Francisella tularensis (F. tularensis) is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the United States and treatment of tularemia is confined to few antibiotics. In this study, we demonstrate that AR-13, a derivative of the cyclooxygenase-2 inhibitor celecoxib, exhibits direct in vitro bactericidal killing activity against Francisella including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as toward the intracellular proliferation of LVS in macrophages, without causing significant host cell toxicity. Identification of an AR-13-resistant isolate indicates that this compound has an intracellular target(s) and that eﬄux pumps can mediate AR-13 resistance. In the mouse model of tularemia, AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4. Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death. Taken together, these data support the translational potential of AR-13 as a lead compound for the further development of new anti-Francisella agents. Francisella tularensis subspecies tularensis (F. tularensis) is a remarkably infectious facultative intracellular bacterium and the etiologic agent of tularemia, a zoonotic disease. Infection can be acquired via various routes including insect bites, aerosols, or contact with mucous membranes or abrasions (F. tularensis can be divided into three major subspecies including tularensis (highly virulent in humans), holarctica, and mediasiatica (Francisella novicida is a separate but closely related species (tularensis (Type A) can result in a fatal infection (holarctica (Type B) is somewhat less virulent and other Francisella subspecies/species are considered non-pathogenic to humans  has been used to protect against tularemia in government personnel and in other countries, there is no currently approved United States vaccine. Fluoroquinolones , aminoglycosides, and tetracyclines comprise the antibiotics most frequently used to treat tularemia  as well as improved delivery of antibiotics in vivo   . In addielivery) , innate elivery) .Salmonella enterica serovar Typhimurium (S. Typhimurium) and F. tularensis  ], has direct antimicrobial activities against Francisella species with distinct modes of action compared with AR-16. The difference between AR-13 and AR-16 lies in the substituents around the 4-[3-(trifluoromethyl)-1H-pyrazol-1-yl] aniline core. While AR-13 consists of a phenanthrene ring system at the C5 position of the heterocycle, AR-16 has a 4-bromobiphenyl group at that position. Furthermore, the aniline has been converted to a sulfamide in the case of AR-13 . While AR-13 displays bactericidal effects, AR-16 exhibits bacteriostatic activities against subsp. holarctica (strain LVS) and subsp. tularensis (strain SchuS4). Examination of AR-13 resistance mechanisms in the LVS strain illustrated that decreased susceptibility to AR-13 in vitro could be mediated by eﬄux pumps, as an eﬄux pump inhibitor sensitized the AR-13 resistant mutant to AR-13. Cytotoxicity studies revealed that AR-13 displays minimal toxicity to the human monocyte-derived macrophages (hMDMs). Finally, in vivo examination of AR-13 in a mouse model of tularemia showed that AR-13 treatment partially protects the LVS-infected mice from death. Combination of AR-13 with a sub-optimal dose of gentamicin provided increased protection against F. tularensis SchuS4 infection.Previous studies showed that AR-12, a small molecule derived from the cyclooxygenase-2  inhibitor celecoxib, but lacking the COX-2 inhibitory activities, displayed broad-spectrum host-directed antimicrobial activity against fungi , Salmonelarensis , as bacts aureus , multidrs aureus , and Frad AR-16) . In thisFigure 1. Gentamicin, kanamycin, ethidium bromine, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma . F. tularensis SchuS4 strain (Type A), and F. tularensis LVS used in this study were described previously (F. novicida mutant strains were obtained from BEI Resources transposon library1 (F. tularensis SchuS4 and LVS) or for 24 h (F. novicida) at 37°C prior to use in all experiments. Experiments involving the LVS and F. novicida strains were performed in a BLS2 environment. Experiments with F. tularensis SchuS4 were performed in The Ohio State University BSL3 select agent facility in accordance with CDC and locally approved BSL3 facility and safety standards.A library of celecoxib and its derivatives was kindly provided by Arno Therapeutics, Inc. . Chemical structures of celecoxib, AR-13 and AR-16 are presented in eviously . When nelibrary1 . The baclibrary1  for 48 hFrancisella to AR-13, AR-16 and other antimicrobials were determined by minimum inhibitory concentrations (MICs) and bacterial killing assays. MIC assays were performed using the microtiter broth dilution method with an initial inoculum of approximately 106 bacteria/ml as described previously  at 37°C. Viable bacteria at different time-points were evaluated by serial dilution and plating on mTSB agar plates or CHA plates.The susceptibilities of eviously , 2012. Mr) mutants in vitro by stepwise selection in broth culture. Briefly, 50 μl from an overnight culture of LVS was exposed to increasing concentrations of AR-13 in 5 ml mTSB broth with 1.25 μg AR-13/ml as a starting concentration at 37°C while shaking. 50 μl of this LVS grown culture was then passed into twofold increasing concentrations of AR-13 in 5 ml mTSB. The process was repeated until LVS was able to stably grow in 20 μg AR-13/ml (approximately 20 passes). Approximately 70 AR-13r clones were selected to determine the MIC to AR-13. Two representative AR-13r mutants were then passed in non-selective AR-13 free mTSB for 10 passes of an overnight culture. The stable AR-13r mutants were chosen for the subsequent studies including genomic sequencing, RNA sequencing, and assays regarding the mechanism of AR-13 resistance.To examine the mechanism of AR-13 action, AR-13 was used to select for spontaneous AR-13-resistant . Samples were sequenced on the Illumina MiSeq instrument using paired-end 2 × 300 bp chemistry, generating more than 3 million read pairs per sample. Churchill2  was run TM rRNA removal kit for Gram-Negative bacteria . To generate directional signal in RNA seq data, libraries were constructed from first strand cDNA using ScriptSeqTM v2 RNA-Seq library preparation kit . rRNA-depleted RNA (50 ng) was fragmented and reverse transcribed using random primers containing a 5′ tagging sequence, followed by 3′ end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. After magnetic-bead based purification, the di-tagged cDNA was amplified by limit-cycle PCR using primer pairs that anneal to tagging sequences have adaptor sequences required for sequencing cluster generation. The AMPure XP System (Beckman Coulter) was used to purify RNA-seq libraries. The Agilent 2200 TapeStation using High Sensitivity D1000 ScreenTape was used to determine the quality of libraries, which were then quantified using the Kappa SYBR®Fast qPCR kit . On average, 21 million paired-end 150 bp RNA-Seq reads were generated using the Illumina HiSeq 4000 platform for each sample (the range was 19 to 24 million). Each sample was aligned to the GCF_000009245.1 assembly of the F. tularensis LVS reference from NCBI7 using version 0.7.5a of BWAMEM8. The GFF file provided with the GCF_000009245.1 assembly from NCBI was used to identify transcript features and raw coverage counts were calculated using HTSeq9. After normalization of the RNA-Seq gene expression data a post-alignment statistical analyses was performed using custom analysis scripts written in R and DESeq210. Normalized read counts were used for comparisons of gene expression and associated statistical analysis of the different bacteria/conditions. Fold change values were displayed as test condition/control condition, where values less than one were expressed as the negative of its inverse . A false discovery rate of 10%  was used in determining transcripts that were significantly differentially expressed.Similarly, the RNA from the wild-type LVS and stable isogenic AR-13 resistant mutants was purified from mid-log cultures in mTSB using the RNAeasy plus mini kit . Using methods described previously , after d2 for 5 days. Teflon wells were chilled on ice to recover the PBMCs, which were then re-suspended in RPMI 1640 with 10% autologous serum. Cells were then allowed to attach in 24-well or 6-well tissue culture plates for 2–3 h at 37°C in a humidified incubator with 5% CO2. After washing to remove the