at the P1 and P3 residues in the scaffold led to the discovery of potent inhibitor 24 (second order rate-constant (kin/Ki): 4305 M−1 s−1). The analogue also exhibits potent growth inhibition against various leukemia cell lines.Analogue development based upon the syringolin B scaffold has also been investigated by several research groups . For exa79 The biological activity of the analogues was further examined through cytotoxicity experiments with numerous neuroblastoma cell lines. The thiasyrbactin scaffold has established itself as a drug-like starting point for inhibition of the immunoproteasome.Thiasyrbactin analogues with a sulfur-for-carbon substitution within the macrolactam ring were synthesized with varying levels of sulfur oxidation. These analogues were reported for their activity towards both the constitutive and immunoproteasomes, more selectively inhibiting the T-L site of the immunoproteasome.Apiospora montagnei Sacc. TC 1093 by Koguchi et al. while the group was screening for 20S proteasome inhibitors.80,81 Among all of the TMC cyclic peptides, TMC-95 A (25) and B (26) were identified as specific inhibitors of the 20S proteasome, displaying activity against both the ChT-L (IC50: 5.4 nM (TMC-95 A) and 8.7 nM (TMC-95 B)) and T-L (IC50: 200 nM (TMC-95 A) and 490 nM (TMC-95 B)) subunits. TMC-95A further demonstrated cytotoxic activities against HCT-116 and HL-60 cells with IC50 values of 4.4 μM and 9.8 μM, respectively. The X-ray crystal structure of the yCP:TMC-95A complex was later solved by Groll et al. (2.9 Å resolution), revealing that the macrocycle reversibly inhibits the 20S CP through non-covalent interactions within the catalytic core.82 The specificity of the natural product for the chymotrypsin-like site is believed to be due to greater interaction with the S3 specificity pocket.Several classes of macrocyclic peptides have been identified for their inhibitory activity towards the proteasome. Perhaps the most explored class has been the TMC cyclic peptides . TMC-95 83–86 The first total syntheses of TMC-95A and B were achieved by Lin and Danishefsky in 2002.83 The presence of sensitive functional groups in addition to the difficulty in accessing the macrocycle has encouraged subsequent analogue development to be focused on the synthesis of simplified molecules. Kaiser et al. synthesized compound 27, a simplified analogue based upon the minimal requirements for binding as determined from the X-ray crystal structure of TMC-95A/proteasome complex.87 The analogue exhibited inhibitory activity against the ChT-L site of the 20S human proteasome (IC50: 1.9 μM) as compared to TMC-95A (IC50: 0.012 μM).88 The same researchers evaluated the effect of additional P′ residues at the C-terminus of TMC-95A in a separate study.89 The Danishefsky group established the importance of the enamide moiety at the C-8 position of the molecule;90 analogues lacking this displayed at least a 1000-fold less potent inhibition towards the ChT-L site as compared to their natural product parent molecule. Furthermore, alteration of the enamide sidechain also affected inhibitory activity; the presence of a propyl substituent in exchange for a propylene or allyl amide sidechain results in diminished activity. In previous studies, the biaryl moiety of TMC-95A was deemed responsible for the induction and stabilization of a β-type peptide backbone conformation while in complex with the 20S proteasome.82 Kaiser et al. implemented the substitution of the biaryl heterocycle using a more accessible biaryl ether moiety with the goal of retaining the similar β-type backbone conformation.91 The biaryl ether analogue 28 retains inhibitory potency towards the ChT-L site of the yCP (IC50: 5.5 μM). Later studies revealed that due to the lack of the oxindole, biaryl ether analogues do not adopt the desired stabilized β-type structure while in complex with the 20S proteasome.92 Wilson et al. later synthesized macrocyclic peptide aldehydes upon the previously established biaryl ether analogues which are nanomolar inhibitors of the ChT-L site.93 Further efforts to access simplified analogues of TMC-95A have eliminated synthesis of the challenging macrocycle altogether through the development of linear peptide mimics of the natural product. The Vidal group—in collaboration with many other research groups—has used this linear TMC-95A approach to synthesize effective inhibitors.94–99 In particular, the linear 3-hydroxyoxindole-containing derivatives exhibited subunit selectivity towards the C-L site).96 Optimization of the scaffold led to the discovery of potent inhibitor 29, which inhibited the ChT-L of the constitutive and immunoproteasomes .99 Dimerized linear TMC-95A mimics using the active derivative 29 scaffold have also been synthesized to evaluate their ability to inhibit multiple active sites at once. Using either PEG spacers100 or oligomers of aminohexanoic and adipic acid as spacers,101 researchers were able to achieve nanomolar inhibition of the ChT-L sites of the 20S proteasome.Due to the biological activity and overall complexity of the TMC-95 natural products, many groups sought to undertake their synthesis.Archangium gephyra by the Hofle group in 2002.102,103 This group of cyclic octapeptides displayed growth inhibitory activity against a variety of mammalian cell lines,102 and became a focus of total synthesis for many groups.104–106 Argyrins A, B, C, D and F (30–34)   were latet al. reported that thiopeptides thiostrepton (35) and siomycin A (36) exhibit inhibitory activity against the 20S proteasome, which in turn contributes to the effects observed on the oncogenic transcription factor Fox1  are cyclic hexapeptides isolated by Sekizawa et al. from Streptomyces sp. MK600-cF7.117 All four natural products exhibit inhibitory activity against the β5 subunit . Because of their stability as cyclic peptides, these natural products were believed to interact with the proteasome through van der Waals and hydrophobic interactions rather than through covalent bond formation. Scytonemide A (41)was isolated from the cultured freshwater cyanobacterium Scytonema hofmannii (UTEX 1834).118 Scytonemide A potently inhibits the ChT-L site of the 20S proteasome (IC50: 96 nM). The presence of an imine moiety within the macrocycle of scytonemide A was hypothesized to be a potential site of attack by the nucleophilic Thr1Oγ of the active site; however, the X-ray crystal structure of this natural product in complex with the 20S proteasome to validate the hypothesis has not been reported. Recently, the cyclic hexapeptide baceridin (42) was isolated from the culture broth of an epiphytic Bacillus strain and underwent total synthesis by the Kalesse group.119 Baceridin inhibits all three catalytic sites of the 20S proteasome and inhibits proliferation in several tumor cell lines.Several macrocyclic peptide scaffolds that have been identified as inhibitors of the proteasome have yet to be further evaluated through SAR studies . Among t43) was isolated by Omura et al. from Streptomyces sp. OM-6519 and displayed biological activity in its initial discovery.120,121 In their 1994 study, Fenteany et al. researchers further evaluated lactacystin and analogues including omuralide for their biological activity. Results suggested the contribution of either the N-acetyl cysteine or β-lactone moieties towards their ability to affect cell cycle progression in Neuro 2A and MG-63 osteosarcoma cells, although the molecular target of these substrates was unidentified at the time.122 A subsequent study indicated that lactacystin inhibits the 20S proteasome.123 Researchers later determined that rather than acting as an inhibitor towards the 20S proteasome, lactacystin acts as a prodrug of its active β-lactone omuralide (44).124,125 The first total syntheses of lactacystin and omuralide were achieved by the Corey group, and many total syntheses and formal syntheses have been recorded since.126–128The β-lactone natural products have also been heavily evaluated for their ability to inhibit the proteasome . Lactacy129 Research conducted by Soucy et al. further complemented these results with derivative 45, which contains an n-propyl group in place of the methyl substituent at C7. Compound 45 as a Kobs/[I] which is greater than two-fold more potent than omuralide (Kobs/[I] = 46 500 M−1 s−1).130 The compact, densely functionalized omuralide scaffold indicated the importance of exploring non-peptidic natural products as potential inhibitors of the 20S proteasome.In an SAR study of the omuralide scaffold, the Corey group determined that while the hydroxy-isobutyl substituent at C9 is necessary for potent inhibition of 20S bovine proteasome, alteration of the methyl substituent at C7 with larger or bulky alkyl groups improves potency towards the target.Salinospora strain CNB-392 and its structure was elucidated by Feling et al.131 The natural product shares its unique fused γ-lactam–β-lactone bicyclic ring scaffold with that of omuralide. Salinosporamide A was tested for its cytotoxicity against HCT-116 human colon carcinoma cells, displaying an IC50 value of 35.1 nM. Due to its structural similarity to omuralide, salinosporamide A (46) was also tested for its inhibitory activity towards 20S proteasome; it