Effective control of an exoskeleton robot (ER) using a human-robot interface is crucial for assessing the robot's movements and the force they produce to generate efficient control signals. Interestingly, certain surveys were done to show off cutting-edge exoskeleton robots. The review papers that were previously published have not thoroughly examined the control strategy, which is a crucial component of automating exoskeleton systems. As a result, this review focuses on examining the most recent developments and problems associated with exoskeleton control systems, particularly during the last few years (2017–2022). In addition, the trends and challenges of cooperative control, particularly multi-information fusion, are discussed. Nowadays, ERs are becoming increasingly popular due to improved robotic technologies and the positive perception of people toward interacting with robots. Man-machine collaboration technology can enhance the exoskeleton's convenience or comfort, as demonstrated in rehabilitation studies  and produce promising results. However, several methods still show a big gap to be filled that may promote high recognition accuracy. Thus, it is urgent to design robust and effective control algorithms to help identify the user's motion intention and optimally produce the correct trajectories with the WRs. Furthermore, it is possible to improve recognition accuracy by combining two physiological data types, such as EEG and EMG. This study aims to summarize and compare the various HMCC methods based on physiology and conventional cooperative control (CC). In addition, the advantages and disadvantages of each strategy will be discussed.Physiological signals and data collected through human-robot interaction (HRI) are essential to the success of human movement intention detection methods in an authentic setting. The method is based on physiological data, which means that sEMG  employing an LSTM neural network is achieved. Through sEMG signals, it is possible to achieve human voluntary joint torque. However, employing a non-linear model approach is complicated and challenging  has been investigated, and the HRCC method with a low-level position monitor and high-level trajectory tracking has been investigated for LLRR to produce the required trajectory  is the main focus of what has been reviewed in this survey. It has been found that the last in-depth survey of the LLERCC was published in 2013 . In addition, Section 7, details the challenges of human-robot cooperative control, while the concluding part of the paper is presented in Section 8.The development of ERs technologies has rapidly increased in recent years; however, most of them are still in their infancy. At present, the control of lower limb robots is a popular topic of research in the United States of America (USA), Europe, China, Japan, and Singapore. The United States, Japan, and Europe are now leading in this field, as these regions already have some fairly mature commercial products on the market  methods that come from EEG signals produced autonomously through external stimulation  and non-restorative  have been applied to replace or rehabilitate function. Tetraplegic users have employed BMIs to control robotic/prosthetic limbs and FES to carry out tasks and grab or grasp objects. BMIs can perform the restoration function process after a stroke. Hence, stroke patients utilized biofeedback of the mu-beta sensorimotor rhythm to shift the hand using a rehabilitation device  combining EEG and EMG data information to achieve promising control over modern prosthetic legs is required. Fusion techniques can be employed in specific detection modalities [primarily electromyography, electroencephalography, and mechanical sensors see ; Mohebbivia the spinal cord and promoting the sensory and motor pathways to function again. An EEG and EMG feature hybridization approach has been investigated to prove the feasibility of combining both bioelectrical data for extending human lifespans  to complete a task, and the wearers are expected to be qualified to exert as much muscle power as possible to initiate the motions. Thus, the controllers must detect the user's force and intensity. These control approaches can be classified as IC, which allows for the modification of the connection force between a human and an assistive robot to provide comfort when the user is being controlled by the robot. On the contrary, in rehabilitation situations, when the patient's capability is not enough to produce adequate force, the controller should produce the force/position pattern. The patient then bases their movements on it, with minimum muscular power.The user–robot combination adaptation is the essential technique that would be considered for further study. Truly, this is the key to achieving or boosting robot-patient cooperative control. In this regard, robots must be harmonized (integrated) with the patient's prompt movements. In addition to this, the implementation of this concept will stimulate the necessity to identify the movement prompts presented by the patient and suitably adapt the support device and coupling forces. Typically, to cope with human ability restoration, it is crucial to focus on control techniques such as active-assisted control, challenge-based control, haptic simulation, and non-contact coaching. Hence, this would result in a promising stage of vital restoration. A human exoskeleton connection platform often operates in a user loop, which consists of three main parts: the user, the user-robot interconnection, and the exoskeleton. The wearable device is a common HRI structure that is usually in charge of sending information and power to the intermediate user and device. The information or data transfer needs a sensing approach  to be aware of the user's movements and send it to the device monitoring or control system. The power transfer comes from the device, which can yield the power demanded to support the user's motion based on the user's intent. Thus, future scholars must pay more attention to the user's desired movement estimation technique, which is vital to achieving a suitable device assistance control method. Again, it will become increasingly significant when improving the durability and practicability of recognition techniques, which could be an essential point of future research.Most of the existing power assist controls are based on impedance control and bioelectrical signals; among them, IC is unsuitable for the support stage. The control-based bioelectrical signal has strong fuzziness, poor anti-interference, and low control precision. Both the IC and the control based on bioelectrical signals divide the gait cycle into the support and swing stages, which are difficult to adapt to different stages of the gait cycle itself. Thus, it is critical to consider a new adaptive control method for further LLRERs based on the needs of patients. According to the different stages of the gait cycle, this method does not need on-demand auxiliary control methods divided into support phase and swing phase control. Also, it does not require a physical therapist to use passive and active rehabilitation training to switch between different modes. However, according to the man-machine system for rehabilitation robots, trajectory tracking and real-time error learning in patients with functional sports ability enable continuous and seamless on-demand auxiliary torque control.According to the principles of modern hemiplegia therapy, whether a patient is different or not, they will not be in good health during rehabilitation. However, the various control algorithms ignore the improvement in the affected limb's condition. Real-time VIAC cannot be achieved by actively adjusting impedance parameters in complex situations based on the patient's health. Hence, the control strategy needs to be further improved and adjusted. In recent years, many studies have explored the LLRT approach and achieved powerful results. However, there are very few robots with full-cycle rehabilitation training (RT), which can promote positive human-machine interaction and boost user performance. In addition, existing LLRRs cannot effectively realize full-cycle gait RT. Thus, further study on HMCC based on the full-cycle RT is indispensable. In this regard, it is particularly important to develop LLRR that can adapt to various RT modes and enhance rehabilitation outcomes. Finally, The virtual reality approach will improve and play a critical role in the further growth or expansion of the lower- or upper-limb rehabilitation control aspect. In this way, the rehabilitation robot should be able to contribute to or support specific motions. The current state of the art can be categorized as “patient-cooperative” or “assist-as-needed” robots, which increase the user's power in rehabilitation by contributing only the minimal assistance required. The following benefits can be achieved during rehabilitation in a virtual reality environment with robotic control concepts. Firstly, this technology can provide direct visual feedback for the patients, making the training process more intuitive and guiding them to complete the movement tasks effectively. Secondly, virtual reality technology can achieve various game- and task-based training strategies to boost rehabilitation training. Beyond that, it can easily enhance the users' motivation to actively participate in sports-related exercise and offer a promising or optimized rehabilitation outcome  centers; and increased motivation of the patient, who can now control the movement of the robot. All of these benefits can truly promote a promising rehabilitation of the CNS . The rehabilitation area  can make extensive use of power assistance for elderly or frail people to help patients resume a normal life and enhance their quality of life. In this examination, the background and development of exoskeletons, actuation control approaches, classifications, and their various applications have been discussed. Thus, two popular control approaches, namely the physiological-based control (sEMG and EEG) and the traditional-based cooperative control for the lower limb exoskeleton have been reviewed, and an in-depth comparison has been presented. Moreover, the benefits and drawbacks of human movement intention detection approaches that rely on physiological signals and human-robot interaction data have been highlighted. We discussed the trends of cooperative control (multiple information fusion) since the combination of two bioelectrical signals can significantly increase recognition accuracy. Finally, based on the reviewed articles, it can be concluded that the virtual reality approach can improve and play a significant role in developing the lower or upper limb rehabilitation control aspect. Thus, future rehabilitation can explore virtual reality as a significant part of a larger rehabilitation setup that provides rich multimodal stimuli and direct dynamic interaction between the user and a smart machine.GM wrote the first draft of the manuscript. XZ contributed to conception and supervision of the study. RD, AA, KH, and EM organized the graphs and tables. All authors contributed to manuscript revision, read, and accepted the submitted version.