lymphocytes, hMDM monolayers were seeded at a density of approximately 2.0 × 105 cells/well for 24-well plates for infection studies.Human monocyte-derived macrophages (hMDMs) were derived from human blood acquired via venipuncture from healthy donors following a Ohio State University Institutional Review Board approved protocol. Written informed consent was provided by study participants. The protocol followed published methods . Briefly5 cells/well with RPMI 1640 supplemented with 20% autologous serum. This medium was replaced with 2% autologous serum in RPMI 1640 containing different concentrations of AR-13 or AR-16 (in 0.1% DMSO) or DMSO vehicle control. Triton X-100 (0.1%) was used as a positive control. After 24 h or 48 h of treatment, supernatants were collected, centrifuged to remove cells, and subjected to the LDH assay (as per manufacturer’s instructions). LDH release was measured (570 nm) and the cytotoxicity was calculated as a percentage of the Triton X-100 treated cells (positive control).Human monocyte-derived macrophages viability was assessed in the presence of AR-13 and AR-16 using a lactate dehydrogenase (LDH) assay  described previously . hMDM ce600 of 0.4 which is approximately 3 × 109 CFU/ml). LVS was added to hMDMs at a multiplicity of infection (MOI) of 50 in the presence of 2% autologous serum in RPMI 1640 plus L-glutamine (Gibco-Life Technologies). Extracellular bacteria were removed 2 h post-infection by addition of 50 μg/ml of gentamicin (Gibco-Life Technologies) for 1 h followed by three washes of the monolayer with pre-warmed RPMI 1640 to remove additional extracellular bacteria. Various concentrations of AR-13 were then added to culture medium (with 2% autologous serum and 10 μg/ml gentamicin). As a control, the parental compound of AR-13, AR-12, which was previously shown to inhibit Francisella growth in macrophages via induction of autophagy, was included. Soluble AR-12 and AR-13 were dissolved in 10 mg/mL in DMSO and diluted in RPMI 1640 containing 2% autologous serum to the appropriate concentrations. At 22 h post-treatment, the infected hMDMs were lysed with 0.1% Triton X-100  in PBS for 15 min. The cell lysates were then serially diluted with PBS, plated onto CHA agar and enumerated after 72 h incubation at 37°C.Live vaccine strain growth in hMDMs was performed as described previously  with a mad libitum to the mice (5 mice/group unless otherwise indicated) in sterile micro isolator cages and allowed to acclimate for 2–3 days prior to challenge. All experimental procedures were carried out in strict accordance with guidelines established by The Ohio State University Institutional Animal Care and Use Committee (IACUC), and all efforts were made to minimize animal suffering.Six–eight weeks old pathogen free female BALB/c mice were purchased from Harlan Sprague . Food and water were provided F. tularensis SchuS4 and LVS was performed as previously described .Intranasal (I.N.) infection of escribed . BrieflyF. tularensis SchuS4 and LVS strains. Mice were infected with F. tularensis SchuS4 or LVS and treated, starting about 1 h post-infection, with different doses  of AR-13 in 200 μl of polyethylene glycol (PEG) PEG400:0.9% saline:ethanol (50:35:15) given by the intraperitoneal (I.P.) route  route  once daiP-values were calculated using one-way ANOVA for multiple comparisons and adjusted with Bonferroni’s correction; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant. A Chi-square test was used for survival analysis. GraphPad Prism 6 was used for statistical analysis.Data are presented as mean ± standard deviation (SD). 11 under the record number GSE100069. Genomic sequences and alignment data have been deposited to the Sequence Read Archive12 under identifier SRP109660.RNAseq data were deposited into the GEO RepositoryFrancisella infection, we screened 64 compounds derived from celecoxib  and F. tularensis SchuS4 (data not shown), and 5 μg/ml for F. novicida (data not shown). To explore the modes of action of these two anti-Francisella agents, we performed bacterial killing assays the presence of 10 μg/ml for each compound in PBS  or mTSB , as well as two Gram-negative bacteria, Pseudomonas aeruginosa strain PAO1 and Salmonella Typhi. The results showed that AR-13 exerted strong antimicrobial activities on the two Gram-positive bacteria, but not on the two Gram-negative bacteria  release in the culture supernatants from hMDMs treated with various concentrations of AR-13 at 24 and 48 h post-treatment  significantly inhibited the growth of LVS in hMDMs, reducing the CFU recovered by ∼0.5 logs. As a control, AR-12, known to inhibit intracellular Francisella growth by the induction of autophagy , 8x MIC (20 μg/ml), and 16x MIC (40 μg/ml) was unsuccessful. Stepwise selection in mTSB broth with increasing concentrations of AR-13 was performed to attempt to select for AR-13 resistant mutants of LVS. After approximately 20 overnight passages in increasing AR-13 selective pressure, AR-13 resistant mutants were obtained and sensitivity to AR-13 was examined by culturing in mTSB with serial concentrations of AR-13 (0.016–10 μg/ml). As shown in Figure 4A, a representative AR-13 resistant mutant was able to grow in the presence of up to 10 μg/ml AR-13 but was inhibited in 20 μg/ml (eightfold increase in MIC) (data not shown). Examination of the stability of an AR-13 resistant mutant was performed by passaging the mutant in AR-13-free mTSB for 10 overnight passages, with several clones then chosen for bacterial killing assays. As shown in Figure 4B, after 10 overnight passages in AR-13-free mTSB, there was no difference in viable bacteria recovered between the initial AR-13 resistant mutant (MT) and that of 10 overnight passages in AR-13-free mTSB (MT-10 passages). These data suggest that Francisella is able to develop resistance to AR-13 and the resistance is relatively stable.Examination of the resistance of microbes to new antimicrobials provides insight into the mechanism(s) of action of the drug as well as drug targets. We sought to examine whether Francisella indicated an alteration in the inherent genomic information of the resistant mutant. Comparative genomic analysis was performed between the AR-13 resistant mutant and its parent wild type strain. This analysis identified three non-synonymous mutations in the AR-13 resistant mutant. Interestingly, two of them were found in genes encoding for outer membrane eﬄux proteins FTL_1107 (FtlC) and FTL_1865 (TolC) (Table 1). TolC is the outer membrane channel component for multidrug eﬄux and type I secretion systems. The other mutation was in gene locus FTL_0600 (wbtH homolog in F. novicida) (Francisella (m-chlorophenyl hydrazone (CCCP). Various concentrations of CCCP were tested against LVS to which dramatic reductions in growth were observed at >∼8 nM . In addition, the AR-13 resistant isolate also conferred decreased sensitivity to ethidium bromide (EtBr) which is mediated by TolC as previously observed , but does not affect sensitivity to kanamycin . In parallel, we examined independent mutants of two TolC orthologs,  and FTL_1865  as well as the O-antigen synthesis locus , which were obtained from a BEI Resources transposon library .Comparative transcriptomic analysis revealed that the AR-13 resistance of this mutant is not due to changes in the RNA levels since no gene transcript was differentially expressed greater than 1.7-fold relative to the parental strain (data not shown). Thus, the observed stable AR-13 resistance in 5 (TolC)  with amiovicida)  that is ncisella . We hypoobserved   and causes minimal cytotoxicity to hMDMs  which indicated that they can tolerate at least total 100 mg AR-13/kg. We next infected mice by the I.N. route with a lethal dose of LVS (3 × 103 CFUs/mouse) and then treated the infected mice, starting about 1 h post-infection, once daily by I.P. injection with 2.5, 5, or 10 mg AR-13/kg/day in 200 μl PEG  from LVS infection . There were no culturable bacteria in the lungs of these surviving mice at 15 days post-infection (data not shown). Because the 5 and 10 mg AR-13/kg/day treatments demonstrated less protective effects than mice given 2.5 mg AR-13/kg/day, but alone showed no toxicity at these doses, it is possible that the combination of LVS infection and these higher doses of AR-13 may be toxic to mice.AR-13 exerts bactericidal effects on 0 μl PEG  for 10 dF. tularensis SchuS4  in the mouse infection model. Our previous publication showed that