exhibits potent inhibitory activity towards the ChT-L site (IC50: 1.3 nM). Chauhan et al. further investigated the bioactivity of salinosporamide A in in vitro and in vivo studies.132 Salinosporamide A inhibits the ChT-L site with an EC50 value of 3.5 ± 0.3 nM as well as the C-L and T-L sites with EC50 values of 430 ± 34 nM and 28 ± 2 nM, respectively. Salinosporamide A also induced apoptosis in multiple myeloma cells which are resistant towards conventional and Bortezomib therapies.Salinosporamide A was first isolated as a product of the ocean sediment-dwelling bacteria 133 the molecule has since become an attractive target for total synthesis, and many syntheses of salinosporamide A have been reported.134–148 Exploration of the structure–activity relationship of the salinosporamide scaffold began with Macherla et al.,149 and has since been a focus of several research groups  in complex with salinosporamide A, implicating the importance of the chloroethyl group in the overall inhibitory activity towards the proteasome.150 Following transesterification of the β-lactone, the resulting hydroxyl group cyclizes upon the chloro substituent to form a tetrahydrofuran irreversibly.Only a year after its discovery, the Corey group reported the first total synthesis of salinosporamide A;h groups . Alteratet al. synthesized salinosporamide A analogues which replaced the chloro substituents with functionalities of varying leaving group potentials to further probe the mechanism of inhibition.151 All analogues potently inhibit the ChT-L site of the 20S proteasome; tosyl analog 47 displays inhibitory activity towards the ChT-L site of the 20S proteasome with an IC50 value of 2.5 ± 0.4 nM. Qualitative dialysis experiments with the analogues indicated that the presence of a leaving group prolongs the duration of inhibition. The researchers also performed kinetics experiments with salinosporamide A to understand the mechanism of its proteasome inhibition, determining that the natural product is a slow tight binding inhibitor which confers its activity through a multi-step inhibition mechanism. Fluorosalinosporamide 48 was later synthesized by Eustáquio and Moore using a combination of genetic engineering and precursor-directed synthesis.152 Unlike salinosporamide A, 48 did not undergo displacement of its halogen to afford an irreversible adduct during inhibition, but rather acts as a reversible inhibitor towards the ChT-L activity of the 20S proteasome with two-fold reduced potency relative to salinosporamide A (IC50: 1.5 ± 0.05 nM). The ability of the fluorine to participate in hydrophobic interactions with the proteasome was cited as a justification of its inhibitory potency.Manam 49).153 Antiprotealide—later discovered as a natural product154—exhibited 2.5-fold more potent inhibitory activity towards the β5 subunit than omuralide, but was less potent than salinosporamide A. Synthesis of the γ-lactam–β-lactone congener (50) of antiprotealide was also successfully achieved by the Corey group. While the analogue displayed slower inactivation towards the 20S proteasome than salinosporamide A and omuralide, its improved stability under physiologic conditions was an advantageous feature for relevant drug development.The Corey group further explored the salinosporamide A and omuralide scaffolds through the synthesis of a γ-lactam–β-lactone hybrid antiprotealide -salinosporamide A and derivative (—)-homosalinosporamide A.145 (—)-Homosalinosporamide A varies from (—)-salinosporamide A only through the length of its C2 sidechain; the compound contains an additional methylene carbon. The researchers proposed that the derivative would behave similarly to salinosporamide A in its mechanism of inhibition towards the human proteasome. (—)-homosalinosporamide A (52) displayed similar inhibitory potency towards the ChT-L activity of the 20S proteasome as compared to (—)-salinosporamide A, with an IC50 value of 0.7 ± 0.04 nM (versus 0.8 ± 0.08 nM). However, X-ray crystallographic evidence of the hCP: (—)-homosalinosporamide A complex illustrated that (—)-homosalinosporamide A does not form a tetrahydropyran during its inhibition of the proteasome.157The length of the chloroalkyl chain at the C2 position of the salinosporamide scaffold is also an important feature with regards to its inhibitory potency. Nguyen Streptomycetes JS360 in 2007 by Stadler et al.158In vitro evaluation of the inhibitory potency of the cinnabaramides towards the human 20S proteasome mirrored results of previously established inhibitors of similar structure. For example, cinnabaramide A (53) (IC50: 1 nM) displayed similar potency as salinosporamide A. Cinnabaramide A is also cytotoxic against colon cancer cell line HCT-116 at a level relative to that of salinosporamide A. Cinnabaramides F (54) and G (55) are believed to act as a prodrug to form the reactive β-lactone in a manner reminiscent to lactacystin; these natural products display potent inhibition towards the human 20S proteasome with IC50 values of 6 nM and 0.6 nM, respectively. rac-Cinnabaramide A was later synthesized by the Romo group.137 In a subsequent study, Rachid et al. accessed chlorinated derivatives of cinnabaramides A–D using mutasynthetic methods.159 15-Chlorocinnabaramide A (56) exhibits greater inhibitory activity towards the β5 subunit of the 20S proteasome than its parent compound, with an IC50 value of 9.3 ± 5.9 nM . Derivative 56 also displays potent cytotoxicity against the HCT-116, RPMI8226, and SW840 cancer cell lines.The structurally related cinnabaramides  were firStreptomyces sp. KY11780 by Asai et al.160 The antiproliferative and antitumor activities of both natural products is attributed to a β-lactone moiety at their C-terminus. Belactosin A contains a cyclopropane ring within its backbone which is absent in that of belactosin C. Belactosin A and C both displayed in vitro antiproliferative activity against HeLa S3 cells with IC50 values of 51 μM and 200 μM, respectively. Belactosin A further demonstrated the ability to halt cell cycle progression in the G2/M stage in tumor cells. Akai et al. reported the molecular mechanism of action of belactosins A (57) and C (58): both demonstrate nanomolar inhibitory activity towards the ChT-L site of the rabbit 20S proteasome, both displaying IC50 values of 0.21 μM.161 Optimization of the belactosin A scaffold was further achieved by benzylation of it carboxylic acid. The first total synthesis of belactosin A was completed by Armstrong and Scutt in 2004.162 An enantioselective total synthesis of belactosins A, C and its homologue homobelactosin C (59) was reported shortly thereafter by Larionov and de Meijere.163 X-ray crystallographic evidence based upon the yCP:homobelactosin complex suggested that the β-lactone moiety undergoes nucleophilic attack by the Thr1Oγ residue within the catalytic site to form an ester through a covalent, irreversible bond.164Belactosins A and C  were fir165–167 The (cis/L-anti) configuration demonstrated promise, as evidenced by compound 60. Compound 60 selectively inhibits the ChT-L site of the human 20S proteasome with an IC50 value of 5.7 ± 1.2 nM, comparable to that of bortezomib. Compound 60 also inhibits the growth of HCT116 cells with an IC50 value of 1.82 μM via the same mechanism as bortezomib . The X-ray crystal structure of yCP:60 complex (2.8 Å), provided researchers with key interactions that contribute to inhibition. Optimization of the transition-state conformation of 60 was subsequently reported.168 Installation of a methyl group at carbon C1′ adjacent to the cyclopropyl group led to analog 61, which is restricted into a predominant syn conformation. Derivative 61 exhibited lower inhibitory activity towards the ChT-L site relative to the parent compound with an IC50 value of 47 ± 2.9 nM. However, when evaluated for its growth inhibitory activity against several tumor cell lines , compound 61 exhibits activity similar to its parent compound.Exploration of the stereochemistry surrounding the belactosin A cyclopropyl ring indicated its contribution to biological activity .165–167 168 and thus sought to improve stability by increasing steric hindrance surrounding the β-lactone ring.169 Analogue 62 not only contains an additional methyl group at the α-position of the lactone carbonyl carbon, but also retains the same methyl group at the C1′ carbon adjacent to the cyclopropyl ring to lock it into the syn conformation. While analogue 62 displays lower activity towards the ChT-L site of the proteasome as compared to parent compound 60 , 62 exhibited comparable growth inhibitory activity towards HCT-116 cells relative to the established inhibitor (IC50: 4.0 μM (62); 1.8 μM (60)). These results provided a novel belactosin analogue with improved biological stability.Researchers hypothesized that poor performance of belactosin analogues under biological conditions and in cell studies may be due to chemical or enzymatic hydrolysis of the reactive β-lactone moiety,et al. using a topology-based scaffold hopping approach.170 These synthetically accessible inhibitors represent novel scaffolds with improved drug-likeness in comparison to past belactosin analogues. Simplified nonpeptidic belactosin scaffolds were further explored through