In patients with castration-resistant prostate cancer (CRPC), clinical resistances such as androgen receptor (AR) mutation, AR overexpression, and AR splice variants (ARVs) limit the effectiveness of second-generation antiandrogens (SGAs). Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. Here, we found and identified a bifunctional small molecule Z15, which is both an effective AR antagonist and a selective AR degrader. Z15 could directly interact with the ligand-binding domain (LBD) and activation function-1 region of AR, and promote AR degradation through the proteasome pathway. In vitro and in vivo studies showed that Z15 efficiently suppressed AR, AR mutants and ARVs transcription activity, downregulated mRNA and protein levels of AR downstream target genes, thereby overcoming AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance in CRPC. In conclusion, our data illustrate the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment. Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death for men in western countries . Advance50 (drug concentration that results in 50% protein degradation) potency up to 1 nM. However, low cell permeability, poor pharmacokinetic properties, and complex chemical structures may restrict the clinical application of PROTAC drugs  site, such as darolutamide . AnotherAC drugs . What’s AC drugs .via binding to AF-1 of the AR to reduce its stability  and PSA-luc were incubated with 5α-dihydrotestosterone (DHT) and 10 μM candidate compounds for 24 h. The cell lysates were collected and AR transcriptional activity was detected by dual-luciferase reporter assay. We identified 19 compounds that showed more than 25% AR transcription inhibition activity, among which compound Z15  of Z15 in LNCaP cells was ~0.22 μM, which was comparable to ENZa  and AR F876L , are key causes leading to antiandrogen resistance. Dual-luciferase reporter assay results indicated Z15 could efficiently inhibit DHT-induced both AR T877A and AR F876L transcriptional activities . Taken tPSA, PMEPA1, and TMPRSS2) by quantitative real-time PCR (qRT-PCR) assay. The results revealed that Z15 significantly inhibited the mRNA expression levels of these genes . Both Z15 and ARV-110 reduced AR, KLK3, and TMPRSS2 protein levels significantly  is the main participant that regulates intracellular protein degradation. To explore whether Z15 promoted AR degradation through UPP, LNCaP cells were treated with Z15 in the presence or absence of proteasome inhibitor MG132. Indeed, Z15 reduced the AR protein levels after 8 hr treatment, while AR protein levels reduction was counteracted by MG132. Similarly, Z15 induced AR protein decline was also counteracted by MG132 in VCaP cells . Further50=1.37 μM) showed comparable proliferation inhibition potency with positive control ARV-110 (IC50=0.86 μM). However, in ARV7-positive 22Rv1 cells, the proliferation inhibition activity of Z15 (IC50=3.63 μM) was much stronger than that of ARV-110 (IC50=14.85 μM). Both Z15 and ARV-110 displayed weak inhibition effects on the proliferation activity of AR-negative PC-3 and DU145 cells  not only correlate with antiandrogen resistance but also relate to poorer prognosis of patients with CRPC . Effortsvia ARV7 . Unfortuvia ARV7 . Neverthvia ARV7 . HoweverHowever, we notice that the AR degradation potency of Z15 is not as stronger as AR-targeted PROTAC molecule ARV-110, suggesting that Z15 is a less specific AR degrader compared to ARV-110. We acknowledge that while Z15 suppresses CRPC progress mainly through targeting AR and ARV7, Z15 may have other mechanisms of action which contribute to its anticancer activity in vitro and in vivo. Nevertheless, Z15 is a novel selective AR and ARV7 degrader that warrants further structure optimization and anti-CRPC mechanisms investigation.The LNCaP, PC-3, 22Rv1, VCaP, and DU-145 human prostate cancer cell lines were purchased from American Type Culture Collection (ATCC). The identity of these cell lines has been authenticated by STR profiling, and all these cell lines tested negative for mycoplasma contamination. LNCaP  and 22Rv1  cell lines were cultured with RPMI-1640 supplemented with 10% fetal bovine serum . PC-3  was cultured with F-12K supplemented with 10% FBS (PAN-Biotech), VCaP , and DU145  cell lines were cultured with DMEM supplemented with 10% FBS (PAN-Biotech).4 cells per well in 24-well plates and subsequently co-transfected with 100 ng PSA-luc, 20 ng wt-AR , and 3 ng Renilla plasmids using Lipofectamine 2000 reagent  following the manufacturer’s protocol. Then, 24 hr after the transfection, the medium was changed to phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped FBS, containing 5 nM DHT (1 µL) and 1 µL test compounds at the designated concentrations. After a further 24 hr incubation, the cells were lysed in 100 µL passive lysis buffer per well, and 20 µL cell lysates were used for detection of the luciferase activity using a Dual-luciferase Assay System  on a plate reader . All experiments were run in triplicate.Plasmid wt-AR is the full-length cDNA of wild-type AR, plasmid AR F876L is the full-length cDNA of mutant AR that harbors F876L mutation, plasmid T877A is the full-length cDNA of mutant AR that harbors T877A mutation, plasmid PSA-luc is a reporter gene plasmid in which firefly luciferase expression is dependent on the PSA promoter, and plasmid Renilla is a Renilla luciferase reporter gene plasmid. The plasmids wt-AR and AR F876L were kindly provided by Dr. J.H. Wu , plasmid PSA-luc was kindly provided by Dr. Hiroyuki , whereas plasmid T877A and plasmid Renilla were constructed in-house. During the reporter assays, 24 hr before transfection, PC-3  cells were seeded at a density of 6–7×105 cells per well in six-well plates. After 48 hr incubation, 4 µL DMSO  or 4 µL test compounds were added to each well at the designated concentrations. After another 24 hr incubation, the cells were lysed with 60 μL radio-immunoprecipitation assay lysis buffer. Then the protein lysis buffer was treated with 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes for western blot analysis. The following antibodies were used for the detection of proteins: rabbit anti-AR , mouse anti-AR , mouse anti-PSA , rabbit anti-cleaved PARP , rabbit anti-GR , mouse anti-HSP90 , mouse anti-CDK7 . Mouse anti-β-actin  was used as a loading control. Proteins were visualized using anti-mouse, anti-rabbit, or anti-goat HRP-conjugated secondary antibodies  and ECL-Plus . The resulting bands were analyzed and quantified using ImageJ v1.49g . Each experiment was repeated at least twice.Cells were seeded at a density of 3–4×104 cells per well in 96-well plates. VCaP and 22Rv1 cells were cultured in a complete RPMI-1640 growth medium, PC-3 cells were cultured in an F-12K growth medium, and DU-145 cells were cultured in a DMEM growth medium. After 24 hr incubation, 1 µL DMSO (Sigma-Aldrich) or 1 µL test compounds were added to each well at the designated concentrations. After 72 hr incubation, 20 µL MTT (Invitrogen) solution (5 mg/mL in PBS) was added per well and incubated for another 4 hr. The MTT formazan formed by metabolically viable cells was dissolved in 100 µL isopropanol. The absorbance was measured at 570 nm wavelength on a plate reader . Experiments were performed in triplicate. The value of the DMSO group was defined as 100%.Cells were seeded at a density of 1×102 cells per well in six-well plates. After 24 hr incubation, 4 µL DMSO (Sigma-Aldrich) or 4 µL Z15 and ARV-110 were added to each well at the designated concentration. The first culture media change occurred after another 48 hr incubation and then repeated every three days for 2 weeks until cell colony formation. Then, the culture media was removed and the cells were washed twice with PBS, 1 mL methanol was added to each well for 10 min, the cells were treated with 1 ml Giemsa stain for 10 min, and each well was washed with water until the background was clean, and then aired for another 30 min before taking pictures of the colonies. To quantify staining, the stained wells were washed with 1 mL 10% acetic acid, and absorbance was detected at a wavelength of 590 nm on a plate reader , The value of the DMSO group was used as control and defined as 100%. Experiments were performed in triplicate.PC-3 and 22Rv1 cells were seeded at a density of 4×10Digest the PCa tissues in 5 mg/ml Collagenase II with 10 µM Y-27632 in a 15 ml Falcon tube at 37 °C on a shaking platform for 4 h. Use 1 ml of 5 mg/ml Collagenase II per ~50 mg minced tissue. The tumor tissue fragments were digested into epithelial cell clusters, washed with ice PBS and centrifuged for 1 min at 300×g. Then the precipitate was suspended in the precooled matrigel. The suspension was inoculated in a 24-well plate and incubated in a 37 ℃ incubator for 30 min until the matrigel became gelatinous. Then, 500 μL medium per well was added, the PCa organoid medium was prepared as follows: Add 1.0 ml B27, 500 µl nicotinamide (1 M in PBS), 125.0 µl N-acetylcysteine (500 mM in PBS), 0.5 µl of EGF (0.5 mg/ml in PBS + 0.1% BSA), 5.0 µl A83-01 (5 mM in DMSO), 50.0 µl Noggin (100 µg/ml in PBS + 0.1% BSA), 50.0 µl R-spondin 1 (500 µg/ml in PBS + 0.1% BSA or 10% conditioned medium), 50.0 µl dihydrotestosterone (1 µM in ethanol), 5.0 µl FGF2 (50 µg/ml in PBS + 0.1% BSA), 5.0 µl FGF10 (0.1 mg/ml in PBS + 0.1% BSA), 5.0 µl prostaglandin E2 (10 mM in DMSO), 16.7 µl SB202190 (30 mM in DMSO) and top up to 50 ml with adDMEM/F12 . After passaging, Y-27632 is added to the culture medium (e.g. add 5.0 µl of 100 mM to 50 ml human prostate culture medium). The culture medium was refreshed every 3 days, 7 days after PCa organoid cultured, add 1 µL DMSO or Z15 at the designated concentration, then cultured for another 7 days.5 cells per well in six-well plates and cultured with RPMI-1640 with 10% charcoal-stripped FBS for 48 hr before treatment with 