without treatment, mice died from days 4 to 6 following intranasal infection with 10 CFUs of F. tularensis SchuS4  the compound was also tested in combination with a sub-optimal dose of gentamicin, one of the primary antibiotics commonly used to treat Francisella infection  or 5 mg AR-13/kg/day , or with 5 mg AR-13/kg/day plus 0.25 mg gentamicin/kg/day (I.P.) once daily for 5 consecutive days. PEG and 0.25 mg gentamicin treated groups were included as controls. As shown in Figures 6B,C, AR-13 treatment prolonged survival of F. tularensis SchuS4 infected mice but did not protect mice from death. All PEG and gentamicin only treated mice died at day 5 or day 6 post-infection, respectively . However, the combination of a sub-optimal dose of gentamicin with 5 mg AR-13/kg/day protected 60% of the infected mice from death , with no CFU recovered from the lungs of surviving mice (data not shown). These data suggest that AR-13 could potentially be used as a combinational therapeutic to control Francisella infection.We then sought to evaluate the ability of AR-13 to control virulent Type A s SchuS4 . Since Anfection . Mice weFrancisella has not been clinically described , and F. novicida (data not shown). M. tuberculosis and S. aureus have been demonstrated to be susceptible to direct killing by AR-13  . These findings suggest that the antibacterial effect of AR-13 is likely not mediated by rapid disruption of membrane integrity, but rather that the target of the compound is intracellular. Unlike the bactericidal effects of AR-13, AR-16, another derivative of cyclooxygenase-2 inhibitor, exerted bacteriostatic anti-Francisella activity . While bacteriostatic agents have a place in the toolkit of compounds to treat bacterial infections, our work here focused on the bactericidal activity of AR-13.The nature of the antimicrobial activity [defined as killing 99.9% of a bacterial inoculum within a 24 h exposure period ] of AR-1Francisella spp., was moderate . This was surprising, as other derivatives of the parental compound, such as AR-12, penetrate cells and kill intracellularly by the induction of autophagy . Comparative genomic sequencing combined with biological assays demonstrated that this resistance is likely mediated by multidrug eﬄux pumps involving genes FTL_1107 and FTL_1865. Amino acid mutations at Leu236Pro and Glu441Lys, respectively, were identified in these eﬄux pumps which presumably increase general or AR-13 specific eﬄux activity. RNAseq data showed no increase in eﬄux pump gene transcription in these mutants, further suggesting increased eﬄux as the mechanism behind the increase in AR-13 resistance. To the best of our knowledge, no information exists about these mutations regarding enhanced activity of eﬄux pumps in Gram-negative bacteria. The role of eﬄux in resistance was confirmed with the use of an eﬄux pump inhibitor or inactivation of the eﬄux pump, both abolishing the observed resistance , which is known to be mediated, in part, by the eﬄux pump TolC  of AR-13 recovered and were healthy at the study endpoint, while none of the vehicle-treated control mice survived. It is noteworthy that higher doses of AR-13 (5 mg/kg and 10 mg/kg/day) resulted in less protective effects, suggesting potential toxicity of the drug at these doses in infected animals . Since the most protective dose of AR-13 for LVS-infected mice was a total of 25 mg/kg, we sought to examine whether this dose could protect mice from a lethal dose of the human pathogen, F. tularensis SchuS4. Treatment with this dose prolonged survival of the infected mice but did not protect the mice from death . However, when combined with the sub-optimal dose of gentamicin, three of five mice were protected while all infected mice treated with the sub-optimal dose of gentamicin died . F. tularensis is primarily an intracellular pathogen of macrophages, and the compound was shown to have limited activity against Francisella in macrophages. This may partially explain why better protection was not observed in mice. However, gentamicin, an antibiotic that has poor cellular penetrating activity, augmented AR-13 activity, likely by enhancing the permeability of the eukaryotic and/or bacterial envelope to AR-13 (the latter during Francisella’s extracellular phase). However, these data provide strong evidence that AR-13, either with or without traditional antibiotic augmentation, is a promising lead candidate as a drug to aid control of tularemia in humans.Using All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. KH and JG conceived and designed the study. KH, HA, JF, DG, PW, and HC performed the experiments and analyzed the data. KH, LS, and JG: wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Back pain is one of the most common chronic diseases in Germany and has a major impact on work ability and social participation. The German Pension Insurance (GPI) is the main provider of medical rehabilitation to improve work ability and prevent disability pensions in Germany. However, over half of the persons granted a disability pension have never used a medical rehabilitation service. Furthermore, evidence on the effects of medical rehabilitation in Germany is inconclusive. Consequently, this study has two aims: first, to determine barriers to using rehabilitation services, and second, to examine the effectiveness of medical rehabilitation in German residents with chronic back pain.n = 5760 expected) will be sent a second questionnaire. Individuals will be eligible for the first survey if they are employed, have neither used nor applied for a rehabilitation programme during the last 4 years and neither received nor applied for a disability pension. The sample will be drawn randomly from the registers of the GPI North  and the GPI Central Germany  and stratified by sex and duration of sickness absence benefits. Barriers to rehabilitation services will be related to socio-demographic and social characteristics, pain and attitudes to pain, health and health behaviour, healthcare utilisation, experiences and cognitions about rehabilitation services and job conditions. Propensity score matched analyses will be used to examine the effectiveness of rehabilitation services. Data on use of medical rehabilitation will be extracted from administrative records. The primary outcome is pain disability. Secondary outcomes are pain intensity and days of disability, pain self-efficacy, fear avoidance beliefs, self-rated health, depression, healthcare utilisation, self-rated work ability and subjective prognosis of employability, sickness absence benefits, and disability pensions.In 2017 a postal questionnaire will be sent to 45,000 persons aged 45 to 59 years whose pension insurance contributions are managed by the GPI North or the GPI Central Germany. In 2019 respondents who report back pain in the first survey .German Clinical Trials Register (The online version of this article (doi:10.1186/s12889-017-4588-x) contains supplementary material, which is available to authorized users. With 12-month prevalence rates of more than 75% . Back paThe World Health Organisation (WHO) deemed rehabilitation essential to enabling people with disabilities or chronic diseases to participate in work and society, but also identified unmet needs . In GermPrevious international research studies have reported various barriers to using rehabilitation services, i.e. factors that prevent persons from using rehabilitation services despite an obvious need for them. For example, a systematic review  identifiDuring the last two decades, a number of studies have attempted to address the problem of unmet rehabilitation needs in Germany, either by asking people eligible for rehabilitation services or important stakeholders about barriers to using them –13 or byThe second research goal relates to the effectiveness of rehabilitation services. Attending physicians, who are important stakeholders when requesting medical rehabilitation, should base their advice about medical rehabilitation on the best current evidence . AlthougAccording to the demands of evidence-based medicine, the lack of evidence may itself be a major barrier to the utilisation of rehabilitation services. It follows that to justify increased utilisation of rehabilitation services in the future we need high-quality investigations into the effectiveness of rehabilitation services. From a health service research perspective there needs to be a focus on rehabilitation under