synthesis of hybridized bortezomib and epoxomicin analogs.170,171 Divergence from the β-lactone moiety of the belactosin scaffold stemmed from an interest to incorporate more stable electrophilic warheads.Flexible achiral nonpeptidic analogues with a β-lactone moiety and aromatic tails were synthesized by Kawamura et al. first sought to optimize the belactosin C scaffold in 2009 through the introduction of a boronic acid moiety like that of bortezomib.172 Compound 63 was identified as a potent inhibitor of the ChT-L (β5) site of the 20S proteasome (IC50: 0.28 ± 0.04 μM) and also exhibited submicromolar growth inhibitory activity against HeLa cells (IC50: 0.35 ± 0.02 μM) in an MTT assay. The de Meijere group expanded their previous synthetic endeavors of belactosin scaffolds through the synthesis of novel belactosin C-based proteasome inhibitors.173 A subsequent report by the same group elected to divert from the dipeptide scaffold of belactosin C to investigate more easily accessible analogues.174 Analogues containing the N-benzyl amido side chain displayed especially potent inhibitory activity towards the β5 subunit. This scaffold was later altered to produce a minimal β-lactone scaffold for selective inhibition of either the β5c or β5i of the human 20S proteasome.175 Analogue 64 contains a pseudo-isoleucine P1 side chain, whereas 65 contains a smaller pseudo-valine moiety. This difference in size of the P1 side chain resulted in selective inhibition. Compound 64 exhibited preference towards the β5i site (IC50: 14.37 nM) over the β5c (IC50: 21.35 nM), whereas 65 preferentially inhibits the β5c (IC50: 26.87 nM) over the β5i (IC50: 83.62 nM).Nakamura Kitasatospora cystarginea NRRLB16505, these natural products contain a dipeptide backbone with a β-lactone moiety.176 Cystargolides A (66) and B (67) exhibited inhibition towards the ChT-L site of the 20S proteasome with IC50 values of 0.36 ± 0.017 μM and 0.93 ± 0.032 μM, respectively. The total syntheses and absolute stereochemistry of cystargolides A and B were later successfully achieved by Tello-Aburto et al.177 Wolf et al. were also able to access the cystargolides and belactosins using biosynthetic methods. The stereochemistry of these natural products is integral to inhibitory activity: maintaining the  absolute stereochemistry contributes to potency of inhibitors.178 Benzylation of the N-terminus also improved inhibitory potency 100-fold, as evidenced by analogues 68 (IC50: 9.2 ± 0.59 nM) and 69 (IC50: 9.0 ± 1.4 nM).177 Subsequent optimization of the cystargolide scaffold was achieved in 2018: benzyl ether 70 inhibits the hβ5 subunit of Jurkat cell lysate with an IC50 value of 3.1 ± 0.2 nM as compared to cystargolide B (IC50: 0.90 ± 0.11 μM), and is also cytotoxic against MCF-7 and RPMI-8226 cells.179Another class of β-lactone containing natural products which display intriguing activity towards the proteasome are cystargolides A and B . OriginaStreptomyces hygroscopicus No. P247-71 (ATCC 53709).180 Eponemycin (71) and its derivative dihydroeponemycin (72) exhibit potent in vitro toxicity against B16–F10 and HCT-116 cells. The natural product also demonstrated itself as an inhibitor of angiogenesis.181 The cellular target of its antitumor activity was later identified by the Crews group as the proteasome with biotinylated eponemycin analogs.182 Dihydroeponemycin inhibits the proteasome in a competitive and irreversible manner, with the highest rate of inhibition towards the ChT-L site (kassoc: 66.4 ± 8.9 M−1 s−1). Kim et al. further explored the structure–activity relationship of dihydroeponemycin and analogues to improve understanding of subunit selectivity within the 20S proteasome.183 The ability of dihydroeponemycin to interact with the immunoproteasome was attributed to its lipophilic C-terminus.The epoxy-ketone containing natural products have become a specific focus to researchers due to their ability to inhibit the 20S proteasome . Eponemy73) was isolated from a soil-dwelling actinomycete no. Q996-17184 and is comprised of a tetrapeptide skeleton with a C-terminal α,β-epoxyketone. Epoxomicin exhibited strong in vitro cytotoxicities against various cancer cell lines including HCT-116 (IC50: 9.0 nM), and additionally exhibits antitumor activity against B16 melanoma. The total synthesis of epoxomicin was first reported by the Crews group in 1999.185 Due to previous reports that indicated peptide α,β-epoxyketones as inhibitors of the proteasome,186 epoxomicin was evaluated for this activity. Epoxomicin selectively inhibits the chymotrypsin-like site of the 20S proteasome (kassoc: 35 400 M−1 s−1). The N-terminal P3 and P4 residues play a major role in the potency of inhibition towards the proteasome.183 As compared to dihydroeponemycin, epoxomicin and its analogues display greater inhibitory potency. The X-ray crystal structure of the yCP:epoxomicin complex was solved by Groll et al. (2.25 Å resolution),187 revealing the basis for its selectivity towards the ChT-L site. The α,β-epoxyketone was initially believed to undergo a reversible nucleophilic addition by the Thr1Oγ to produce a hemiacetal; subsequent attack of the epoxide by Thr1N forms a morpholino adduct irreversibly. Recently, the mechanism of proteasome inhibition by epoxyketones was revised based on X-ray crystallographic data by Schrader et al.188 Instead of forming a morpholino adduct, epoxyketones are believed to react with the Thr1 residue of the catalytic site to form a 1,4-oxepane ring.The natural product epoxomicin (74).189In vitro studies indicate that YU-101 selectively inhibits the ChT-L site of the 20S proteasome with a kassoc value of 166 000 (5–12 nM), greater than that of epoxomicin and bortezomib. Further optimization of this analogue led to carfilzomib  by the biotech company Proteolix.190 Carfilzomib exhibits selective, potent inhibitory activity towards the ChT-L site of the proteasome, with an IC50 value of 6 nM; the compound also performed well in in vivo studies. In 2012 carfilzomib was approved by the FDA for the treatment of refractory multiple myeloma;191 further alteration of this compound led to the orally available oprozomib (76).192 Subsequent optimization of the epoxyketone scaffold has focused on developing analogues which can overcome bortezomib and carfilzomib-resistant multiple myeloma. Kim et al. recently reported a novel epoxyketone 77, which not only inhibits the ChT-L activity of proteasome from RPMI8226 cell lysates (IC50: 2.1 ± 0.9 nM), but also inhibits proteasome activity (ChT-L) nearly three-fold more potently in RPMI8226-Cfz resistant cells than carfilzomib itself (IC50: 106.2 ± 28.9 nM).193 Other recent advances of the epoxyketone moeity include the design of selective inhibitors for the β1i,194,195 β2i,196,197 and β5i subunits.198Further optimization of the epoxomicin scaffold has been achieved by several groups . The CreSymploca sp.199 These tripeptides contain a lipophilic N-acylated tail at their N-terminus and a leucine-derived epoxyketone at their C-terminus. A scalable total synthesis of both carmaphycins was also conducted and both were evaluated for their ability to inhibit the yeast 20S proteasome. Carmaphycins A (78) and B (79)   exhibite81) and B (82) are isolated from the fermentation broth of Streptomyces sp. TC 1084.202,203 Both display similar inhibitory potency towards the ChT-L site with IC50 values of 5.1 μM and 1.1 μM, respectively. TMC-86A and B also exhibited nanomolar cytotoxicity against various tumor cell lines. TMC-96 (83) was isolated from the fermentation broth of Saccharothrix sp. TC 1094,202,203 and contains a branched N-acylated terminus and a leucine-derived α,β-epoxyketone moiety similar to dihydroeponemycin. TMC-96 exhibits inhibitory activity towards the ChT-L and C-L sites of the 20S proteasome , and additionally displays nanomolar cytotoxic activity against a variety of tumor cell lines. TMC-89A (84) and B (85) are α,β-epoxyketone-containing tripeptides which were isolated from the fermentation broth of Streptomyces sp. TC 1087 by Koguchi et al. in their search for new proteasome inhibitors of bacterial origin.204 When tested for their proteasome inhibitory activity, both displayed equipotent micromolar activity towards the ChT-L site of the 20S proteasome, with IC50: 1.1 μM. TMC-89A and B also exhibit slight selectivity towards the T-L site of the 20S proteasome, with IC50 values of 390 nM and 510 nM, respectively. In vitro cytotoxicity studies reveal that in comparison to many other α,β-epoxyketones, TMC-89A and B are not remarkably cytotoxic against tumor cell lines. Subsequent optimization of these natural products has not been reported but may benefit from the addition of N-terminal lipophilic residues to improve cell permeability.Several other epoxyketone natural products were also isolated and evaluated for their ability to inhibit the proteasome . TMC-86ACystobacter fuscus MCy9118.205 Macryanone A (86) contains an α,β-epoxyketone moiety which made it an intriguing compound to test for proteasome inhibition. Macyranone A inhibits the ChT-L activity of the yCP (IC50: 5.9 nM) and the human constitutive proteasome and immunoproteasome, with IC50 values of 21 nM and 15 nM, respectively. X-ray crystallographic analysis of the