4 µL DMSO (Sigma-Aldrich) or 4 µL test compounds added to each well at the designated concentrations. After another 24 hr incubation, the total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The total RNA (1 μg) was used for complementary DNA synthesis using a cDNA reverse transcription kit . Real-time PCR was performed in triplicate using gene-specific primers on the PCR instrument . The mRNA levels were normalized to GAPDH. The gene-specific primers are listed in Cells were seeded at a density of 3–4×10via EZLink NHS-Biotin Reagents  at a ratio of 1:1 for 1 hr at 25 °C. A duplicate set of Super streptavidin-coated biosensors were incubated in PBS, then moved to wells containing 0.02% Tween-20 PBS solution with 50 µg/mL biotinylated AR protein for 10 min. Non-specifically bound AR was removed with a 1 min wash step, then the baseline was detected at the same wells for 1 min, and AR-coated biosensors were moved to wells containing 250 µM Z15 and 0.02% Tween-20 PBS solution for the association. The biosensor was then moved back to the baseline solution and dissociation of Z15 from the biosensors was detected. The baseline-association-dissociation steps were repeated for the adjacent wells containing 125, 62.5, 31.25, and 15.6 µM Z15, respectively. A negative control of 1% DMSO was also used during these steps. Association and dissociation of samples and the negative control to coated biotinylated AR sensors were measured for 1 min. Data analysis on the ForteBio Octet RED instrument was performed using a reference well subtraction in the ForteBio data analysis software. Binding between ENZa and AR was performed by the same method.The bio-layer interferometry measurements were conducted using a ForteBio Octet RED96 instrument  equipped with super streptavidin biosensor chips (ForteBio). For the AR LBD binding analysis, purified AR protein  was biotinylated For the AR AF1 binding analysis, the AR-AF1 domain (110–486 aa) was cloned into the Pet21a vector and transformed into BL21-competent cells. Expression of the protein was induced with 0.5 mM IPTG when the optical densitometry reached 0.6 and then incubated at 37 °C for 5 hr. Cells were harvested and lysed in a lysis buffer . The AR-AF1 protein was first purified with affinity chromatography (Ni Sepharose excel) using the ÄKTA chromatography system . Peak fractions were pooled and further purified with anion exchange (Q Sepharose Fast Flow) chromatography. Binding affinities between AR AF1 and ENZa, Z15, and UT-34 were performed by the same method as described above.5 cells per well in 12-well plates cultured with RPMI-1640 media supplemented with 10% FBS. After 48 hr incubation, RPMI-1640 media was removed and cells were washed with PBS, then cultured with phenol-free RPMI-1640 media supplemented with 2% BSA mixed with 1 nM [3H] DHT  and 5 µL test compounds at the designated concentrations. Thereafter, 5 µL 10 µM R1881 was transferred to the assay plate for complete competitive binding (background) and DMSO was used for total binding (hole coverage). After incubating at 37 ℃ for 4 hr, the cells were washed twice with ice-cold Tris-buffered saline and then lysed with a solution of 200 µL 0.2 M NaOH. Isolated cell samples were counted using a scintillation counter, with appropriate standards of total activity and blank controls. The ‘Inhibition [% Control]’ was calculated using the equation: %Inhibition = ×100. Experiments were performed in triplicate.LNCaP cells were seeded at a density of 2×10AF1 was cross coupled with EDC and NHS on CM5 sensor chip to obtain ~7000 RU AF1 immobilized on chip surface. The experiment was conducted on method model, and performed at 30 µL/min, 25 ℃. Three buffer blanks were added to the beginning, followed by the compound samples. Association and dissociation time were set to 120 s. The needle was extra washed with 50% DMSO in every cycle, DMSO effects between channels was normalized with 1% DMSO controls.Sensorgrams for all binding interactions were recorded in real time and analyzed after subtracting that from reference channel and internal blanks. Then, sensorgrams were fitted with kinetics or steady-state affinity model. A set of predefined models for kinetic and steady-state affinity are provided with Biacore 8 K Evaluation Software. KD was fit by Biacore 8 K Evaluation Software.LNCaP cells were grown on the glass cell culture dish in phenol red-free RPMI-1640 medium containing 10% charcoal-stripped FBS for 24 h, and then treated with either DMSO, 5 nM DHT, 5 µM ENZa with 5 nM DHT, or 5 µM Z15 with 5 nM DHT, respectively for further 4 hr. Cells were fixed with 4% (vol/vol) paraformaldehyde and permeabilized with 0.2% Triton X-100. Then the cells were incubated with AR antibody . The secondary antibody, goat anti-rabbit with FITC (Santa Cruz) was used at a ratio of 1:100. The counterstain DAPI was used to visualize the cell nucleus. The images were detected under an UltraVIEW vox spinning disc confocal scanning system (Perkin Elmer) on an Olympus Ⅸ81 microscope.7 22Rv1 cells. When the average tumor volume reached 50–100 mm3, the mice were castrated under Nembutal anesthesia and then randomized into three groups (n=3). Oral treatments with Z15 (10 or 20 mg/kg) or vehicle were administered daily and continued for 3 weeks. The tumor volume was monitored every other day, the body weight was monitored every three days, and the tumor volume was calculated according to the formula W 2 ×L/2 (mm3), where W was the short diameter and L was the long diameter. All animal experiments have been approved by the Ethics Committee of Nanjing Lambda Pharmaceutical Co., Ltd (Reference number: IACUC-20210902).BALB/c nude male mice  were subcutaneously injected with 1×102O2 for 10 min, blocked with serum-free protein block, and incubated with the primary antibodies  for 2 hr at 25 °C, followed by incubation with HPR-conjugated secondary antibody for 1 hr at 25 °C. The immunoreaction products were visualized with 3, 3-diaminobenzidine/H2O2 solution.Formalin-fixed paraffin-embedded sections were stained with hematoxylin and eosin. IHC staining was performed with antibodies against Ki67  and PSA . Generally, the rehydrated slides were microwave-heated for 20 min in citrate buffer  for antigen retrieval. Then, the slides were incubated in 1% Hg for 10 min. The supernatants were incubated with 5 µL antibody to AR  overnight at 4 ℃, with 40 µL protein A/G and rocked for 4 hr at 4 ℃. The protein A/G beads were pelleted and washed twice with ice-cold PBS. The precipitates were resolved on an SDS-polyacrylamide gel electrophoresis gel and subjected to Western blot analysis.22Rv1 cells co-transfected with pRBG4-CW7-myc-ubiquitin  were treated with or without 5 µM Z15 in the presence of 5 µM MG132 for 12 hr. Then, the cells were washed with ice-cold PBS and collected in NP-40 buffer. The lysates were lysed for 1 hr in an ice bath, then centrifuged at 12,000 6 cells in T25 culture flask. After 48 hr incubation, 5 µL test compounds were added to each flask at the designated concentrations. After another 8 hr incubation, the cell samples were lysed and the proteins were digested according to the manufacturer’s instructions.LNCaP Cells were seeded at a density of 1.5×10The tryptic peptides were dissolved in solvent A , and directly loaded onto a homemade reversed-phase analytical column . Peptides were separated with a gradient from 6% to 24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24% to 35% in 14 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 450 nL/min on a nanoElute UHPLC system .The peptides were subjected to capillary source followed by the timsTOF Pro  mass spectrometry. The electrospray voltage applied was 1.60 kV. Precursors and fragments were analyzed at the TOF detector, with a MS/MS scan range from 100 to 1700 m/z. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode. Precursors with charge states 0–5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle. The dynamic exclusion was set to 30 s.The resulting MS/MS data were processed using MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the human SwissProt database (20422 entries) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in first search and 5 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification, and acetylation on protein N-terminal and oxidation on Met were specified as variable modifications. FDR was adjusted to <1%. The cut-off values for identification of potential hits were set at Fold Change <0.67 or Fold Change >1.5 and p-value <0.05.Enrichment of pathway analysis: Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected p-value <0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner The AR antagonist common molecular characteristic pharmacophore model and the antagonistic form AR-LBD were constructed as previously described . About 7p-TsOH  in DCM (10 mL) at 0 °C. Heat the reaction mixture at reflux for 4 hr. The reaction was monitored by TLC. After completion of the reaction, it was quenched by water (20 mL) and extracted with ethyl acetate (3×20 mL). The organic layer was dried over Na2SO4 and concentrated to give a residue. The residue was purified by a silica gel column eluted with pet ether and ethyl acetate to give the yellow liquid 2. Yield 63%.Add a solution of 2-Acetylthiophene (10 mmol) in CH2Cl2 (4 mL) dropwise to a solution of N-bromosuccinimide  and Add 2-bromo-1-(2-thienyl)-ethanone (5 mmol) to a solution of thiourea  in EtOH (5 mL) at RT. Heat the reaction mixture at reflux for 2 hr. Reaction mixtures changed from faint yellow to orange during the course of the reaction, with the formation of a precipitate. The mixture was then allowed to cool to RT. After reaching RT, the mixture was filtered with a Buchner funnel. The precipitate was then rinsed with ethyl acetate, in order to remove excess thiourea, which generated a yellow solid. Yield 90%.2SO4 and concentrated to give a residue. The residue was purified by a silica gel column eluted with pet ether and ethyl acetate to give the pellucid liquid 6. Yield 92%.To a solution of 3,5-Dimethyl-4-hydroxymethylisoxazole (10 mmol) in anhydrous DCM (10 mL) was added dropwise phosphorus tribromide  at 0 °C. Heat the reaction mixture at RT for 3 hr. The reaction was monitored by TLC. After completion of the reaction, it was quenched by Saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (3×20 mL). The organic layer was dried over Na2O 9:1 (5 ml). Heat the mixture under reflux for 12 hr. The mixture was then allowed to cool to RT. The mixture was acidified by 20% HCl solution with white precipitate formed. The mixture was filtered with a Buchner funnel and rinsed with 95% EtOH, which generated a white solid 8. Yield 60%.Add KOH  and 4-(bromomethyl)–3,5-dimethyl-1,2-oxazole  to a solution of 4-hydroxybenzoic acid  in EtOH:H3N  in DCE (30 mL) at RT. Heat the reaction mixture at reflux for 24 hr. The reaction was monitored by TLC. After completion of the reaction, it was then allowed to cool to RT and quenched by Saturated brine (20 mL). The mixture was extracted with DCM (3×20 mL). The organic layer was dried over Na2SO4 and concentrated to give a residue. The residue was purified by a silica gel column eluted with pet ether and ethyl acetate to give the white solid Z15. Yield 66%.Add 4-(2-thienyl)–2-thiazolamine (3 mmol), 1.0equiv to a solution of 4-[ methoxy]-benzoic acid , EDCI , HOBT , DMAP  and Et1H NMR  δ 12.71 , 8.19 , 7.59 , 7.56 , 7.54 , 7.20 , 7.17–7.13 , 5.07 , 2.47 , 2.27 . HR MS (ESI), m/z: 412.0789 [M+H]+.Z15. White solid (813.8 mg), t-test, and the differences were considered statistically significant for P<0.05.All statistical analyses were performed using Prism v5.01 . Except where specified, comparisons between the groups were performed using a two-tailed Student’s  The present study reports the discovery and preclinical evaluation of a novel therapeutic agent for the treatment of castration-resistance prostate cancer through inducing degradation of androgen receptor. The major strength of this study is the identification of a novel lead compound and its interesting in vitro and in vivo activities in prostate cancer models. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wafik El-Deiry as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Frank Cackowski (Reviewer #1).Thank you for submitting your article "Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The data has novel and interesting findings with the small molecule for potential further development in castrate-resistant prostate cancer but the reviewers cautioned against over-interpreting or over-selling the proposed mechanism. The manuscript requires extensive proofreading for english language.Reviewer #1 (Recommendations for the authors):Despite having the ability to grow in the presence of low concentrations of androgens, most cases of castration resistant prostate cancer retain activity of signaling through the androgen receptor (AR) and employ one or more of several resistance mechanisms including amplification of the AR gene, point mutations in the AR gene, or splice variants such as ARv7 which lack the AR ligand binding domain and signal in a ligand independent fashion. Therefore, a key therapeutic approach in the field is to develop methods of inhibiting these mutant or amplified forms of AR. A particularly active area of work is the development of selective androgen receptor degraders, which not only inhibit AR, but cause its destruction. Some molecules are in clinical trials, but more are needed. Here the authors discovered a new molecule, named Z15, which they propose binds to the AR ligand binding domain and causes AR destruction by way of the proteasome; therefore acting as a selective androgen receptor degrader (SARD). ;Strengths: The discovery of Z15 and its efficacy are quite promising:1. Z15 is effective against AR expressing cell lines in vitro including cell lines expressing mutant forms of AR. In support of the specificity of the molecule, it has minimal activity against prostate cancer cell lines which do not express AR.2. Z15 is active in a mouse xenograft prostate cancer model and is tolerable to the mice, as it does not cause weight loss.3. IC50 and Kd values are similar to the commonly used anti-androgen, enzalutamide.Weaknesses: The proposed mechanism of action of Z15 is not supported by the data. Therefore, Z15 cannot accurately be called a selective androgen receptor degrader. However, Z15 does appear to act through the androgen receptor in some, as of yet, unknown fashion.1. Despite proposing that Z15 binds to the AR ligand binding domain, it appears to have similar efficacy against wild type AR and the splice isoform ARv7 which lack the AR ligand binding domain. For example, please see figure 2E. Z15 has similar effects on wild type AR (top band) and ARv7 (bottom band). Multiple high profile publications have reported lack of the ligand binding domain in ARv7. For example, see Antonarakis et al. N Engl J Med 2014; 371:1028-1038.2. The data that Z15 affects AR protein stability as opposed to AR protein expression by some other means is not convincing. For example, see the cyclohexamide experiment in figure 5D. There is minimal difference between the cyclohexamide minus conditions  and the cyclohexamide containing conditions .I think Z15 is a useful molecule, but please do not over-sell the data. I do not think that it acts through your proposed mechanism. However, Z15 is specific to AR expressing cell lines, and efficacious in vivo. I suggest only publishing that much of the story and leaving the downstream mechanism to a subsequent publication.The manuscript is in need of English language editing.Reviewer #2 (Recommendations for the authors):The manuscript reports the discovery of a novel selective androgen receptor degrader (SARD) targeting the androgen receptor ligand binding domain (AR-LBD) via virtual screening and bioassays. However, the potential targets and binding sites for the degradation of AR and ARVs have not been clarified because the binding of AR-LBD may not induce the degradation of ARVs. For instance, Ponnusamy et al. (10.1158/0008-5472.CAN-17-0976) reported SARD not only binds to AR LBD but also binds to the amino-terminal transcriptional activation domain (AF-1) of AR. Overall, this paper is not suitable to be published on this journal.1. The authors demonstrate that Z15 is a SARD with glucocorticoid receptor (GR) transcription inhibition activity. The authors should test the transcription inhibition activities of Z15 against progesterone receptor (PR) and mineralocorticoid receptor (MR) due to their high structural similarities.2. Lines 110-111. The authors state 'The results indicated that Z15 could inhibit DHT-induced transcriptional activities of both exogenous and endogenous AR in a dose-dependent manner .' However, it seems Z15 inhibits VCaP not in a dose-dependent manner .3. The authors describe and interpret their data using the word 'significantly' for the Western blot result. Quantitative results for all Western blot results in the manuscript should be presented, such as using the densitometric analysis of the band intensities.4. The authors should quantitatively analyze the AR nuclear localization results in Figure 3.5. Positive control should be added in Figure 6A-F, such as Enzalutamide.6. The loading controls (β-action) are uneven, indicating the samples are not quantified before Western blot analysisReviewer #3 (Recommendations for the authors):The present study reports the discovery and preclinical evaluation of a novel therapeutic agent for the treatment of castration-resistance prostate cancer through inducing degradation of androgen receptor.The major strengths of this study is the identification of a novel lead compound and its interesting in vitro and in vivo activities in prostate cancer models. The major weaknesses with this study are that since the concept of induced AR degradation is not new, additional data are needed to demonstrate some major advantages of the agent reported by the authors over current AR degraders and its potential for clinical translation.The authors have identified a good lead compound but further optimization and studies are needed to make a true impact for the filed of developing AR degraders for the treatment of castration-resistance prostate cancer.This study reported identification of a novel selective androgen receptor degrader molecule (SERD), which the authors named Z15. The authors have provided data to demonstrate the following for Z15:1) inducing degradation of wild-type AR and AR-V7 in a proteasome-dependent manner;2) displaying fairly potent cell growth inhibition of AR+ LNCaP, VCaP and 22RV1 cell lines and some selectivity over AR- PC-3 and DU-145 cell line;3) binding to AR ligand binding domain (LBD) directly with a Kd value of 21 μm and blocking the binding of AR agonist with AR LBD;4) inhibiting 22RV;1 tumor growth.eLife publication.Overall, this is an interesting study but does not rise to the impact of an A number of groups have pursed the development of SARD molecules in the last 15+ years and some SARD molecules have been advanced into clinical development. The novelty of this present study is the discovery of a structurally novel SARD molecule with a modestly potent in vitro activity. There is no direct comparison of Z15 with current best SARD molecules to demonstrate any major advantages.There are some specific deficiencies that need to be addressed.1. Since Z15 induces AR degradation after 12 h treatment time, the authors should rule out that induced AR degradation is specific. It is possible that the levels of other proteins may also be reduced and reduction of AR protein is one of many mechanisms for the cell growth inhibition of Z15;2. While Z15 binds to AR LBD with a Kd value of 21 uM, this level of binding could be an artifact, not necessarily a real binding. Therefore, the binding assay needs to be further optimized;3. Z15 induces degradation of AR in cells and effectively inhibits tumor growth in the 22RV1 xenograft model in mice, AR degradation in tumor tissues needs to be demonstrated.eLife. Your revised article has been evaluated by Wafik El-Deiry (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Reviewer #1 (Recommendations for the authors):I thank the authors for their continued work and find the revised manuscript to be much improved. The work was particularly strengthened by the data showing binding of Z15 to both the AR ligand binding domain and the transactivation domain – which makes their other data make much more sense.I have one significant area where I suggest further improvement. In lines 226-244, the authors describe the LNCaP prostate cancer cell line as castration resistant. I and most investigators generally consider LNCaP cells to be castration sensitive. Conversely, the sub-line of LNCaP, C42B, is generally considered to be castration resistant but retains expression of the androgen receptor.Reviewer #3 (Recommendations for the authors):The authors have performed additional experiments to address concerns raised in the previous review. Despite their great efforts, the following