real-life conditions and its long-term effects through comparisons with patients who do not receive rehabilitation services. Large-scale, randomised controlled trials with long-term follow-up periods are not feasible due to the legal foundations of rehabilitation, so appraisals of the outcomes of medical rehabilitation must be based on data from cohort studies. To our knowledge, this study is the first attempt to investigate the effects of German medical rehabilitation services for persons with CBP, under routine conditions and via a large cohort study using propensity score matching.The study protocol has been prepared according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist .The study is designed as a cohort study. Persons with back pain will be identified via a preliminary survey and then followed up. Back pain patients who complete a medical rehabilitation will be compared with back pain patients who do not participate in a medical rehabilitation programme. Propensity score matching will be used to match controls to treated patients and to estimate unbiased effects of medical rehabilitation.Baseline data will be assessed to identify persons with back pain and to analyse barriers to using rehabilitation services. Follow-up data will be assessed at the beginning of the third year of the study to determine the effectiveness of medical rehabilitation. Administrative data from the GPI registers will also be used. Given the observational design no-one will be blinded before, during or after the trial.Participants in the control group receive no medical rehabilitation and are identified through propensity score matching.Participants in the treatment group will take part in a medical rehabilitation programme based on current treatment standards and guidelines for the rehabilitation of back pain . The medApplications to use rehabilitation services, the related decisions, and the utilisation of these services are documented in the GPI registers and data relating to the first year of the study will be extracted from these records.n = 22,500) and GPI Central Germany . The samples will be stratified by sex in a ratio of 1:1 and by sickness absence benefits in a ratio of 2:1 (less than 1 week of sickness absence benefits vs. at least 1 week of sickness absence benefits). In the first quarter of the first year, a baseline survey will be performed to assess the 3-month prevalence of back pain . The localisation of back pain will be represented on a pain drawing [Employed persons aged 45 to 59 years will be included. The exclusion criteria are having applied for or used medical rehabilitation services during the last 4 years and previous requests for or receipt of disability pension benefits. Samples will be drawn in equal proportions from the populations of the GPI North ( drawing . Only peEstimates for baseline response (40%), consent to linking survey with administrative data (80%), back pain (40%) , utilisaAfter the sample has been drawn potential study participants will be contacted by post by the GPI. The first mailing of the baseline survey will include a personalised cover letter, information about the study, a consent form, and two envelopes . The prepaid envelope is to be used to send the completed questionnaire and the envelope containing the consent form to the university. The project staff at the university will collect the consent form envelopes and pass them on to the relevant GPI employees without opening them, as they contain personal data. The GPI employees will check off those who have provided informed consent against a study list and inform the scientists at the university by transferring the identification numbers for consenting participants. Participation is voluntary and participants are free to withdraw their consent at any time without any consequences.www.rehab-bp.de) which will inform about the study and its findings.One, four and five weeks after the initial mailing of the questionnaire reminders will be sent to potential study participants. The first reminder takes the form of a short thank-you letter to remind the recipient about the study and the questionnaire they received recently. The second reminder is sent 3 weeks after the first reminder and contains all of the above-mentioned study material. This reminder is only sent to persons who have not yet returned their questionnaire. The third and final reminder is sent 1 week after the second reminder, once again it highlights the importance of the recipient’s participation in the study and thanks them in advance for their support. The final reminder also contains a link to the study website . Questionnaire and administrative data can be linked via the unique study identifier.The authors of the protocol will be responsible for data management in the study. Data access will be limited to the authors and the team of research assistants.The questionnaire used in this study will include items on various barriers to rehabilitation utilisation, primary and secondary outcomes and some additional variables. Data on additional outcomes will be extracted from administrative records . These administrative data will be provided by the GPI North and GPI Central Germany. A complete list of all measured constructs, measurement points and expected scaling is shown in Table Pain disability will be the primary outcome in our analyses of the effectiveness of medical rehabilitation and will be assessed at baseline and follow-up. Three items from the German version of the Chronic Pain Grade Questionnaire will be used to assess pain disability , 49. TheThe secondary outcomes of our analyses of the effectiveness of rehabilitation will be assessed at baseline and follow-up.Four items from the Chronic Pain Grade Scale will be used to assess pain intensity (three items) and days of disability (one item) , 49. TheSelf-efficacy with respect to coping with back pain will be assessed with the German adaptation of the Pain Self-efficacy Questionnaire  , 51. TheThe German version of the Fear Avoidance Belief Questionnaire (FABQ) , 53 compGeneral health will be assessed with one item from the Copenhagen Psychosocial Questionnaire with an 11-point scale  , 55.Depressive symptoms will be measured using the 8-item depression module of the Patient Health Questionnaire (PHQ-8), which is recommended for research purposes , 57. IteSelf-rated work ability is assessed using the Work Ability Score (WAS) . This 11Three items will be used to assess whether participants believe they will remain at work until retirement, whether they assume that their health is permanently jeopardised and whether they intend to request a disability pension . DichotoSingle items will be used to assess use of three types of pharmaceuticals over the last 3 months: pain-reducing medication, mood-enhancing medication, medication for other health complaints. Response categories are ‘no’ (0 points), ‘regularly’ (e.g. daily) (1 point) and ‘as needed’ (1 point). Outpatient visits to physicians will be assessed as the number of visits in the last 12 months. The utilisation of outpatient therapies (e.g. physiotherapy) will be assessed as the number of therapies units used over the last 12 months. Hospitalisation within the last 12 months will be captured as a dichotomous variable (0: ‘no’ and 1: ‘yes’). The number of days of sick leave during the last 12 months will also be assessed.Data on days in receipt of sickness absence benefits, days in regular employment, applications for and granting of a disability pension will be extracted from the GPI registers during the third year of the study. These variables are the secondary outcomes in our analyses of the effectiveness of medical rehabilitation.Data on other variables will be collected at baseline in order to provide a description of the study sample, to determine potential barriers to the use of rehabilitation services, to gain variables for estimating the propensity score, and to identify variables that might influence the effect of rehabilitation services.Comorbidity will be assessed using an adapted version of the German version of the Self-Administered Comorbidity Questionnaire , 61, usiPhysical exercise, cigarette smoking, body mass index and membership of a self-help group or organisation will be assessed as indicators of health-related behaviour.Physical job demands will be measured using five 4-point items that yield a total score ranging from 0 to 15 points . PsycholMotivational factors