yCP: macyranone complex (2.8 Å resolution) revealed that it reacts irreversibly with the catalytic sites of the proteasome in a similar mechanism to epoxomicin. Despite its promising performance in in vitro enzymatic testing, macyranone A displayed poor cytotoxicity against mammalian cell lines including HCT-116, THP-1 and HL-60 cancer cell lines.The macyranones are a group of six linear peptides which were produced by the myxobacteria et al. The group utilized molecular evolutionary analyses of complex metagenomes to target and isolate the previously unidentified clarepoxcin and landepoxcin natural products (206 Clarepoxcins A–E (87–91) were produced by S. albus:AR456, while landepoxcins A (92) and B (93) were produced by S. albus:AR412. Clarepoxcins A–D are potent low nanomolar inhibitors of the human 20S proteasome, with an IC50 ∼ 6.9–15.1 nM (ChT-L). The clarepoxcins also exhibited potent cytotoxic activity against HCT-116 cells. While clarepoxcin E did not display inhibitory activity towards the 20S human proteasome, it did exhibit potent cytotoxicity against HCT-116. The authors proposed that in cells, clarepoxcin E acts as a prodrug to form the reactive α,β-epoxyketone moiety. Landepoxcins A and B displayed higher nanomolar inhibitory activity towards the human 20S proteasome, however landepoxcin A did exhibit impressive cytotoxicity against HCT-116 cells.The most recently discovered epoxyketone natural product inhibitors were reported by Owen products .206 Clar50 value of 19.8 μM.207 Epoxyphomalins A and B were first isolated as products of the marine-derived fungus Phoma sp. by Mohamed et al.208 Epoxyphomalin A displayed cytotoxicity towards several human tumor cell lines. The intracellular target of epoxyphomalins A and B was later identified as the 20S proteasome;209 both epoxyphomalins A (94) and B (95) exhibit dose-dependent inhibition of the 20S human proteasome subunits  and strongylophorines 15/16 (97) exhibited the most potent inhibitory activity towards the ChT-L site of the 20S proteasome with an IC50 of 2.1 μM and 3.6 μM, respectively (Noda ectively .98) was isolated from the New Caledonian marine sponge Petrosaspongi nigra among the other petrosaspongiolides A–R. The petrosaspongiolides are sesterterpenes all containing a cheilantane skeleton, and were first identified as inhibitors of the preparation of phospholipase A2.211 Petrosaspongiolide M was later identified by Margarucci et al. as a proteasome inhibitor using chemical proteomics.212 The natural product inhibited the C-L and ChT-L activity of the proteasome in a cell-based assay with IC50 values of 0.85 ± 0.15 μM and 0.64 ± 0.15 μM, respectively. Petrosaspongiolide M was later reported to exhibit potent inhibitory activity towards the ChT-L and C-L activities of the proteasome with submicromolar values in an in vitro fluorometric enzyme assay.213 Optimization led to analogues 99 and 100, which both feature a benzothiophen-2-yl substituent at the C4 position of the butenolide ring. Analogue 99 exhibited potent inhibitory activity against the ChT-L and C-L site of the 20S proteasome, with IC50 values of 0.06 ± 0.009 and 0.22 ± 0.03 nM, respectively. The desbrominated analogue 100 also displayed potent inhibitory activity towards the ChT-L and C-L sites, with IC50 values of 0.07 ± 0.01 nM for both sites (Petrosaspongiolide M (th sites .101) was identified by West and Faulkner as a metabolite of the marine sponge Acanthodendrilla sp. in 2008.214 Acanthosulfate (50: 4.5 μM) but lacked selectivity and potency when tested for activity against the BMS Oncology Diverse Cell Panel (ODCA).Several other terpenoid natural products have also been identified as inhibitors of the 20S proteasome. The merosesterterpene acanthosulfate (osulfate  exhibite102) is a quinone methide triterpene which is extracted from the “Thunder God Vine” (Tripterygium wilfordii Hook F.) and has been recognized for its biological activity. Dou et al. discovered the inhibitory activity of celastrol towards the 20S proteasome in 2006.215 Celastrol selectively inhibits the ChT-L activity of the proteasome with an IC50 value of 2.5 μM. The compound also demonstrated the ability to inhibit the proteasome of prostate cancer cells in a cell-based assay at 1–5 μM. Celastrol further demonstrated antitumor activity in an in vivo study. Researchers reported that treatment of PC-3 containing mice with the natural product resulted in tumor reduction. Because celastrol has many intracellular targets,216 focus on optimization of the celastrol scaffold has involved improving its solubility and physiological properties. Recently, celastrol has been reported as cytotoxic against human multiple myeloma cells.217 Further investigation of compounds from the extract of the “Thunder God Vine” (Tripterygium wilfordii F. Hook) focused on the diterpene tri-epoxide lactone triptolide (103). Originally discovered in 1972, researchers reported its antileukemic activity thus providing impetus for further biological evaluaton.218,219 Although triptolide does not inhibit the ChT-L site of purified 20S proteasome, it was able to inhibit proteasomal activity within cancer cells. This interesting result suggests that triptolide acts as a prodrug, and one of its metabolites—perhaps an oxidized ketone—acts instead as the biologically active molecule.Celastrol  contains an identical scaffold to that of celastrol except for the carboxylic acid moiety, which is instead replaced by a methyl ester. Pristimerin selectively inhibits the ChT-L activity of the 20S proteasome with an EC50 value of less than 125 nM. Researchers further indicated in a cell-based experiment that pristimerin suppresses NF-KB activity through proteasome dependent and independent pathways. Pristimerin also demonstrated promising activities in cytotoxicity and in vivo studies. The steroidal lactone withaferin A (105) is a natural product which was isolated from the Indian Winter Cherry Withania somnifera Dunal, a plant which is popular for its use in Ayurvedic medicine.221 The steroid contains two conjugated ketones and bears structural similarity to celastrol. Withaferin A had previously been recognized for its various biological activities, though the mechanism of action by the natural product had not been elucidated. Dou et al. identified the 20S proteasome as an intracellular target of withaferin A.222 Withaferin A inhibits the ChT-L activity of the 20S proteasome with an IC50 value of 4.5 μM and also exhibits proteasomal inhibition within PC-3 cells. The natural product also performed well in in vivo tumor studies conducted on mice. In silico docking studies in addition to kinetic studies suggested that the conjugated ketone moieties of withaferin A may interact covalently with the Thr1Oγ within the β5 active site to confer inhibition of the 20S proteasome.Tiedemann Trichoderma sp. 1212-03, and have recently been evaluated for their biological activities.223–225 Neomacrophorin I demonstrated cytotoxicity against human colorectal cancer COLO 201 cells, though its mode of action for this cytotoxicity was initially unclear. An MTT assay of the neomacrophorins with promyelocytic leukemia HL60 cells revealed that neomacrophorins I–VI (106–111) inhibit growth of the HL60 cells. The molecules were also responsible for inducing apoptotic cell death in HL60 cells, and further demonstrated in vitro inhibitory activity towards the 20S proteasome. Neomacrophorins I and IV demonstrated the most potent inhibitory activity towards the ChT-L site as compared to the other neomacrophorins, with IC50 values of 5.7 ± 1.0 and 5.3 ± 1.2 μM, respectively. The quinone moiety was deemed an important feature for the inhibitory proteasome activity and cytotoxicity.The neomacrophorins  were ideet al. isolated the aaptamine natural products (Aaptos suberitoides as part of their efforts to identify novel proteasome inhibitors from marine invertebrate extracts and marine-derived fungi cultures. Aaptamine (112), isoaaptamine (113) and demethylaaptamine (114) inhibit the ChT-L and C-L sites for both rat and human 20S proteasomes (IC50: 7.0–20.2 μM). When tested for cytotoxicity against HeLa cells, the aaptamines did not exhibit a correlation between cytotoxicity and proteasome inhibition.Various alkaloids have also been identified as inhibitors of the proteasome. Tsukamoto products  from theXestospongia cf. vansoesti and evaluated for their proteasome inhibitory activity.226 Salsolinol (115) and its derivative norsalsolinol (116) inhibit the ChT-L site with IC50 values of 279.0 and 193.7 μM, respectively. Cytotoxicity studies indicated that salsolinol is cytotoxic against several cell lines; norsalsolinol displayed cytotoxicity against HeLa cells (IC50: 42.4 μM) but was not tested against other cell lines.The tetrahydroisoquinoline salsolinol alkaloids were later isolated from the marine sponge et al. , neo-kauluamine (118) and pre-neo-kauluamine (119) exhibit potent inhibitory activity towards the ChT-L site of the 20S proteasome with IC50 values of 2.0, 0.13 and 0.34 μM, respectively. Acanthomanzamine D (120) also exhibits nanomolar inhibitory activity towards the chymotrypsin-like site of the 20S proteasome (IC50: 630 nM), albeit with less potency than the kauluamines. The presence of