points are still not being addressed.1. While it is clear that Z15 reduces the levels of wild-type AR and ARV7, the proposed biochemical mechanism of action is not convincing, The main issue is that Z15 binds to AR LBD with Kd = 21 μm and to AR AF1 domain with Kd = 15 uM, it has a biological activity at sub-μM ranges, much stronger than its direct binding to AR LBD and AR AF1. Based upon its chemical structure, Z15 should have a high plasma protein binding and would require higher concentrations in cells to achieve its biological activity if the proposed direct binding, followed by induced degradation is true. Therefore, it is very likely that Z15 reduces the levels of wild-type AR and ARV7 through a different mechanism, other than those proposed by the authors.2. While Z15 reduces the levels of wild-type AR and ARV7 proteins in cells and in tumor tissues, it is still unclear if Z15 also reduces the levels of many other proteins in cells and in tumor tissues. The authors have examined three other proteins in the revision and showed that Z15 does not reduce these other three proteins. However, in order to be certain that Z15 specifically reduces the levels of wild-type AR and ARV7 proteins in cells, a more global proteomic analysis would be needed.eLife. Your revised article has been evaluated by Wafik El-Deiry (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Please see the comments of reviewer #3 below.In addition, the manuscript still needs significant English language proofreading due to errors throughout and awkward sentences. Some examples include:Abstract: "restrict the second-generation antiandrogens benefit"Lines 47-51: "Clinical studies have shown that the FDA approved second-generation antiandrogens, including AR antagonists  and androgen-synthesizing enzyme inhibitors (abiraterone) benefits patients with CRPC in overall survival time and prostate-specific antigen (PSA) decline"Lines 51-53: "However, 30~40% of patients with CRPC fail to respond to ENZa or abiraterone, even the remaining patients developed resistance after a few months of the initial response"Lines 53-56: "To date, multiple possible mechanisms for the resistance development have been identified, among which the mutations in the AR LBD, AR amplification, AR splice variants (ARVs) expression, and intra-tumoral de novo synthesis of androgens had been broadly observed in the clinic"Line 72-73: "While, current AR PROTACs cannot induce the degradation of ARVs that lack LBD."Lines 116-118: "In 22Rv1 cells which naturally express full-length AR and ARV7, DHT-induced PSA-luc activity was also dose-dependently blocked by Z15 ."Lines 120-122: "We also detected the selectivity of Z15 in GR-positive PC-3 cells co-transfected with the MMTV-luc expression vector plasmid, which were incubated with dexamethasone (Dex) and different concentrations of Z15 for 24 h."Line 147-148: "We further checked AR and PSA protein levels in LNCaP cells under Z15 and DHT treated by western blot."Line 159-160: "Next, 4D-lable free proteomics were performed to analysis the influence of Z15 on global protein levels in LNCaP cells."Line 169-170: "Western blot analysis indicated that Z15 have no influence on GR, HSP90 and CDK7 protein levels in 22Rv1 cells ."Lines 178-181: "As shown in Figure 3A-B, DHT-binding promoted the importing of AR into the nuclear relative to the DMSO-treated group, while both ENZa and Z15 partially reversed the DHT-induced AR nuclear localization."Line 192-194: "In line with the BLI assay consequence, Z15 (IC50 = 45.32 nM) still exhibited similar AR binding affinity to ENZa (IC50 = 78.95 nM) ."Line 201-202: "AR AF1 was previously considered as an important molecule target region of AR."Line 213-214: "To confirm this hypothesis, we investigated the AR protein and mRNA levels without DHT in LNCaP cells under Z15 treated."Lines 235-239: "As a result, the proliferation of both VCaP and 22Rv1 cells were significantly inhibited by Z15 in a dose-dependent manner, while Z15 (IC50 = 1.37 μM) showed comparable proliferation inhibition potency with positive control ARV-110 (IC50 = 0.86 μM) in VCaP cells, the 22Rv1 cells proliferation inhibition potency of Z15 (IC50 = 3.63 μM) was greater than ARV-110 (IC50 = 14.85 μM)."Lines 251-254: "What's more, Z15 treatment also promoted the apoptosis of AR positive CRPC cell lines  but not AR negative DU145 cells both in dose-dependent and time-dependent manners ."Lines 257-259: "Therefore, we designed and optimized the synthesis route of compound Z15  and prepared the gram level of the compound for an in vivo anti-tumor assay in nude mice."Line 259-260: "Particularly, we investigated the efficacy of Z15 in vivo by 22Rv1 xenografts in male BALB/c nude mice."Line 260-261: "A total of 5 ×106 22Rv1 cells were injected into the left flank of the male mice and allowed to develop tumors volumes of ~100 mm3."Lines 268-270: "Western blot analysis indicated that the tumor AR, ARV7, and PSA protein levels were significantly declined in both the 10 and 20 mg/kg Z15 treated group ."Lines 275-276: "Since Z15 showed inspiring CRPC inhibition potential, we next performed a chemical structure similarity search in SciFinder and the ZINC database to seek Z15 analogs."Lines 279-283: "As a result, most of these compounds pronouncedly inhibited 280 DHT-induced AR transcriptional activity at a concentration of 1 μM except for ZL-1 , suggesting that the dimethyl isoxazole group played an indispensable role in the AR inhibitory activity of Z15 and its analogs."Lines 304-306: "Moreover, Z15 could also degrade AR potentially through the proteasome pathway, which supports Z15 to be dual function AR inhibitor and degrader molecule ."Lines 306-308: "Importantly, several Z15 analogs from the structural similarity search exhibit stronger AR inhibitory and downregulation potency than Z15, which suggest Z15 is a promising lead compound for further optimization."Line 311-312: "which meant AR antagonizing methods were too faint to overcome the ARamplification".Line 334: "but none of these compounds had been gained clinical success".Please provide a detailed Figure legend for Figure 9 that outlines the mechanism. At present, it is just a one-sentence title for the figure.Reviewer #3 (Recommendations for the authors):In the revised manuscript, the authors have performed several experiments to confirm the direct binding of Z15 to AR. These new experiments included measuring the binding of Z15 to proteins from cytosolic lysates of LNCaP cells by competing with radio-labeled DHT and the surface plasmon resonance assay, which provide additional evidence that Z15 binds to AR. In addition, the authors have performed global proteomics experiments to show that AR, as well as several AR-regulated gene products, were down-regulated. Although it is still very likely Z15 has a complex cellular mechanism of action, the proposed mechanism action by the authors (i.e. direct binding to AR and induced degradation of AR and ARV7) is responsible in part for the anti-tumor activity of Z15 in prostate cancer in vitro and in vivo. It is recommended that the authors make the following revisions before the publication of the manuscript.1. Use the binding from the surface plasmon resonance assay as the primary biochemical evidence that Z15 binds to AR, due to the limitations of the BLI assay as the authors indicated in their letter.2. Provide a Table for all those proteins down-regulated by ARV-110 and Z15 and compare those proteins commonly down-regulated by both compounds, as well as additional proteins, which are differentially regulated by ARV-110 and Z15.The proteomic data further showed that while AR is obviously the protein most profoundly reduced by ARV-110, AR is only among those proteins reduced by Z15, suggesting that Z15 is a much less specific degrader of AR, as compared to ARV-110. The authors should add discussions to acknowledge that while down-regulation of AR and ARV7 appears to be a major mechanism for the antitumor activity of Z15, Z15 may have other mechanisms of action which contribute to its anticancer activity in vitro and in vivo.Overall, Z15 is an interesting compound but is not a specific degrader of AR and ARV7. The cellular mechanism of action for Z15 is much more complex than the authors tried to portray. Essential revisions:The data has novel and interesting findings with the small molecule for potential further development in castrate-resistant prostate cancer but the reviewers cautioned against over-interpreting or over-selling the proposed mechanism. The manuscript requires extensive proofreading for english language.Reviewer #1 (Recommendations for the authors):1. Despite proposing that Z15 binds to the AR ligand binding domain, it appears to have similar efficacy against wild type AR and the splice isoform ARv7 which lack the AR ligand binding domain. For example, please see figure 2E. Z15 has similar effects on wild type AR (top band) and ARv7 (bottom band). Multiple high profile publications have reported lack of the ligand binding domain in ARv7. For example, see Antonarakis et al. N Engl J Med 2014; 371:1028-1038.Thank you very much for this recommendation. ARV7 which lacks the ligand-binding domain but remains constitutively active as a transcription factor, the works of Antonarakis et al and other researches indicated the ARV7 expression was associated with the resistance of CRPC to enzalutamide or abiraterone. As Z15 displays both the full length-AR and the ARV7 inhibition activity, while ARV7 does not possess LBD region, we wonder if Z15 could also bind to other region of AR and then induce ARV7 degradation. The AF1 of AR was considered as an important molecule target region of AR previously, and our BLI assay found that AR AF1 inhibitor UT-34 could bind to AR AF1 directly with KD value of 9.3 μM , however, UT-34 could not induce ARV7 degradation in 22Rv1 cells in our western blot analysis . Interestingly, Z15 was also detected binding to AR AF1 with KD value of 15.0 μM . Not surprisingly, we didn’t find any binding affinity between AR AF1 and ENZa even at a high concentration of 200 μM . These data illustrated that Z15 potently inhibits AR-V7 by directly binding to AR AF1. Moreover, that Z15 was also identified binding to the AR LBD. Therefore, Z15 displays the similar efficacy against wild type AR and th AR-V7.2. The data that Z15 affects AR protein stability as opposed to AR protein expression by some other means is not convincing. For example, see the cyclohexamide experiment in figure 5D. There is minimal difference between the cyclohexamide