related to application for rehabilitation services will be assessed using a set of questions that was recently developed for the Third German Sociomedical Panel of Employees . The queAge and sex will be derived from the GPI registers. Data on net income, job position, and educational level will be self-reported. Assessment of migrant status will be assessed based on Schenk’s recommendations . Partnert tests and analyses of variances for continuous variables, Mann–Whitney U tests for ordinal variables and chi-squared tests for binary variables. The second step will consist of logistic regression analyses to identify the most important predictors of rehabilitation intention, rehabilitation application, acceptance onto a rehabilitation programme and use of rehabilitation. In the third step we will estimate generalised structural equation models to describe the direct and indirect effects of potential barriers.To identify barriers to the utilisation of medical rehabilitation services we will compare persons with back pain who complete a medical rehabilitation with persons with back pain who do not. We will identify barriers to use of rehabilitation services across the entire course of the rehabilitation process, from recognition of need for rehabilitation and applying for a medical rehabilitation programme, to applying successfully for a grant, participating in a rehabilitation programme and completing the programme. Different barriers apply to each step and these will be identified by analysing group differences in baseline values of the outcome and control variables at each stage of the process. The first step in the analysis will be univariate analyses: Rehabilitation participants and untreated subjects will be matched by propensity scores to achieve a balanced sample and enable calculation of unbiased estimates of effectiveness –72. The The propensity score will be estimated using a logistic regression model including all variables that are associated with treatment allocation. For every treatment case three persons with a similar propensity score will be selected from the larger pool of non-treatment cases. Re-sampling with replacement will be used to reduce bias. If it is necessary to achieve a balanced sample, a caliper of one-quarter of the propensity score will be used during re-sampling. Additional matching schemes will be used in the sensitivity analysis.Standardised percentage bias will be calculated as an indicator of bias before and after matching due to differences in observed sample characteristics. This is the difference between sample means in the intervention and control groups relative to the square root of the average of the sample variances in both groups . MultiplThe average treatment effect for the treated cases will be estimated with weighted linear regression models in the case of continuous outcomes and generalised linear models in the case of binary outcomes (see above: primary outcome and secondary outcomes) . Robust z-standardised [Moderator analyses will be performed to clarify whether individual characteristics modify the treatment effect. The moderating effects of individual characteristics will be modelled in terms of multiplicative interaction terms for the treatment indicator and potential moderators. In the case of continuous moderators, these variables will be dardised . ModeratOur cohort study examines barriers patients with CBP face in attempting to access rehabilitation; it addresses the lack of prospective, longitudinal studies in this area of research. Barriers to accessing and using rehabilitation services are analysed at several time points. Furthermore this study appraises the effectiveness of routine German medical rehabilitation services for patients with back pain. The findings of this study will be published in peer-reviewed journal articles and presented at conferences.Recruitment has started and is ongoing Additional file 2Is the letter telling potential participants about the study’s objectives. (DOCX 15 kb)


Action research (AR) has an important role to play in educating physical therapists. Increasing efforts should be encouraged to instigate AR programs in physical therapy practice and clinical education. Such programs commonly require considerable effort and understanding by clinical instructors, and require adoption of new educational methods. AR programs can lead physical therapists and clinicians to be more questioning and reflective in evaluating practical questions regarding patient therapy and education. The purpose of this article is to educate the readers on the importance of AR and to provide a few relevant references on that topic. A specific study is described in this paper in which physical therapy clinical instructors participated in a structured workshop designed to demonstrate the values of AR and how such values can be incorporated in teaching their students. AR can lead to improved therapist-patient interaction and help solve specific practical problems arising during therapy sessions.


Cancers initially reviews the role of AR in advanced prostate cancer, and then explores the potential importance of AR signaling in other epithelial malignancies. The first few articles focus on the use of novel AR-targeting therapies in castration-resistant prostate cancer and the mechanisms of resistance to novel antiandrogens, and they also outline the interaction between AR and other cellular pathways, including PI3 kinase signaling, transcriptional regulation, angiogenesis, stromal factors, Wnt signaling, and epigenetic regulation in prostate cancer. The next several articles review the possible role of androgens and AR signaling in breast cancer, bladder cancer, salivary gland cancer, and hepatocellular carcinoma, as well as the potential treatment implications of using antiandrogen therapies in these non-prostatic malignancies.The notion that androgens and androgen receptor (AR) signaling are the hallmarks of prostate cancer oncogenesis and disease progression is generally well accepted. What is more poorly understood is the role of AR signaling in other human malignancies. This special issue of  Cancers is to shed more light on the clinical significance of androgen/AR signaling, not just in prostate cancer, but also in other epithelial malignancies.Androgens and androgen receptor (AR) signaling are the hallmarks of prostate cancer oncogenesis and disease progression. While the medical literature is saturated by studies examining the role of androgens/AR in prostate cancer, less attention has been given to the potential importance of the AR pathway in other human malignancies. The goal of this special issue of This theme issue begins with a thoughtful summary by Schweizer et al.  introducThe second series of articles begins to address the role of AR signaling in other human cancers, with a focus on potential therapeutic implications. Rahim et al.  begin wiCancers, that they become informed about the role of androgens and AR signaling in the context of multiple different cancer types, and that this treatise will ignite further clinical research and therapeutic trials aiming to modulate the AR pathway in various human malignancies.We hope that the readership enjoys this this special issue of