the eight-membered ring in addition to the β-carboline appeared to be necessary for proteasome inhibition.Specific manzamines have been identified as inhibitors of the 20S proteasome by Tsukamoto et al. .227,228 Acanthostrongylophora ingens and evaluated them for their biological activity  and tetradehydrohalicylamine B (122) differ only in their level of unsaturation; whereas the latter contains a pyridinium ring, the former contains a substituted tetrahydropyridine ring. The halicyclamines were tested for cytotoxic activities against HeLa cells which showed that tetradehydrohalicyclamine B exhibited low micromolar cytotoxicity (IC50: 12 μM), whereas halicyclamine B is not cytotoxic. Halicyclamine B and tetradehydrohalicyclamine B demonstrated inhibitory activity towards both the cCP and iCP.The Tsukamoto group also isolated two halicyclamines from activity .229 Hali123) and its analogs  inhibitors of the caspase-like activity of the 20S proteasome. In silico docking suggested an interaction of the N5 substituents with a Tyr residue  for their selectivity. Introduction of a large, flexible hydrophobic residue at the C1 position led to the discovery of sixteen derivatives with micromolar (IC50: 2–5 μM) activity towards the β1 subunit.231In silico docking indicated that these hydrophobic moieties bind within the primed substrate binding channel of the β1 active site to confer selectivity. Replacement of the carbonyl at C4 with a benzylamino moiety further improved the inhibitory activity of the scaffold to lead to β1-selective nanomolar inhibitor (124) (IC50: 600 nM).232Cerpegin . Discovered by Scheuer et al. in 1993,234 researchers reported that palau ‘amine displayed cytotoxic activity. The first total synthesis of (±)-palau’amine was achieved by the Baran group in 2010,235 followed by an enantioselective synthesis of (—)-palau’amine shortly thereafter.236 Due to its reported cytotoxicity, palau’amine and its relatives (±)-dibromophakellin (126) and (±)-dibromophakellstatin (127) were evaluated as inhibitors of the ChT-L activity of the cCP and iCP.237 (—)-Palau’amine displayed 2-fold improved inhibitory activity towards the constitutive human 20S proteasome relative to its racemate (IC50: 2.5 (±0.7) vs. 5.5 (±1.5) μM, respectively), indicating that the (—) enantiomer is responsible for inhibitory activity towards the proteasome. The presence of a cyclic urea or guanidine ring is integral to inhibitory activity. Researchers synthesized several indole analogs238 as potential inhibitors based on the (±)-dibromophakellin scaffold using the same strategy as Hewlett and Tepe in their total synthesis.239 (±)-Indolophakellin analog 129 exhibited potent and specific inhibitory activity towards the β5c of the 20S proteasome (IC50: 3.5 ± 0.7 μM) at a comparable potency to (±)-palau’amine. An X-ray crystal structure of the yCP:129 complex (2.5 Å resolution) revealed that the analogue confers its inhibition through solely non-covalent interactions with the S3 subpocket of the β5 subunit, including H-bonding and halogen bonding interactions. Since this study, newly discovered pyrrole–imidazole alkaloids have frequently been tested for their proteasomal inhibitory activity. For example, 5-bromopalau’amine (128) was recently isolated among several bromopyrrole alkaloids from the Dictyonella sp. marine sponge and exhibited inhibition towards the ChT-L site of the 20S proteasome (IC50: 9.2 ± 3.2 μM).240Pyrrole–imidazole alkaloids have also been investigated for their ability to inhibit the proteasome . Pyrrole130), (—)-ECG (131), (—)-GCG (132) and (—)-CG (133)50 values 86–194 nM, (—)-EGCG being the most potent among them.241 Catechins lacking the ester moiety do not display this same inhibitory potency. Subsequent cell-based studies using (—)-EGCG demonstrated the compound's ability to inhibit the 26S proteasome in Jurkat T cells; 10 μM (—)-EGCG inhibited ∼70% of the proteasomal ChT-L activity. EGCG also inhibits the ChT-L activity in breast (MCF-7) and prostate (PC-3 and LNCaP) cancer cells. Enantiomeric analogues of natural catechins, (+)-EGCG and (+)-GCG inhibit the ChT-L activity of the proteasome in both in vitro and in vivo studies to a similar potency of the natural catechins. However, global protection of the hydroxy groups of (+)-EGCG with benzyl moieties renders the compound inactive, implicating that the presence of at least one hydroxyl group is necessary for inhibitory activity.242 The mechanism of proteasome inhibition by catechins was later postulated by Smith et al.243 (—)-EGCG irreversibly inhibits the ChT-L activity of the 20S proteasome in a time-dependent manner, indicative of covalent bond formation within the active site. Docking studies carried out using AutoDock indicated this potential interaction. When docked with the ChT-L site, the ester carbonyl of (—)-EGCG is within 3.18 Å of Thr1Oγ for nucleophilic attack. The hydrophobic A ring of (—)-EGCG also sits within the S1 subpocket of the ChT-L site to confer selectivity between subunits. Due to instability of (—)-EGCG under biological conditions, subsequent studies of the tea polyphenols for proteasome inhibition focused upon improving analogue stability under physiologically-relevant conditions. Several studies by the Dou group have been conducted to generate bioactive analogues of (—)-EGCG: examples of these include peracetate esters244,245 and fluorinated analogues.246Many flavonoids have been isolated and evaluated for their ability to inhibit the 20S proteasome based upon their intriguing bioactivities. The green tea-derived catechin polyphenols have been among these flavonoids of interest, and include epigallocatechin-3-gallate ((—)-EGCG), epicatechin ((—)-EC), epigallocatechin ((—)-EGC), gallocatechin-gallate ((—)-GCG), and catechin gallate. The ester moiety present in of some of these flavonoids was thought to contribute to proteasome inhibition, as the ester-containing catechins (—)-EGCG (134) was first reported in 2003 by Kazi et al. for its inhibitory activity towards the ChT-L site of the 20S proteasome in a cell-free assay with an IC50 value of 26 μM.247 Docking studies suggested that unlike tea polyphenol (—)-EGCG, genistein does not covalently interact with the Thr1 residue of the active site. Genistein was also isolated in addition to proteasome inhibitors isoliquiritigenin (135) and 7-hydroxyflavanone (136) from the plant Spatholobus suberectus.248 The three compounds exhibited low micromolar inhibition towards the β5-site of the 20S proteasome, with IC50 values ranging from 4.88 (±1.55) to 9.26 (±1.2) μM. Apigenin (137), quercetin (138), kaempferol (139) and myricetin (140) were later evaluated by Chen et al. for their ability to inhibit the 20S proteasome.249 All four compounds inhibited the ChT-L site of the 20S proteasome in a cell-free assay, with apigenin displaying the most potent activity (IC50: 1.8 ± 0.03 μM). The compounds displayed similar inhibitory activity towards the 26S proteasome in intact Jurkat cells. Apigenin was still the most potent of the four compounds. In silico docking studies suggested that the carbonyl at C-4 is the site of nucleophilic attack by the Thr1Oγ residue of the active site. Additionally, the hydroxyl group at C-3 was believed to interfere with binding of the flavonoids to the active site. Further studies by the same group evaluated the structure–proteasome–inhibitory activity relationship of flavonoids chrysin (141), luteolin (142), naringenin (143), and eriodictyol (144) in comparison to apigenin.250 The 2,3 double-bond featured within the flavanones chrysin, apigenin and luteolin was deemed necessary for of inhibition towards the ChT-L site of the proteasome. A subsequent study by Wu and Fang reported that the inhibition of chrysin, apigenin and luteolin is selective for ChT-L and T-L catalytic activities of the proteasome within tumor cells.250In addition to the tea polyphenols, several other flavonoids have been identified for their ability to inhibit the proteasome 145) and 7-methoxydichamanetin (146) were recently isolated in a bioassay-guided fractionation of the Piper sarmentosum plant by Pan et al.251 These flavanones exhibited inhibitory activities toward the ChT-L site of the human 20S proteasome, with IC50 values of 2.87 ± 0.26 and 3.45 ± 0.18 μM, respectively. The flavonoid glycosides baicalin (147) and scutellarin (148) were isolated from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz by Wu et al. These compounds exhibited the ability to preferentially inhibit ChT-L activity in A549 and HL60 cancer cells.252 The biflavonoids morelloflavone (149) and talbotaflavone (150) were isolated from the stem bark of Garcinia lateriflora by Ren et al., and exhibited inhibition against the ChT-L site of the 20S proteasome, with IC50 values of 1.3 μM and 4.4 μM, respectively.253 The prenylated flavonoid sanggenon C was also reported as an inhibitor of the 20S proteasome.254 Huang et al. indicated that the natural product inhibits the ChT-L activity of the 20S proteasome in enzymatic studies and also in H22 cell lysate in a dose-dependent manner.Pinocembrin (151) is a symmetric polyphenol which was also reported as a proteasome inhibitor through in vitro and in vivo studies.255,256 Curcumin inhibits the ChT-L