minus conditions  and the cyclohexamide containing conditions .Thank you for this suggestion. We performed the densitometric analysis of the band intensities in Figure 5D, and the AR/β-actin quantitative results showed that Z15 significantly accelerated AR degradation in LNCaP cells treated with CHX  compared to Z15 blank conditions  .I think Z15 is a useful molecule, but please do not over-sell the data. I do not think that it acts through your proposed mechanism. However, Z15 is specific to AR expressing cell lines, and efficacious in vivo. I suggest only publishing that much of the story and leaving the downstream mechanism to a subsequent publication.Thank you for this insightfully suggestion. In the revised manuscript, we tried our best to explain why Z15 induced both AR and ARV7 degradation, and found that the main reason was that Z15 could bind to both LBD and AF1 domain of AR . While we also think that the exhaustive mechanism about Z15 induced AR and ARV7 positive prostate cancer cells inhibition activity is worth to be explored.The manuscript is in need of English language editing.Thank you for the kindly suggestion. The language of the manuscript has been polished by the English writing agency in the revised manuscriptReviewer #2 (Recommendations for the authors):The manuscript reports the discovery of a novel selective androgen receptor degrader (SARD) targeting the androgen receptor ligand binding domain (AR-LBD) via virtual screening and bioassays. However, the potential targets and binding sites for the degradation of AR and ARVs have not been clarified because the binding of AR-LBD may not induce the degradation of ARVs. For instance, Ponnusamy et al. (10.1158/0008-5472.CAN-17-0976) reported SARD not only binds to AR LBD but also binds to the amino-terminal transcriptional activation domain (AF-1) of AR. Overall, this paper is not suitable to be published on this journal.D value of 9.3 μM . By the same method, Z15 was also detected potently binding to AR AF1 with KD value of 15.0 μM . Not surprisingly, we didn’t find any binding affinity between AR AF1 and ENZa even at a high concentration of 200 μM . These data illustrated that Z15 potently inhibits ARV7 by directly binding to AR AF1.Thank you for this insightfully suggestion. We referenced the works of Ponnusamy et al. to investigated the mechanism of Z15 induced ARV7 degradation. As AR AF1 was considered as an important molecule target region of AR previously, and Ponnusamy et al. reported SARD not only binds to AR LBD but also binds to the AR AF1. Our BLI assay found that previous reported AR AF1 inhibitor UT-34 could binding to AR AF1 directly with K1. The authors demonstrate that Z15 is a SARD with glucocorticoid receptor (GR) transcription inhibition activity. The authors should test the transcription inhibition activities of Z15 against progesterone receptor (PR) and mineralocorticoid receptor (MR) due to their high structural similarities.50 of Z15 was 0.41 μM , while both GR and ER transcription inhibition IC50 of Z15 were over 20 μM , the PR transcription inhibition IC50 of Z15 was 9.29 μM , these results suggested that Z15 was a highly selective AR inhibitor.Thank you for this suggestion. As we found Z15 showed powerful AR transcription inhibition activity at the concentration of 10 μM, while its glucocorticoid receptor (GR) transcription inhibition activity was quite feeble . Z15 hardly inhibited dexamethasone (Dex) induced GR transcription activity comparing to the GR antagonist mifepristone (Mif) . We further compared the steroidal receptor transcription inhibition activity of Z15 in AR, GR, estrogen receptor (ER) and progesterone receptor (PR) dual-luciferase reporter assays, the exogenous AR transcription inhibition IC2. Lines 110-111. The authors state 'The results indicated that Z15 could inhibit DHT-induced transcriptional activities of both exogenous and endogenous AR in a dose-dependent manner .' However, it seems Z15 inhibits VCaP not in a dose-dependent manner .Thank you for this suggestion. We changed the description in the revised manuscript as: To further test the AR inhibition potency of Z15, we next performed dual-luciferase reporter assay in several human prostate cancer cell lines including wt-AR-transfected PC-3, LNCaP, and 22Rv1 cells. The results indicated that Z15 could inhibit DHT-induced transcriptional activities of both exogenous and endogenous AR in a dose-dependent manner . In VCaP cells which overexpress AR, we found that 5 μM enzalutamide (ENZa) could not inhibit AR transcription activity, while Z15 showed potently AR transcription inhibition activity even in low concentration .3. The authors describe and interpret their data using the word 'significantly' for the Western blot result. Quantitative results for all Western blot results in the manuscript should be presented, such as using the densitometric analysis of the band intensities.Many thanks for the suggestion. the densitometric analysis of the band intensities for all Western blot results in the revised manuscript were presented in supplementary materials .4. The authors should quantitatively analyze the AR nuclear localization results in Figure 3.Thank you for this suggestion. The quantitatively analysis of the AR nuclear localization results in Figure 3A was presented in Figure 3B.5. Positive control should be added in Figure 6A-F, such as Enzalutamide.50 = 3.63 μM) was greater than ARV-110 (IC50 = 14.85 μM) , Z15 strongly decreased 22Rv1 cells colony numbers compared to both untreated group and ARV-110 treated group , Z15 treatment also showed the stronger apoptosis induction activity of AR positive CRPC cell lines  than ARV-110 .Thank you for this suggestion. As AR LBD targeted degrader ARV-110 showed potently prostate cancer inhibition activity in clinical trials, we chose ARV-110 as positive control to analysis whether Z15 induced AR inhibition and degradation could lead to the CRPC cell growth inhibition. We found that the 22Rv1 cells proliferation inhibition potency of Z15 (IC6. The loading controls (β-action) are uneven, indicating the samples are not quantified before Western blot analysisThank you for this comment. We checked all the loading controls (β-action) presenting in our manuscript, we found some loading controls (β-action) truly are uneven, such as Figure 5D, Figure 8C, as we performed densitometric analysis of the band intensities for all Western blot results in the revised manuscript , we think this problem could not influence our judgment to the Western blot results.Reviewer #3 (Recommendations for the authors):The present study reports the discovery and preclinical evaluation of a novel therapeutic agent for the treatment of castration-resistance prostate cancer through inducing degradation of androgen receptor.The major strengths of this study is the identification of a novel lead compound and its interesting in vitro and in vivo activities in prostate cancer models. The major weaknesses with this study are that since the concept of induced AR degradation is not new, additional data are needed to demonstrate some major advantages of the agent reported by the authors over current AR degraders and its potential for clinical translation.Thank you for this insightfully suggestion. Based on our data presented in the revised manuscript, there are some advantages of Z15 compared to current reported AR degraders. Compared to AR PROTAC ARV-110, Z15 could not only bind to AR LBD but also AR AF1 , thus that ARV7 positive enzalutamide resistant 22Rv1 cells proliferation inhibition potency of Z15 (IC50 = 3.63 μM) was greater than ARV-110 (IC50 = 14.85 μM) , Z15 strongly decreased 22Rv1 cells colony numbers compared to both untreated group and ARV-110 treated group , Z15 treatment also showed the stronger apoptosis induction activity of AR positive CRPC cell lines  than ARV-110 . Compared to AR AF1 inhibitor UT-34 which was reported possess ARV7 degradation activity, Z15 has similar mechanism, as the AR DC50 of Z15 in LNCaP cells was 1.05 μM , in 22Rv1 cells, the AR DC50 was 1.16 μM, the ARV7 DC50 was 2.24 μM , however, UT-34 could not induce ARV7 degradation in 22Rv1 cells in our western blot analysis , which indicated that Z15 may has stronger ARV7 degrade activity than UT-34. Based on PCa patient tumor tissues, we further cultured PCa organoids and treated organoids with 1 μM Z15 for 7 days. The results showed that Z15 significantly inhibited PCa organoid proliferation compared to the control group , which showed the potential of Z15 for clinical translation.The authors have identified a good lead compound but further optimization and studies are needed to make a true impact for the filed of developing AR degraders for the treatment of castration-resistance prostate cancer.Thank you for this suggestion. Seven compounds  with more than 80% similarity with Z15 were finally purchased for the further bioactivity evaluation. ZL-2 and ZL-4 exhibited the most AR transcription inhibitory potency, while others also showed comparable AR inhibition activity compared to Z15 . In addition, Western blot analysis revealed that these active molecules could reduce AR and PSA protein levels in a dose-dependent manner. Notably, most of them showed stronger AR down-regulation activity than Z15 . Together, these active Z15 analogs indicate that through the chemical structural modification of Z15, we may find out more novel selective androgen receptor degraders with better AR inhibition activity in a shorten future. Moreover, we have designed the synthesis route of Z15, and prepared more than one gram of product for the in-vivo assay. It would be conveniently to expand the compound space to optimize Z15 via the design route.This study reported identification of a novel selective androgen receptor degrader molecule (SERD), which the authors named Z15. The authors have provided data to demonstrate the following for Z15:1) inducing degradation of wild-type AR and AR-V7 in a proteasome-dependent manner;2) displaying fairly potent cell growth inhibition of AR+ LNCaP, VCaP and 22RV1 cell lines and some selectivity over AR- PC-3 and DU-145 cell line;3) binding to AR ligand binding domain (LBD) directly with a Kd value of 21 μm and blocking the binding of AR agonist with AR LBD;4) inhibiting 22RV;1 tumor growth.Overall, this is an interesting study but does not rise to the impact of an eLife publication.A number of groups have pursed the development of SARD molecules in the last 15+ years and some SARD molecules have been advanced into clinical development. The novelty