GARS, which encodes glycyl-tRNA synthetase (GlyRS), mediate selective peripheral nerve toxicity resulting in Charcot-Marie-Tooth disease type 2D (CMT2D) is still largely unresolved. The transmembrane receptor protein neuropilin 1 (Nrp1) was recently identified as an aberrant extracellular binding partner of mutant GlyRS. Formation of the Nrp1/mutant GlyRS complex antagonises Nrp1 interaction with one of its main natural ligands, vascular endothelial growth factor-A (VEGF-A), contributing to neurodegeneration. However, reduced extracellular binding of VEGF-A to Nrp1 is known to disrupt post-natal blood vessel development and growth. We therefore analysed the vascular system at early and late symptomatic time points in CMT2D mouse muscles, retina, and sciatic nerve, as well as in embryonic hindbrain. Mutant tissues show no difference in blood vessel diameter, density/growth, and branching from embryonic development to three months, spanning the duration over which numerous sensory and neuromuscular phenotypes manifest. Our findings indicate that mutant GlyRS-mediated disruption of Nrp1/VEGF-A signalling is permissive to maturation and maintenance of the vasculature in CMT2D mice.The mechanism by which dominantly inherited mutations in the housekeeping gene  GARS is a widely and constitutively expressed gene that encodes glycyl-tRNA synthase (GlyRS), which serves to covalently attach glycine to its transfer RNA (tRNA), priming it for protein translation1. Dominant mutations in GARS cause Charcot-Marie-Tooth disease type 2D , a currently incurable disorder of the peripheral nerves with the principal clinical symptom of muscle wasting originating in the extremities2. A battery of evidence indicates that these GARS mutations cause a toxic, gain-of-function in mutant GlyRS8, and that this is at least partially reliant upon the surface exposure of protein regions usually buried in wild-type GlyRS9. Recently published work suggests that these neomorphic regions cause mutant GlyRS to aberrantly bind to the transmembrane receptor neuropilin 1 (Nrp1)10, amongst other proteins8. Nrp1 functions in both the nervous and vascular systems by modifying multiple signalling pathways11. Mutant GlyRS was shown to specifically interact with the extracellular region of Nrp1 integral to VEGF-A165 binding (the b1 domain)12, and thereby antagonise Nrp1/VEGF-A signalling  hindbrains phenocopy the facial motor neuron migration defects of mice deficient in VEGF-A164  or Nrp113. Moreover, genetically reducing Nrp1 levels enhances the severity of CMT2D pathology, whereas providing additional VEGF-A165, via lentiviral intramuscular injections, improves the phenotype10.ing Fig. 10. CMT2D15, and a mild reduction in VEGF-A causes progressive motor neuron degeneration in mice16, partially explaining why antagonisation of Nrp1/VEGF-A signalling could be contributing to the neuronal pathology in CMT2D10. However, VEGF-A is also critical to blood vessel formation (vasculogenesis), expansion (angiogenesis), and luminal growth of arteries (arteriogenesis), with the latter two processes requiring VEGF-A signalling through Nrp1 and VEGFR218. Nrp1 additionally regulates vascular development by promoting extracellular matrix remodeling downstream of integrins19 and by suppressing excessive TGF-β signalling20. Although mutating the VEGF-A-binding site of Nrp1 does not phenocopy the severity of full Nrp1 knockout mice, it causes a post-natal impairment in angiogenesis and arteriogenesis22, resembling the milder phenotype of mice expressing VEGF-A isoforms lacking the Nrp1-binding site23. This work suggests that Nrp1 has both VEGF-dependent and VEGF-independent functions in blood vessels, and that Nrp1/VEGF-A signalling is perhaps more important for vascularisation post- than pre-birth21. Consistent with this, blocking VEGF-A binding to Nrp1 with a monoclonal antibody impairs vascular remodeling in the peri-natal mouse retina24.VEGF-A functions in diverse neuronal processes10, 2) GlyRS is secreted25 and found circulating in serum of humans and mice25, and 3) vascular impairment can contribute to neurodegeneration26, we decided to assess the effect of mutant GlyRS on the vascular system of the GarsC201R/+ murine model of CMT2D.Given that 1) mutant GlyRS competes with VEGF-A for Nrp1 bindingGARS have been shown to alter protein conformation – five CMT2D-associated mutants9 and P234KY corresponding to murine P278KY10, which is the spontaneous mutation found in GarsNmf249/+, the more severe CMT2D mouse model4. Neomorphic Nrp1 binding and disturbance of Nrp1/VEGF-A signalling has been confirmed for a number of GlyRS mutants, including P234KY10. In order to verify a common mechanism in the mild GarsC201R/+ mouse27, we performed co-immunoprecipitation assays in NSC-34 cells expressing V5-tagged wild-type, P234KY, L129P, and C157R  GlyRS. GlyRSL129P was included because it is one of the GARS mutants with the best evidence linking it to human neuropathy28. As expected, wild-type GlyRS did not bind Nrp1, while GlyRSC157R did, albeit at a lower affinity than the other two mutant proteins  muscles from one and three month old wild-type mice. These muscles are thin and flat, permitting the visualisation of the entire vascular and nervous systems within the tissue36. In these muscles at both time points, Nrp1 (white) did not specifically localise to SV2/2H3+ (green) motor neurons or S100+ (blue) Schwann cells   Figs . Nrp1 coed) Figs  and myeled) Figs , suggestin vitro  and VEGFR2 (red) protein levels and localisation in lumbrical and TVA muscles, but found no obvious discrepancies between genotypes  with either glyceraldehyde 3-phosphate dehydrogenase (Gapdh) or endothelium-specific platelet endothelial cell adhesion molecule 1 (Pecam1) as the reference gene 14, and genes integral to angiopoietin-Tie signalling 39. We saw no difference between genotypes in the expression of any of these genes  capillary network in one and three month old retinas .Nrp1 is indispensable for vascularisation of the mouse central nervous system, but is thought to be less critical, although still importantnas Fig.  using annas Fig. . Gars miers Fig. , densitiers Fig. , and braers Fig.  as wild-ers Fig. . SimilarNrp1 mutation that abrogates VEGF-A binding42. By adulthood, vascular extension has recovered, but artery number remains diminished22. To determine whether mutant GlyRS binding to Nrp1 causes similar nascent phenotypes, we dissected retinas from P6-7 wild-type and GarsC201R/+ mice and stained them with IB4 , vascular extension and artery number are reduced in mice carrying a knockin ion Fig. . Furtherion Fig. , which iion Fig. .10. We therefore assessed parameters of the capillary network in E13.5 hindbrains of wild-type and GarsC201R/+ mice. Mutant brains showed no differences in blood vessel structures between genotypes  was used instead of IB4 due to superior staining of the vasculature in this tissue , but not wild-type, was capable of aberrantly binding to Nrp1 .Mutant GlyRS appears to compete with VEGF-A for extracellular binding to the transmembrane receptor protein Nrp1 contributing to the peripheral nerve pathology observed in CMT2Drp1 Fig. . This in44, but its post-natal function and localisation remains less well defined. Motor neuron-specific ablation of Nrp1 using the Olig2 promoter is reported to cause post-natal motor axon loss and muscle atrophy45, but Nrp1 was deleted developmentally46 rather than at or post-birth. It is possible that low levels of post-natal Nrp1 expression in motor neurons allows aberrant mutant GlyRS binding and signalling sufficient to drive peripheral nerve degeneration in CMT2D mice. However, given that embryonic defects are observed in Gars mice10, and that Nrp1 expression is developmentally downregulated in neuronal tissue48, we should not rule out that mutant GlyRS-mediated inhibition of Nrp1/VEGF-A signalling during development may be predisposing the motor system to the subsequent degeneration observed at later, post-natal stages49.The fundamental requirement for Nrp1 in motor nervous system development is undisputed31, we decided to assess capillary architecture in a range of tissues covering the developmental to late symptomatic period. Capillary diameter, density, and branching were unaltered by mutant GlyRS in both distal and proximal skeletal muscles  according to the manufacturer’s instruction.NSC-34 cell line was purchased from ATCC  and grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin-streptomycin . Cells were split every two to three days using 0.25% (v/v) trypsin-EDTA  and cultures maintained at 37 °C in a 5% (v/v) CO2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4), scraped into PBS, pelleted, re-suspended in Pierce IP Lysis Buffer  for 30 min, and the extract cleared for 7 min at 12,000 × g. Protein G beads (Invitrogen) were pre-incubated with rabbit anti-Nrp1  or rabbit IgG  for 30 min before mixing with cell lysates overnight. The beads were then washed three times with buffer  Triton X-100, 5% (v/v) glycerol), and immunoprecipitates probed by western blot. Immunoprecipitates were fractionated by 4-12% (v/v) Bis-Tris-Plus SDS-PAGE gels (Invitrogen) and transferred to PVDF membranes using the iBlot Dry Blotting System (Invitrogen). Membranes were blocked for 1 h with Tris-buffered saline with 0.1% (v/v) Tween 20 containing 5% (w/v) non-fat dry milk. Wild-type and mutant GlyRS proteins were detected using mouse anti-V5  and Nrp1 using the same antibody as for co-immunoprecipitation (1/1000). After primary antibody incubation, membranes were washed and probed with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies , followed by detection using ECL western