activity of the 20S mammalian proteasome with an IC50 value of 1.85 μM, demonstrates the ability to inhibit the 26S proteasome in human colon cancer HCT-116 and SW480 cell lines, and also induces apoptosis. In silico docking studies suggests that the ketone moieties are susceptible to nucleophilic attack by the Thr1Oγ of the chymotrypsin-like site. A recent study by Zhang et al. introduced an α-aminoboronic acid electrophile to the scaffold to improve potency.257 These compounds displayed impressive growth inhibitory activity against HCT-116 cells.The promiscuous active agent curcumin (152) was recently identified as an inhibitor of the 26S proteasome in the HEK293A-luciferase-cODC cell line. Emodin inhibited luciferase-ODC degradation with an EC50 of 6.33 μM and also exhibited inhibitory activity against the ChT-L and C-L sites of the proteasome, with IC50 values of 1.22 and 0.24 μM, respectively.258In silico docking with the catalytic sites of the proteasome indicated that the carbonyls are susceptible to nucleophilic attack by the Thr1Oγ much like the other carbonyl-containing flavonoids.Emodin (153) was isolated from the traditional Chinese medicine Zi Cao (gromwell) and reported as an inhibitor of ChT-L activity of 20S rabbit proteasome (IC50: 12.5 μM).259 The natural product also demonstrates inhibitory activity towards the 26S proteasome in cell studies (PC-3 and murine hepatoma H22). A later study by Wada et al. complemented these results, demonstrating the ability of shikonin to induce apoptosis in various multiple myeloma cells including bortezomib resistant cells KMS11/BTZ (SHK at 2.5–5 μM).260In silico docking studies suggest that the quinone carbonyls interact with the ChT-L site in such a way that they became highly susceptible to the catalytic site's nucleophilic Thr1 residue.Polyketides represent another class of natural products which have been scrutinized for their biological activity 154) was identified as a weak inhibitor of the proteasome.261 Kendomycin exhibited the ability to weakly inhibit the activity of the ChT-L site of the proteasome (IC50: 67.9 μM). X-ray crystallographic analysis of the yCP: kendomycin complex indicates that it does not interact with the inner chamber of the core particle, but rather covalently attaches to β2-H141Nγ along the outside of the 20S proteasome. Kendomycin sits within the surface-exposed pocket formed by the interface of the β2–β7′ subunits.Recently, the cytotoxic macrocycle kendomycin  was isolated alongside known mycalolide A (156) and 30-hydroxymycalolide A (157) from a marine sponge of the genus Mycale  which was collected from a Chicago city parkway. The most potent cylindrocyclophanes A4–A2 (158–160) inhibit the ChT-L activity at low micromolar concentrations . Cylindrocyclophanes A4–A2 also display potent cytoxicity against HT-29 cells, with EC50 values of 2.0, 0.5 and 1.7 μM, respectively. Researchers suggested that in addition to a dichloromethyl moiety, the presence of hydroxyl group at C14 was also important for inhibitory activity. However, the inhibitory potency results did not correlate to cytotoxicity results, suggesting that the main reason for cytotoxicity was not due to proteasome inhibition Several macrocyclic cylindrocyclophanes were also reported as proteasome inhibitors by Chlipala 161), its analogues, and its acyclic analog seco-rapamycin (162) have also been identified as allosteric inhibitors of the 20S proteasome by Osmulski and Gaczynksa 264 Rather than bind competitively within the catalytic sites of the 20S proteasome, these compounds bind to specific grooves on the α-ring to confer inhibition. Rapamycin inhibits the ChT-L and T-L activities of the 20S proteasome in a reversible manner, with IC50 values of 1.9 and 0.4 μM, respectively. Identification of a minimal binding pharmacophore led researchers to the discovery of analog 163, a pipecolic ester carbamate which inhibits the ChT-L site of the 20S proteasome .265The macrolide rapamycin  scaffolds, which retain the key components of their parent molecule. Modification of natural product scaffolds by researchers has led to more potent, physiologically relevant, and selective inhibitors as starting points for the treatment of disease. Thus far, the clinical impact of proteasome inhibitors has been significant with several agents currently clinically used to treat multiple myeloma and mantle cell lymphoma.There are no conflicts to declare.


Hyper-activated melanocytes are the major cause of skin hyper-pigmentary disorders, such as freckles and melasma. Increasing efforts have been made to search for materials with depigmenting activity to develop functional cosmetics. As a result, numerous materials have been reported to have depigmenting activity but some of them are known to cause unwanted side effects. Consequently, anti-pigmentary natural compounds without concern of toxicity are in great demand. Virtually all sorts of natural sources have been investigated to find anti-pigmentary natural compounds. This review summarizes recently reported anti-pigmentary natural compounds and their mode of action from the ocean, plants, and bacteria. Tyrosinase (TYR), Tyrosinase-related protein-1 (TRP-1), and Tyrosinase-related protein-2 (TRP-2), under the orchestration of the principal melanogenesis regulating factor, Microphthalmia-associated transcription factor (MITF) [Melanin plays a pivotal role in protecting the skin against UV radiation and oxidative stress from various pollutants, but its overproduction is the main cause of human skin hyperpigmentation . Melaninr (MITF) .Dopaquinone (DQ) and/or L-dihydroxyphenylalanine (L-DOPA) to DQ by Tyrosinase activation. DQ reacts with cysteine to form 5- or 3-cysteinyl DOPAs, which then oxidize and polymerize to ultimately make pheomelanin. Alternatively, DQ cyclizes to form a DOPA chrome, which is spontaneously decarboxylated to 5, 6-dihydroxyindole (DHI). DHI rapidly oxidizes and polymerizes to yield DHI-melanin. In case TRP-2 is available, DOPA chrome tautomerizes to form DHI-2-carboxylic acid (DHICA), which can be oxidized and polymerized by TRP-1 to yield DHICA-melanin. The availability of substrates and enzymes decides the type(s) of melanin produced [There are two kinds of melanin pigment: brownish-black eumelanin and reddish-yellow pheomelanin . The synMITF is a crucial transcription factor stimulating the expression of Tyrosinase, TRP-1, and TRP-2. Multiple signaling pathways are involved in MITF regulation. UV exposure induces the activation of p53 and results in increased expression of proopiomelanocortin (POMC), which is then cleaved into small peptides including α-melanocyte-stimulating hormone (α-MSH). The POMC-derived α-MSH binds to the Melanocortin-1 receptor (MC1R) on the surface of melanocytes, leading to the production of cAMP through the activation of adenylyl cyclase. cAMP activates the Protein kinase A (PKA), which translocates to the nucleus and phosphorylates the cAMP response element-binding protein (CREB), causing the upregulation of MITF [ of MITF ,8.MITF is controlled by the Mitogen-activated protein kinases (MAPKs), which are composed of three subtypes: Extracellularly responsive kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 [MITF and eventually stimulates melanogenesis. On the contrary, JNK or ERK activation induces MITF degradation and downregulates melanin synthesis [MITF is also activated by the Wnt/β-catenin pathway. Wnt is a cysteine-rich glycoprotein that couples with the G protein-coupled receptor to inhibit the glycogen synthase kinase-3β (GSK-3β). It causes the accumulation of β-catenin in the cytosol, increasing the upregulation of MITF [Additionally,  and p38 . The pho of MITF .When melanin production is completed, melanin contained in the melanosome is transferred to keratinocyte through melanocyte dendrites. Transfer of melanin is an important process in skin pigmentation, although much about the process is yet to be understood .Tyrosinase activity [Efforts to discover new plant extracts with skin-whitening efficacy have been made using various kinds of melanoma cells. In vivo evaluations using animal models such as mice or zebrafish have also been performed to confirm skin-whitening efficacy. Various parts of ginseng including the roots, seeds, and fruit have been widely used in both food and pharmaceutical products in East Asia for over 1000 years. Recently, a lot of research has been conducted to identify the skin-whitening ingredient from ginseng. Korean Red Ginseng (KRG) extract was verified as possessing inhibitory effects on melanin production in B16F10 cells through the suppression of activity .Panax ginseng calyx is the peduncle between the berry and root of ginseng. This study showed Panax ginseng calyx ethanol extract (Pg-C-EE) suppressed TRP1, TRP-2, Tyrosinase, MITF, p38, ERK, and CREB levels dose-dependently in B16F10 [Panax ginseng seed was non-cytotoxic to Melan-A cells at a concentration of 100 ppm, at which it significantly (p < 0.01) suppressed cellular melanin production by inhibiting Tyrosinase activity [P. ginseng berry also inhibited melanin production and MITF protein expression in melan-A cells [P. ginseng, inhibited the melanin transfer on MNT-1/HaCaT coculture system and three-dimensional (3-D) human skin equivalent [n B16F10 . Ethanolactivity . Floralg-A cells . Additiouivalent .Aster spathulifolius extract (ASE) inhibited melanin production at concentrations of 50, 100, and 200 μM in B16F10 cells. Western blot analysis revealed that ASE extract activated both MAPK/ERK and Akt/GSK3β phosphorylation, promoting the ubiquitin-dependent degradation of MITF. The whitening effect of ASE was also observed on C57BL/6J mice, in which hyper-pigmentation was induced by 3, 6, and 9 weeks of UV irradiation. A significant reduction in melanin content was identified in the epidermis at a dose of 140 mg/kg [The extract from Lonicera japonica and, upon testing, inhibited melanogenesis in Melan-A cells at 300 μM without cytotoxicity through decreasing Tyrosinase, TRP-1, and TRP-2 expression. It has been shown that this effect occurred in a dose-dependent manner by promoting the Akt and ERK signaling pathways. In addition, sweroside was proven to be effective in vivo by reducing melanin spots in zebrafish embryos [MITF by activating the ERK signaling pathways, decreasing the expression of Tyrosinase, TRP-1, and dopachrome tautomerase (DCT) in SK-MEL-2 cells. Furthermore, ISL inhibited the melanocyte dendricity and melanin transport in cocultured SK-MEL-2 cells and HaCaT by suppressing the expression of Rab27a and Cdc42 proteins [Pueraria thunbergiana significantly inhibited melanogenesis in B16F10 cells via anti-Tyrosinase activity. Phosphorylation of GSK-3β was decreased by P. thunbergiana in a dose-dependent manner, but the amount of phospho-ERK had no change, suggesting that Pueraria thunbergiana inhibits MITF through the Akt/GSK-3β pathway [Juglans mandshurica (1–10 μM) suppressed Tyrosinase and MITF in B16F10 cells and primary human epidermal melanocytes (PHEMs). The extract was proved to be non-toxic up to 10 μM. The mechanism of suppression was revealed as a result of markedly enhanced phospho-ERK [Sweroside was isolated from  embryos . Isoliquproteins . The eth pathway . Leaf anspho-ERK .Sophora flavescens root inhibited the transfer of melanin to keratinocytes via regulating both RAC1 in SK-MEL-2 cells and PAR2 in HaCaT cells. RAC1 promotes dendrite and lamellipodium formation in melanocytes. PAR2 stimulates the phagocytosis rate of keratinocytes and increases melanin transfer. The levels of RAC1, PAR2, and MITF mRNAs were suppressed following the treatment with S. flavescens extract [Morus alba leaves has an anti-melanogenesis effect via inhibiting Tyrosinase activity in α-MSH-activated B16-F10 cells in a dose-dependent manner. It inhibited MITF, Tyrosinase, and TRP-1 by downregulating CREB and p38 signaling pathways [MITF in Mel-Ab cells [Camellia oleifera extract was shown to inhibit melanin synthesis in B16F10 by suppressing Tyrosinase and TRP-2 production [Camellia sinensis water extracts also significantly (p < 0.001) decreased Tyrosinase protein expression in a concentration-dependent manner [Phyla nodiflora reduced cellular melanin content and Tyrosinase activity in B16F10 cells and had no cytotoxic effects. The mechanism was associated with the downregulation of CREB and MITF. It also induced the phosphorylation of ERK1/2 and p38 MAPK [The ethanol extracts of  extract . A 70% epathways . StudiesAb cells . Camellioduction . Camellit manner . Eupafolp38 MAPK . The folTyrosinase inhibitors [The ocean has enormous biodiversity, with more than 250,000 species listed and many other species yet to be discovered. Marine resources have the properties of photoprotective, anti-aging, antioxidant, and moisturizing activities, attracting great attention from the cosmetic industry . Besideshibitors . The folTyrosinase inhibitory activity of four types of extract, Endarachne binghamiae, Schizymenia dubyi, Ecklonia cava (EC), and Sargassum silquastrum (SS), was measured at two temperatures. Schizymeniadabyi (SD) and Sargassum silquastrum (SS) extracts showed 90.75% and 70.75% inhibitory activity at 20 °C, respectively. Endarachnebinghamiae (EB) and Ecklonia cava (EC) extracts showed 81.26% and 73.22% inhibitory activity at 70 °C, respectively. To investigate the inhibitory activities in vivo model, a zebrafish embryo was used. Eckloniacava (EC) and Sargassum silquastrum (SS) extracts showed 48% and 50% inhibitory effects, respectively [Tyrosinase catalytic activity competitively. [Eckloniastolonifera, OKAMURA, phloroglucinol, eckstolonol, eckol, phlorofucofuroeckol, and eckol, were treated to mushroom Tyrosinase. The inhibitory kinetics showed that phloroglucinol and eckstolonol were competitive inhibitors, and the remaining three compounds were noncompetitive inhibitors [Ishigeokamurae showed a 78.73% inhibition against mushroom Tyrosinase. It binds non-competitively to the enzyme and changes the shape of the enzyme, which leads to inhibit the binding of substrate to the active site [Eisenia bicylis is a noncompetitive inhibitor of Tyrosinase and blocks IBMX-induced melanin formation [Turbinariaconoides is a brown alga that produces zinc oxide. Inhibition activities were measured on Tyrosinases from potato and L-TYRosine. The combination of crude extracts of Turbinariaconoides and zinc oxide nanoparticles as a comparator showed 75% and 56% inhibition activity [Sargassum plagyophyllum and Eucheuma cottonii were treated with L-tyrosine substrate and L-DOPA substrate. IC50 values for L-tyrosine substrate (monophenolase) were 2691.478 μg/mL and 2195.206 μg/mL. IC50 values for L-DOPA substrate (diphenolase) were 2631.648 μg/mL and 1769.336 μg/mL, respectively. IC50 values for diphenolase are less than those for monophenolase, which means the extracts have more significant effects on dephenolase [Symphyocladialatiuscula exerts a strong hydrogen bond interaction with Arg268 and Per404 of Tyrosinase. Bromophenol only binds to free enzymes and works as a competitive inhibitor. Furthermore, it significantly reduced Tyrosinase expression levels [Eckloniacava are slow-binding inhibitors. Both compounds interacted with His85 and Asn260 on the Tyrosinase active site, but each had different binding mechanisms. The former slowly binds to the active site following a single step-binding mechanism. The latter binds to the enzyme and induces a new conformational state of enzyme that inhibits substrate to make a complex with Tyrosinase [Ishigefoliaceae inhibited melanin synthesis and Tyrosinase activity through the ERK pathway-mediated suppression of MITF, Tyrosinase, TRP-1, and TRP-2 in α-MSH-stimulated B16F10 cells [The ectively . Fucoidaitively. . Five cohibitors . Diphlorive site . Fucofurormation . Turbinaactivity . Methanohenolase . Bromophn levels . Phlorof10 cells .Undaria pinnatifida (UPEF), a phlorotannin-rich extract of Ecklonia cava (ECE), and glycosaminoglycans (GAGs). Each component alone inhibited mushroom Tyrosinase in α-MSH-stimulated B16F10 cells but was cytotoxic. However, the combination of three components showed a stronger whitening effect without cytotoxicity. A mixture of compounds in a ratio of 4:5:1 (UEG-451) showed 71.1% whitening activity with the highest cell viability. Additionally, a UEG-451 combination downregulated melanogenesis-related proteins, including Tyrosinase, TRP-1, and TRP-2, in α-MSH-stimulated B16F10 cell by regulating the expression of MITF [Stichopus japonicus extracts were evaluated by measuring the inhibition of mushroom Tyrosinase and melanogenesis in B16F10 melanoma cells. The results indicated that the extracts markedly inhibited Tyrosinase activity and melanin synthesis. MITF expression was also suppressed by the phosphorylation of ERK, leading to reduced melanogenesis-related gene expression [Streptomyces extracts, Trichostatin A (TSA) showed the strongest Tyrosinase inhibition activity. TSA is a mixed-type inhibitor that can bind to the enzyme with or without substrates being bound [MAPKs (mitogen-activated protein kinase) and PKA (Protein kinase A) signaling pathways, resulting in the inhibition of Tyrosinase activity, melanin secretion, and melanogenesis-related gene expression [Pandina boryana extracts regulated the phosphorylation of ERK and inhibited the expression of melanogenesis-related protein via the MAPK pathway [Sargassum siliquosum extracts with mushroom Tyrosinase revealed that 125 μg/mL of the extracts show 73% inhibition activity. The extracts have a phenol group; thus, the hydroxyl group of phenol makes a hydrogen bond with the active site of Tyrosinase that interrupts substrate binding with the enzyme and causes a conformational change in the enzyme [Sargassum serratifoliu extracts, Sargaquinoic acid (SQA), was investigated with mouse melanoma cells. SQA reduced cAMP by hydrophobic interaction with the binding site between cAMP and PKA. Reduced cAMP causes PKA inactivation, leading to the inhibition of CREB and MITF signals. Besides, the downregulation of MITF led the inhibition of melanogenesis-related protein expressions including TRP-1, TRP-2, and Tyrosinase. Additionally, SQA inhibited Tyrosinase by MITF degradation through the phosphorylation of ERK1/2 [Cladiella australis  exhibited a skin-whitening effect in B16-F10 cells (in vivo). MITF, Tyrosinase, TRP-1, TRP-2, and Gp100 were significantly reduced by Cladiella australis-4-(phenylsulfanyl) butan-2-one compared to