of this present study is the discovery of a structurally novel SARD molecule with a modestly potent in vitro activity. There is no direct comparison of Z15 with current best SARD molecules to demonstrate any major advantages.50 = 3.63 μM) was greater than ARV-110 (IC50 = 14.85 μM) , Z15 strongly decreased 22Rv1 cells colony numbers compared to both untreated group and ARV-110 treated group , Z15 treatment also showed the stronger apoptosis induction activity of AR positive CRPC cell lines  than ARV-110 . Compared to AR AF1 inhibitor UT-34 which was reported possess ARV7 degradation activity, Z15 has similar mechanism, as the AR DC50 of Z15 in LNCaP cells was 1.05 μM , in 22Rv1 cells, the AR DC50 was 1.16 μM, the ARV7 DC50 was 2.24 μM , however, UT-34 could not induce ARV7 degradation in 22Rv1 cells in our western blot analysis , which indicated that Z15 may has stronger ARV7 degrade activity than UT-34. Based on PCa patient tumor tissues, we further cultured PCa organoids and treated organoids with 1 μM Z15 for 7 days. The results showed that Z15 significantly inhibited PCa organoid proliferation compared to the control group , which showed the potential of Z15 for clinical translation.Thank you for this suggestion. The most interesting properties for Z15 are that Z15 could inhibit both FL-AR and AR-V7, as well as its degradation potency for both FL-AR and AR-V7. Based on our data presented in the revised manuscript, there are some advantages of Z15 compared to current reported AR degraders. Compared to AR PROTAC ARV-110, Z15 could not only bind to AR LBD but also AR AF1 , thus that ARV7 positive enzalutamide resistant 22Rv1 cells proliferation inhibition potency of Z15  and cyclin-dependent kinases 7 (CDK7) as controls. Western blot analysis indicated that Z15 have no influence on GR, HSP90 and CDK7 protein levels in 22Rv1 cells 2. While Z15 binds to AR LBD with a Kd value of 21 uM, this level of binding could be an artifact, not necessarily a real binding. Therefore, the binding assay needs to be further optimized;D) is about 10-4 – 10-7 M, thus, we think that the ForteBio Octet detected binding affinity between Z15 and AR LBD with a KD Value of 21 μM is generally convincing. Moreover, the competitive AR LBD binding assay was performed to demonstrate the direct interaction between Z15 and AR LBD. Synthetic androgen R1881 displayed strong binding potency to AR with IC50 value of 0.30 nM, which indicated this assay system's feasibility. In line with the BLI assay consequence, Z15 (IC50 = 45.32 nM) still exhibited similar AR binding affinity to ENZa (IC50 = 78.95 nM) . Besides, our fluorescence polarization (FP) assay also demonstrated Z15 could compete with androgen binding to AR LBD . These data suggested that Z15 could antagonize AR by targeting the ligand binding domain directly.Thank you for this comment. According to the manual of ForteBio Octet RED96 instrument , the ForteBio Octet detected protein-small molecule (MW < 2 kDa) general affinity .The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Reviewer #1 (Recommendations for the authors):I thank the authors for their continued work and find the revised manuscript to be much improved. The work was particularly strengthened by the data showing binding of Z15 to both the AR ligand binding domain and the transactivation domain – which makes their other data make much more sense.I have one significant area where I suggest further improvement. In lines 226-244, the authors describe the LNCaP prostate cancer cell line as castration resistant. I and most investigators generally consider LNCaP cells to be castration sensitive. Conversely, the sub-line of LNCaP, C42B, is generally considered to be castration resistant but retains expression of the androgen receptor.50 = 1.37 μM) showed comparable proliferation inhibition potency with positive control ARV-110 (IC50 = 0.86 μM) in VCaP cells . What’s more, Z15 treatment also promoted the apoptosis of VCaP cells .Thank you very much for this recommendation. LNCaP should exactly be a castration sensitive cell line. As we have some difficulties in purchasing or asking C4-2B cells from ATCC or other labs, we chose VCaP cells for further investigation which are also generally considered to be castration resistant but retains expression of the androgen receptor. As a result of MTT assay, the proliferation of VCaP cells was significantly inhibited by Z15 in a dose-dependent manner, Z15 (ICReviewer #3 (Recommendations for the authors):The authors have performed additional experiments to address concerns raised in the previous review. Despite their great efforts, the following points are still not being addressed.1. While it is clear that Z15 reduces the levels of wild-type AR and ARV7, the proposed biochemical mechanism of action is not convincing, The main issue is that Z15 binds to AR LBD with Kd = 21 μm and to AR AF1 domain with Kd = 15 uM, it has a biological activity at sub-μM ranges, much stronger than its direct binding to AR LBD and AR AF1. Based upon its chemical structure, Z15 should have a high plasma protein binding and would require higher concentrations in cells to achieve its biological activity if the proposed direct binding, followed by induced degradation is true. Therefore, it is very likely that Z15 reduces the levels of wild-type AR and ARV7 through a different mechanism, other than those proposed by the authors.50 = 1.37 μM) showed comparable proliferation inhibition potency with positive control ARV-110 (IC50 = 0.86 μM) in VCaP cells . What’s more, Z15 treatment also promoted the apoptosis of VCaP cells .Thank you very much for this recommendation. LNCaP should exactly be a castration sensitive cell line. As we have some difficulties in purchasing or asking C4-2B cells from ATCC or other labs, we chose VCaP cells for further investigation which are also generally considered to be castration resistant but retains expression of the androgen receptor. As a result of MTT assay, the proliferation of VCaP cells was significantly inhibited by Z15 in a dose-dependent manner, Z15  exhibited similar AR binding affinity to ENZa (IC50 = 78.95 nM) . Besides, our fluorescence polarization assay also demonstrated Z15 could compete with androgen binding to AR LBD .1) The competitive AR binding assay was performed to demonstrate the direct interaction between Z15 and AR LBD, whereby compounds in competition with the radioligand The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Please see the comments of reviewer #3 below.Reviewer #3 (Recommendations for the authors):In the revised manuscript, the authors have performed several experiments to confirm the direct binding of Z15 to AR. These new experiments included measuring the binding of Z15 to proteins from cytosolic lysates of LNCaP cells by competing with radio-labeled DHT and the surface plasmon resonance assay, which provide additional evidence that Z15 binds to AR. In addition, the authors have performed global proteomics experiments to show that AR, as well as several AR-regulated gene products, were down-regulated. Although it is still very likely Z15 has a complex cellular mechanism of action, the proposed mechanism action by the authors (i.e. direct binding to AR and induced degradation of AR and ARV7) is responsible in part for the anti-tumor activity of Z15 in prostate cancer in vitro and in vivo. It is recommended that the authors make the following revisions before the publication of the manuscript.1. Use the binding from the surface plasmon resonance assay as the primary biochemical evidence that Z15 binds to AR, due to the limitations of the BLI assay as the authors indicated in their letter.Thank you for this kind comment. We have redrawn Figure 4 and Figure 4—figure supplement 2, the AR competitive binding assay result was used as primary biochemical evidence that Z15 binds to AR LBD , the surface plasmon resonance assay result was used as primary biochemical evidence that Z15 binds to AR AF1 . The BLI assay results were mainly shown in supplementary materials .2. Provide a Table for all those proteins down-regulated by ARV-110 and Z15 and compare those proteins commonly down-regulated by both compounds, as well as additional proteins, which are differentially regulated by ARV-110 and Z15.Thank you for this helpful suggestion. Supplementary file 1d and Supplementary file 1e were re-arrangement to list all those proteins down-regulated by ARV-110 and Z15. The proteomic data reveals that there are 19 proteins commonly down-regulated by both ARV-110 and Z15, these proteins account for 55.9% (19 among 34) in ARV-110 down-regulated proteins (Supplementary file 1d) and 27.5% (19 among 69) in Z15 down-regulated proteins (Supplementary file 1e). We think these two re-arrangement tables showed ARV-110 and Z15 down-regulation proteins situation more clearly to the readers.3. The proteomic data further showed that while AR is obviously the protein most profoundly reduced by ARV-110, AR is only among those proteins reduced by Z15, suggesting that Z15 is a much less specific degrader of AR, as compared to ARV-110. The authors should add discussions to acknowledge that while down-regulation of AR and ARV7 appears to be a major mechanism for the antitumor activity of Z15, Z15 may have other mechanisms of action which contribute to its anticancer activity in vitro and in vivo.Thank you for your insightful suggestion, this suggestion combined with the previous recommendation about the mechanisms of action of Z15 gives us many inspirations, and we are very grateful for this. In the revised manuscript, we discussed that Z15 may have other mechanisms of action which contribute to its anticancer activity in vitro and in vivo. It is described as follows:“However, we notice that the AR degradation potency of Z15 is not as stronger as AR-targeted PROTAC molecule ARV-110, suggesting that Z15 is a less specific AR degrader compared to ARV-110. We acknowledge that while Z15 suppresses CRPC progress mainly through targeting AR and ARV7, Z15 may have other mechanisms of action which contribute to its anticancer activity in vitro and in vivo. Nevertheless, Z15 is a novel selective AR and ARV7 degrader that warrants further structure optimization and anti-CRPC mechanisms investigation.”