blotting substrate (Thermo Fisher) using the FluorChem M imager .For cell lysate preparation, 36 h post-transfection NSC-34 cells were washed twice in phosphate-buffered saline  Act (1986), and approved by the University College London (UCL) – Institute of Neurology Ethics Committee.37. Eyes were removed and fixed in 4% (w/v) paraformaldehyde  for 2 h, before retinas were dissected and stained as reported previously55. Sciatic nerves were dissected from mice transcardially perfused with 4% PFA, post-fixed for 2 h, and 10 μm sections processed and stained as previously described8. E13.5 hindbrains were dissected and stained using published protocols56.All steps were performed at room temperature, apart from overnight incubations conducted at 4 °C. Lumbrical and TVA muscles were dissected and immunohistochemically labelled as previously describedThe following primary antibodies were used for immunofluorescence studies: mouse anti-Mbp , mouse anti-neurofilament , goat anti-Nrp1 , goat anti-Pecam1 , rabbit anti-S100 , mouse pan anti-synaptic vesicle 2 , and rabbit anti-VEGFR2 . When appropriate, tissues were also incubated with 1 mg/ml IB4 biotin conjugate  in PBS at 1/50 (retinas) or 1/250 . The following day, combinations of Alexa Fluor-labelled secondary antibodies  at 1/250, 2 mg/ml streptavidin Alexa Fluor 488 conjugate  at 1/250, fluorophore-conjugated α-bungarotoxin  at 1/1000, and Cy3-conjugated anti-αSMA  at 1/500 in PBS were used for 2 h, before the tissues were washed and mounted in fluorescent mounting medium  for imaging.https://imagej.nih.gov/ij/). All samples were imaged and analysed blinded to genotype. No samples were excluded from the analyses once imaged. IB4-stained muscles, retinas, and hindbrains, and Pecam1-stained sciatic nerve sections were used for capillary analyses as performed previously57. Maximal Z-resolution stacked images across the entire depth of wholemount tissues were obtained at 40x (for muscles) and 20x (for retinas and hindbrains) magnification and 1024 × 1024 pixel resolution. Sciatic nerve sections were single plane images taken at 20x and 2812 × 2812 pixel resolution. Three non-overlapping fields of view were imaged per lumbrical muscle, and four lumbricals analysed per animal . Eight non-redundant Z-stack images were obtained per TVA and retina, and four per hindbrain, and analysed for each animal. Similar tissue regions were imaged across samples. Five different sections were analysed per sciatic nerve. For measuring capillary diameters, a uniform grid (area per point: 500 μm2) was overlaid onto 3D-projected (Max Intensity) images, and every capillary found at a grid intersection measured (except those that were branching), with repeated measures of the same capillary accepted  and images analysed using ImageJ software  as per the manufacturer’s instructions. SuperScript IV  was used as instructed to reverse transcribe 1 μg RNA. Copy DNA was diluted in water (1/10), before qPCR analyses were performed using PowerUp SYBR Green  and a CFX96 Real-Time System  as previously described58. Cycling conditions were as follows: 50 °C for 2 min, 95 °C for 2 min, 40x . Primers were used at 500 nm and their sequences are detailed in Table Tibialis anterior muscles were dissected from one month old wild-type and Data were assumed to be normally distributed unless evidence to the contrary was provided by the D’Agostino and Pearson omnibus normality test. GraphPad Prism 5  was used for all analyses. Means ± standard error of the mean are plotted, except for individual capillary diameter data, which represent mean and standard deviation. All analyses were performed on animal mean values.Supplementary Information


In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis.Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants.  PSA is an androgen-regulated gene that is dependent upon AR transactivation. Therefore, a rising PSA level despite castrate serum levels of androgen suggests continued AR transactivation. One probable AR mechanism of resistance to hormone therapies associated with increasing PSA levels is expression of constitutively active AR splice variants that lack the LBD.Approximately 20–40% of patients treated for localized prostate cancer will have recurrence. The current therapy for these patients is hormone therapy such as orchiectomy or luteinizing hormone-releasing hormone analogs followed by antiandrogens and 17α-hydroxylase/17,20-lyase (CYP17) inhibitors. Current hormone therapies all target the ligand-binding domain (LBD)123I-EPI-002  region, which is essential for transcriptional activities of both full-length AR (FL-AR) and constitutively active AR splice variants lacking the LBD 13. AF-1 c-EPI-002 . The proDysidea sp.  is a natural compound isolated and purified from the marine sponge idea sp. . Interesin vitro . Here thvar567es have been described , PSA  and anti-His  were from the sources indicated. β-Actin antibody  and anti-biotin  were from Abcam. STAT3  was from Cell Signaling Technology. Anti-streptavidin  was from LI-COR Biosciences. SINT1 is a natural compound . LNCaP cells were authenticated by short tandem repeat analysis and tested to ensure that they were mycoplasma-free by DDC Medical. LNCaP95 cells expressing FL-AR and AR-V7 were provided by Dr. Plymate (University of Washington). These cells were maintained in RPMI 1640 medium supplemented with 10% dextran-coated charcoal-stripped FBS.2.NOD-SCID (6–8 week-old) male mice were maintained in the Animal Care Facility at the British Columbia Cancer Research Centre. All animal studies adhered to the relevant regulatory standards protocols approved by the University of British Columbia Animal Care Committee . Metacam  was injected subcutaneously prior to any surgery. Animals were anesthetized using isoflurane anesthesia and euthanized by COm SINT1, 10 μm bicalutamide, or vehicle for 1 h before addition of the corresponding ligand . After 48 h of treatment, cells were lysed and analyzed for luciferase activities.LNCaP cells were transfected with the reporter plasmid PSA(6.1kb)-luciferase, PRE-luciferase, or GRE-luciferase and expression vector for progesterone receptor (PR)-β or glucocorticoid receptor (GR)-α in serum-free, phenol red-free medium using Lipofectin (Invitrogen). Sixteen hours later, the cells were pretreated with 10 μm modified SINT1  or the vehicle dimethyl sulfoxide (DMSO). 25 μm EPI-053  was used as positive control methyl]amine (Sigma), 1 mm tris(2-carboxyethyl)phosphine, 100 μm biotin-azide reagent, and 1 mm CuSO4. Samples were dialyzed in 50 mm HEPES, pH 8.0, 150 mm NaCl, 0.1% SDS, and 1% Triton X-100 to remove excess biotin-azide reagent. Biotinylated soluble receptors were captured with streptavidin-agarose resin. Protein-SINT1 complexes were separated by 10% SDS-PAGE and subjected to Western blotting analysis using AR-N20 (1:1000) and biotin (1:1000) antibodies. Proteins were visualized using ECL detection reagent (Amersham Biosciences).LNCaP cells were treated for 24 h with 20 μm HEPES, pH 7.9, and 100 mm NaCl at 37 °C for 3 h. LPY probes were labeled with biotin by copper-catalyzed click chemistry reaction at 25 °C for 1 h (Histidine-tagged AR AF-1 recombinant protein was expressed and purified as described . The binm) was incubated with 1 nm FluormoneTM AL Green to achieve ∼80% saturation. The reactions with serial dilutions of test compounds were completed in 40-μl aliquots in triplicates. Displacement of Fluormone AL Green from AR LBD by the test compounds was measured by the change in fluorescence polarization using Infinite® M1000 (Tecan) with excitation at 470 nm and emission at 535 nm.The Androgen Receptor Competitor Assay (Green) kit (Invitrogen) was used. AR LBD -Gal4 DBD prior to pretreatment with 20 μnal 24 h , 8.