the control group. Since there were no cytotoxic, carcinogenic, or teratogenic properties, the research suggested that 4-phenylsulfanyl-butan-2-one could be a safe and effective skin-whitening agent [The skin-whitening effects of a marine-derived mixture from sea squirt skin were investigated. The mixture included a fucoidan-rich extract of  of MITF . Stichoppression . Among Sng bound . Jeju mapression . Pandina pathway . Investie enzyme . One of f ERK1/2 . Chitosa:5:1 UEG-1 showed ng agent .Acetobacter, Aspergillus, and Penicillium [Tyrosinase in a non-classical manner [Saccharomyces cerevisiae extract had inhibitory effects on melanogenesis and melanosome transfer.5-hydroxy-2-hydroxymethyl-4-pyrone, also known as Kojic acid, is naturally derived from fungal metabolites produced by species of icillium . Kojic aicillium . Furtherl manner . Thus, kTyrosinase activity was blocked by the extract without cytotoxicity in B16F10 cells. However, the extracts had no effects on the expression of the MITF protein, which means they may indirectly inhibit Tyrosinase activity, but through post-transcriptional control. In addition, they inhibited PAR-2, melanosome transfer-related protein, so that melanosome transportation would be suppressed [Bifidobacterium adolescentis on melanin contents and Tyrosinase activity were measured. Melanin content of a B16F10 melanoma cell treated with 7.5% B.adolescentis was 55.1% (% of control), and that of the positive controls was 86.4% (kojic acid) and 70.7% (arbutin). Tyrosinase activity of mushroom Tyrosinase treated with 7.5% B.adolescentis was 19.1%, and that of kojic acid and arbutin was 93.6% and 89.4%, respectively. These results indicated that B.adolescentis has a potent anti-melanogenesis activity superior to that of the positive controls, kojic acid, and arbutin [Angelica dahurica root (ADR) was fermented with four different bacteria: Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus acidophilus, and Lactobacillus brevis. IC50 for the Tyrosinase inhibition of extracts of ADR fermented by these four bacteria was 0.57, 2.80, 0.07, and 0.52 mg/m. Among the four bacteria, L. acidophilus-fermented ADR showed the highest activity. The IC50 of non-fermented ADR extract was 8.20 mg/mL, meaning that Tyrosinase inhibition activity of ADR has increased after fermentation [Lactobacillus rhamnosus spent culture supernatant (Lr-SCS) for cosmetic applications. The Tyrosinase inhibitory activity of 100% Lr-SCS was 71.3% compared to 43.6% and 83.6% of 2 mM and 10 mM kojic acid. In this study, the lactic acid in Lr-SCS appeared to suppress tyrosine activity in a dose-dependent manner [Lactobacillus plantarum (pLTA). pLTA decreased melanin synthesis by 57.9% compared to the control group. While pLTA did not inhibit Tyrosinase directly, it decreased intracellular activity of Tyrosinase and suppressed Tyrosinase-related proteins TRP-1 and TRP-2. Furthermore, pLTA induced the phosphorylation of ERK and AKT, leading to MITF degradation [Lactobacillus acidophilus (AL) is a popular probiotic strain, and the study investigated the effects of tyndallized lactobacillus acidophilus on melanogenesis. AL significantly inhibited melanin secretion and intracellular melanin content in α-MSH-treated melanocytes. Specifically, in α-MSH activated melanoma cells, AL inhibited the phosphorylation of PKA and CREB and suppressed the expression of Tyrosinase and MITF. However, when AL was treated on mushroom TYRosine to investigate Tyrosinase inhibition activity, there was no observed effect. Instead, AL reduced the mRNA expression of Tyrosinase, TRP-1, and TRP-2. Therefore, AL does not inhibit Tyrosinase directly but it can modulate melanogenesis through the regulation of the cAMP signaling pathway [Rhodobacter sphaeroides (lycogen) was treated to B16F10 melanoma cell to evaluate anti-melanogenesis activity in vitro. The relative melanin content of lycogen-treated cells was significantly lower than the control group. To identify the mode of action of lycogen, the expressions of Tyrosinase and MITF were examined. Lycogen increased the ERK signaling pathway, which degraded MITF, leading to an anti-melanogenesis effect [ppressed . The eff arbutin . Angelict manner . Lipoteiradation . Lactobas effect .Tyrosinase activity of three Bifidobacterium bifidum-fermented Chinese herb extracts—walnut, Moutan cortex radices, and asparagus root—was investigated. B. bifidum–fermented extracts showed higher Tyrosinase inhibition activity than nonfermented extracts, suggesting that fermentation improved some biochemical activities of the three herb extracts. As a result, the highest Tyrosinase inhibition activities of the three fermented extracts were 58.4% , 66.5% (moutan cortex), and 92.8% (asparagus root) [Gordonivater urolithincaciens and Gordonivater pamelaeae, the urolithin-producing bacteria. Cytotoxicity and Tyrosinase inhibitory effects of UA and UB were evaluated in B16F10 melanoma cells. Cell viability was not affected by up to 10 μM of concentration. The melanin content of B16F10 cells treated with UA10 μM was 56.5%, which is comparable to that of the positive control (kojic acid). The result showed that, while neither Urolithin A (UA) or Urolithin B (UB) affected the mRNA expression of Tyrosinase, both competitively inhibited the enzymatic activity of Tyrosinase [Tyrosinase inhibition activity of nonfermented Rhodiola rosea and Lonicera japonica extracts was 10 mg/mL and 6.93 mg/mL, respectively. However, the Tyrosinase inhibition activity of Alcaligenes piechaudii-fermented extracts was observed to be 0.78 mg/mL and 4.07 mg/mL, suggesting that the whitening effects of fermented R. rosea increased about 12 times, and that of L. japonica increased 70% compared to nonfermented extracts [Lactobacillus helveticus NS8-fermented milk (NS8-FS) was examined for hypopigmentation. By measuring the melanin content and Tyrosinase activities in B16F10 cells, the whitening activity of NS8-FS was investigated. The color of the B16F10 cells was lightened and melanin content was reduced by nearly 40%. Tyrosinase activity was inhibited, and the gene expression of TYRosine-related protein was also downregulated. Therefore, NS8-FS could be both the enzymatic inhibitor and the gene expression regulator of Tyrosinase [Magnolia officinalis bark (MOB) extracts after Aspergillus niger fermentation was investigated. The highest anti-Tyrosinase activity was shown with 3 days fermentation, which increased from 52.8% (before fermentation) to 93.6% (after fermentation). Additionally, the melanin content of human epidermal melanocytes was reduced. Melanin content was almost completely depleted at 200 μg/mL of fermented extracts [Asparagus cochinchinensis extract fermented with Aspergillus oryzae was evaluated for its effects on cell viability and Tyrosinase inhibition. Cytotoxicity effects on HEMs, HaCaT, and A375.S2 cells were not significant. Anti-Tyrosinase activity of both fermented and unfermented extracts was evaluated in mushroom Tyrosinase and human epidermal melanocytes. Anti-Tyrosinase activity of the fermented extract was significantly lower in HEMs than in mushroom Tyrosinase, indicating some discrepancy in Tyrosinase inhibitory activity between mushroom Tyrosinase and human melanocytes. Additionally, with the fermented extract, the inhibition of melanin production was higher than Tyrosinase inhibition activity, suggesting that the fermented extract may downregulate not only Tyrosinase but also the Tyrosinase-related protein MITF. In contrast, the unfermented extract directly inhibited Tyrosinase activity [The anti-us root) . Urolithextracts . Lactobarosinase . The antextracts . Asparagactivity ,49,62. TMITF, Tyrosinase, TRP-1, and TRP-1. A few plant-derived skin-whitening agents promoted ERK signaling pathways, resulting in accelerated MITF degradation and regulating the length of melanocyte dendrites, which may block melanin transportation. Ocean-derived agents mostly suppressed melanin production by inhibiting the enzymatic activity of Tyrosinase, either competitively or noncompetitively. Bacteria-derived skin-whitening agents suppressed melanin production by regulating Tyrosinase activity and melanogenic proteins. Interestingly, fermented extracts showed higher inhibitory activity than non-fermented extracts, which may be owed, at least in part, to the combined effects of plant- and bacteria-derived active ingredients. Of note, most natural skin-whitening agents exhibited lower toxicity than existing skin-whitening agents, including arbutin and kojic acid. Since melanogenesis is complex and a variety of factors and pathways are involved, further research on the whitening activity of natural compounds is needed. Furthermore, although the whitening activity of these natural compounds has been well established in vitro, it is difficult to ensure efficacy in humans, since there are many confounding factors for manifesting whitening effects in human skin [In this review, we investigated plant-, ocean-, and bacteria-derived natural compounds with whitening effects and their modes of action. Most plant-derived skin-whitening agents suppressed melanin production by modulating the expression of melanogenesis-related proteins, such as man skin ,64. Thusman skin .