Gait parameters can measure risks of falling and mortality and identify early stages of frailty. The use of walking aid changes gait parameters. The aim of this study was to describe differences in gait parameters among healthy adults when walking on different surfaces and under different conditions, with and without a rollator.Ten healthy participants walked first without and then with a rollator upslope, downslope and on flat surface, on bitumen and gravel respectively. Step length, walking speed and sideway deviation was measured using an inertial measurement unit. Walking up a slope using a rollator generated the longest step length and walking down a slope using a rollator the shortest. Fastest walking speed was used when walking up a slope with rollator and slowest when walking down a slope with rollator. Sideway deviation was highest when walking down a slope and lowest when walking on gravel, both without rollator. Highest walk ratio was found when walk up a slope without rollator and lowest when walking down a slope with rollator. Data from this study provides valuable knowledge regarding gait parameters among healthy individuals, useful for future clinical research relevant for rehabilitation and public health. Mobility is a prerequisite for the vast majority of everyday activities, and being able to move around outdoors contributes to societal participation , 2, and Among persons with reduced ability to walk, walking aids are often used to enable activity and participation . Even ifThus, the aim of this study was to describe differences in gait parameters among healthy adults when walking on different surfaces and under different conditions, with and without a rollator.The study was an experimental, cross-sectional pilot study. Participants was recruited among students and staff at Lund University, using the following inclusion criteria: 18 years or older; consider themselves healthy; and without known health conditions that could impact their mobility. No other inclusion or exclusion criteria were used. 10 participants were included; seven women and three men, aged 23–62 years (Median 35 years).www.infonomy.com). The IMU has shown good validity and reliability for measuring postural sway [Gait was measured with a nine-axis Inertial Measurement Unit (IMU), worn on the right thigh. The IMU includes an accelerometer, magnetometer, and gyroscope that can detect and measure movement patterns when changing body positions in the frontal, sagittal and mediolateral planes and analyze gait. The IMU is comprised by an STM32 microprocessor coupled to an LSM6DSM accelerometer/gyroscope combination, sampling apparent acceleration and rotational velocity at 50 Hz. The IMU also comprises a GPS and a clock. IMU:s is a practical and portable way of collecting this type of data  and can ral sway , for detral sway  and goodral sway . The harral sway , 18, altGait was measured using four parameters: step length, walking speed, sideway deviation and walk ratio. The direction of measured progression was forward. Step length is defined as step in cm, which is a half gait cycle (right heel strike to left heel strike). Step length is estimated from the angle of the thigh in each completed stride-cycle. In this approximation, a 10 degrees angle in the knee when the heel strikes the floor is assumed . WalkingThe gait parameters were measured during walk under two different conditions: with and without the use of a four wheeled rollator. In addition, gait was measured for each condition on different surface characteristics: up-slope, down-slope, bitumen, and gravel paths. All up-slope and down-slope sections were paved with bitumen, while all gravel paths were flat.The outdoor walk consisted of a lap that partly went in parallel to a bicycle route, and several streets were crossed, including tramrails and traffic lights. Two observants noted the exact time when the environmental condition changed, for example the beginning and end of a gravel part of the lap. Each lap was walked twice, first without rollator, and secondly with rollator. Before starting the lap with rollator, the participants walked for a few minutes to get accustomed to it. They were also informed about the properties of the rollator, and it was adjusted to each participants height. The lap was in total 2.5 km long, with a gravel part of approx. 600 m in the first half the lap. The slope was 100 m long, with an inclination of 1:40. Each lap took 30–45 min to finish. The participants had a convenience break between the two laps, approx. 5–10 min.Data were collected during February–March 2021. Thus, the weather conditions varied from dry to wet conditions, with a temperature between 0–15 degrees Celsius.IBM SPSS version 25 was used for statistical analyses. The data generated by the IMU were recorded each second and analyzed according to the different environmental characteristics in the outdoor walk, with and without rollator. Since not all variables were normally distributed and sample size was small, both mean (M) and standard deviation (SD) as well as median (Md) and min–max values were used. Since repeated measures was applied, an analysis of variance was performed, using repeated measures two-way ANOVA. A p ≤ 0.05 was used for determining statistical significance .Calculating with minimal clinical statistical difference of 0.1 m/sec in walking speed , with a Both the longest and shortest steps were taken while walking with a rollator, with the longest step length used when walking upslope  and shortest step length was used when walking downslope . The highest and lowest walking speed was also identified when the rollator was used. The highest speed was measured when walking upslope with rollator  and the lowest speed when walking downslope with rollator . Highest sideway deviation was found when walking downslope, without rollator  and lowest sideway deviation was found when walking on gravel without rollator . Highest walk ratio was found when walking up a slope without a rollator M = 4.60, Md = 4.59) and lowest when walking down a slope with a rollator . Mean and median values and SD and min–max values for the different variables when walking in the different terrains, with and without rollator are displayed in Table For step length, the analysis of variance during the different conditions showed that walking downslope with a rollator differed significantly from five of the other conditions (p ≤ 0.00–0.01) and walking without a rollator on gravel did not differ from any of the other conditions Table .Table 2MFor walking speed, the analysis of variance showed that the walking conditions did not differ, except between walking with a rollator on gravel and with a rollator on bitumen (p = 0.03) Table .For sideway deviation, the analysis showed that walking downslope without a rollator differed significantly from four of the other conditions (p ≤ 0.00–0.04) and walking upslope without a rollator and on bitumen with a rollator did not differ from any of the other conditions Table .For walk ratio, the analysis showed that walking down a slope with a rollator differed significantly from all of the other conditions (p = 0.00–0.03) Table .This pilot study showed that step length, walking speed, sideway deviation and walk ratio can differ when walking in different terrains and with and without walking. Walking up a slop using a rollator demanded longest step length and walking down a slope using a rollator the shortest. Fastest walking speed was used when walking up a slope using a rollator and slowest when walking down a slope using a rollator while sideway deviation was highest when walking down a slope and lowest when walking on gravel, both without rollator. Walk ratio was highest when walking up a slope without a rollator and lowest when walking down a slope with a rollator. Walking down a slope with rollator differed the most between the other conditions in step length and in walk ratio, walking downslope without rollator differed the most from the other conditions in sideway deviation. There were few differences between the conditions in walking speed.Other research has shown an increase in step length among older persons when walking with a four-wheeled rollator on plain ground . We therWalking speed has a relationship to mortality and can be used as a marker of physical performance and therefore be clinically useful , 29. OurStep length, speed and sideway deviation during walking can differ depending on rollator use, walking surface and environmental characteristics, at least among healthy individuals. Walking downslope with a rollator generated most differences in step length compared to the other conditions and sideway deviation was largest when walking downslope without a rollator. Walking speed however showed very few differences between the conditions. Also, walking upslope with a rollator generated the longest steps (52.44 cm) and walking downslope with a rollator the shortest (43.77 cm). That means that pushing the rollator upslope demands more energy and therefore both step length and walk ratio possibly is increased, and vice versa going down the slope: to slow down speed downslope also demands more energy and step length as well as walk ratio therefore has to be reduced. This implies that walking up and down slopes can be challenging for balance and therefore important to include in rehabilitation programs directed towards improving balance. The type of gait parameters used in this study can be easily measured using wearable devices, such as IMU:s.Our findings provide valuable knowledge regarding gait parameters among healthy individuals, useful for future clinical research relevant for rehabilitation planning and evaluation. The findings are also valuable for the design of pedestrian routes in urban areas to support physical activity and participation in the population.The study has several limitations. To be able to compare walking in the different conditions people who considered themselves healthy were enrolled in this study. This approach has been used before, since it is difficult, or even unethical, to ask persons who are dependent on a walking aid, to walk without it . Gait veWalk ratio was included as a post-hoc analysis and is therefore not normalized for height, since height was not measured on the participants. This must be taken into account when interpreting our results on walk ratio.Even if sufficient for the study design applied, the sample size was small , 32, whiIn future research, the use of several IMU’s can extend the type of data that can be collected in this type of experimental studies and thereby expand knowledge on which type of movements that are necessary for a person to manage to be able to walk outdoors. In this aspect, research regarding gait parameters among older people and people with disabilities is also important.