-Gal4 DBDm), SINT1 (20 μm), or DMSO (vehicle control) for 1 h before treatment for 6 h with 50 ng/ml IL-6. Whole cell lysates were prepared from harvested cells in modified radioimmune precipitation assay buffer. Cell lysates were precleared with 2 μg of normal mouse IgG and 20 μl of Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) to remove nonspecific bead binding. Total STAT3 was immunoprecipitated with 10 μg of antibody against STAT3 (124H6) or 10 μg of normal mouse IgG as a negative control followed by probing the immunoblot with anti-AR antibody  and anti-STAT3 antibody  and normalizing to levels of β-actin  as loading controls. The input lanes were loaded with 5 μg of the starting cell lysate. The relative density of AR for each immunoprecipitated sample was quantified by ImageJ software and normalized to levels of STAT3 pulled down.LNCaP cells were preincubated with EPI-002 (35 μm), EPI-002 (0–31 μm), or a combination with a constant ratio of 1:1.3 for EPI-002/SINT1 concentration. Cells were then incubated with 1 nm R1881 for 48 h, and a luciferase assay was performed. IC50 values for SINT1 for the three reporters were calculated using GraphPad Prism . The combination index of SINT1 and EPI-002 was calculated using CalcuSyn software. Combination indices >1, 1, and <1 indicate antagonism, additive affect, and synergy, respectively.LNCaP cells were transfected with AR-responsive PSA (6.1 kb)-luciferase, PB-luciferase, and ARR3-luciferase reporters. Forty-eight hours later, cells were pretreated for 1 h with SINT1  using AR-N20 antibody (1:1000) and goat anti-rabbit HRP-conjugated secondary antibody .COS-1 cells (AR-negative) were co-transfected with the PB-luciferase reporter and the expression plasmid ARm R1881 (LNCaP) under serum-free and phenol red-free conditions and then incubated for another 48 (LNCaP95) or 72 h (LNCaP). Cells were pulse-labeled with 10 μm BrdU for 2 h. Cells were fixed, and BrdU-labeled cells were identified with anti-BrdU-peroxidase (Roche Applied Science). BrdU incorporation was measured at 570 nm using a VersaMax ELISA microplate reader (Molecular Devices). At least three independent experiments with six technical replicates were analyzed, and average values are indicated.LNCaP and LNCaP95 cells were pretreated for 1 h with SINT1, EPI-002, bicalutamide, enzalutamide, or vehicle before addition of 0.1 nm SINT1, 10 μm bicalutamide, 10 μm enzalutamide, or vehicle. Cells were exposed to 1 nm R1881 for an additional 2 h, fixed with paraformaldehyde, and mounted in Vectashield antifade mounting medium with DAPI . Cellular localization of AR was examined by fluorescence microscopy using an Axiovert 200 microscope equipped with a YFP filter . Images were taken using a Axiocam MR camera and the AxioVision 4.4 software .LNCaP cells seeded on coverslips were transiently transfected with an expression vector for AR-YFP for 24 h prior to pretreatment for 1 h with 10 μm bicalutamide, 10 μm SINT1, or 5 μm enzalutamide under serum-free and phenol-free RPMI 1640 medium conditions for 1 h before adding 1 nm synthetic androgen R1881 or vehicle (EtOH). At 48 h, total RNA was isolated using a PureLink RNA Mini kit (Invitrogen). For in vivo expression, tumors were harvested 3 days after last treatment, and RNA was extracted using TRIzol. Prior to cDNA generation, 4 μg of RNA were DNase-treated using DNase I . DNase-treated RNA was split into two tubes (+RT and −RT), and cDNA was generated using the High Capacity RNA-cDNA kit (Applied Biosystems). Once complete, both reactions were adjusted to 5 ng/μl and stored at −20 °C. Approximately 5 ng of diluted cDNA and gene-specific primers were mixed with Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen). The transcripts were measured using an ABI PRISM 7900 Sequence Detection System (Invitrogen). For all quantitative RT-PCR experiments, each sample was tested in triplicate, and gene expression levels were normalized to the reference gene RPL13A. Neuron-specific enolase (ENO2)  and synaptophysin (SYP)  human primers were from Sigma Life Science. All other primers were described previously  every 3 days for a total of 15 days. Animal behavior and body weight were observed throughout the study period for signs of toxicity. Tumor volumes were calculated by the formula length × width × height × 0.5236. Tumors were excised 3 days after the last injection and prepared for immunohistochemistry or gene expression.Male NOD-SCID mice bearing subcutaneous LNCaP or LNCaP95 tumors were castrated when the tumor volume reached ∼100 mm2O2 for 10 min followed by incubation in Clear Vision Histostaining Blocking Solution  for 1 h at room temperature. Sections were incubated with AR-N20 (1:500 dilution) and PSA  primary antibodies. Samples were then incubated for 30 min at room temperature with secondary antibodies and mounted in Permount mounting medium .Xenograft sections were stained with hematoxylin and eosin and for Ki67 expression by Wax it Histology Inc. Cells that were positive for Ki67 were counted in sections from three xenografts for each treatment. For PSA and AR staining, dewaxed sections were microwaved for 10 min on full power in prewarmed Antigen Unmasking Solution . The endogenous peroxidase activity was blocked by incubation in 3% Hp values less than 0.05.One-way ANOVA Dunnett's multiple comparison test was performed using GraphPad software unless stated otherwise, and differences were considered statistically significant at A), which was consistent with a previous report (A). The antiandrogen bicalutamide inhibited both AR and PR transcriptional activities as reported previously . SINT1 and EPI-002 (negative control)   did not C) contrary to antiandrogens that induced nuclear translocation as reported (11–D). These data suggest that the mechanism of action of SINT1 to decrease androgen-induced AR transcriptional activity was not by preventing nuclear translocation of AR. The fact that SINT1 does not bind to the LBD is consistent with it not inducing nuclear translocation in the absence of androgens like known LBD ligands that include androgens and antiandrogens.AR signaling involves its nuclear translocation upon binding of ligand to the LBD. Antiandrogens are LBD ligands that can induce nuclear translocation of AR in the absence of androgen. In the absence of androgen, AR remained cytoplasmic in cells treated with SINT1 C contrarreported , 11–13. orted 11–D. These A). The EPI-002 click chemistry probe EPI-053 was used as a positive control . Cell lysates were used for immunoprecipitation with streptavidin to isolate those proteins bound to biotinylated probes. Immunocomplexes were split with one aliquot used for Western blotting analysis using an antibody to biotin  and the other aliquot for Western blotting analysis using an antibody to AR . Antibodies against biotin revealed a band corresponding to AR only with cells treated with active analogs EPI-053, LPY30, and LPY31 (very faint band) but not in those exposed to inactive analog LPY19 or the DMSO vehicle control . Consistent with these biotin bands corresponding to AR, the AR was immunoprecipitated with streptavidin only in those immunocomplexes that were both positive for biotin  and biologically active against AR transcriptional activity .To reveal whether SINT1 binds to endogenous AR, LNCaP cells were incubated with modified SINT1 click chemistry probes  but does not affect ligand binding to AR LBD (B). Therefore, we next evaluated whether SINT1 binds the AF-1 region in AR NTD. Purified recombinant AF-1 protein (AF1-His tag) was incubated under cell-free conditions with LPY19, LPY30, and LPY31 prior to click chemistry to add a biotin to the LPY probe . Western blotting analysis using streptavidin revealed binding of the biotinylated LPY30 and LPY31 probes to AF-1 but no binding of the inactive negative control LPY19 . Using an antibody to His tag revealed equal loading of AF-1 protein to each lane . These data show that SINT1 click chemistry probes that inhibit AR transcriptional activity bind to the AR AF-1 region, whereas inactive probe does not bind AF-1.SINT1 binds to endogenous FL-AR B but doeo AR LBD B. TherefTo test whether SINT1 can inhibit transactivation of AR NTD, LNCaP cells were co-transfected with an expression vector for AR NTD encoding amino acids 1–558 fused to the Gal4 DBD and a reporter construct with Gal4 DBD-binding sites. Transactivation of AR NTD is induced by forskolin or IL-6 .A, left). Surprisingly, SINT1 had no significant effect in blocking transactivation of AR NTD induced by IL-6, whereas EPI-002 was a potent inhibitor  as reported (B). Together these data suggest that SINT1, similarly to EPI, binds the AR AF-1 region, but SINT1 may have a different binding site on AF-1 compared with EPI compounds. This is because, unlike EPI, SINT1 had no effect on IL-6 transactivation of AR NTD and did not block STAT3 interaction with AR; however, it significantly inhibited forskolin-induced transactivation of AR NTD.Forskolin-induced transactivation of AR NTD was significantly inhibited by SINT1 and EPI-002 (positive control) A, left. nhibitor A, right reported . STAT3 ireported . Consistreported B. Togeth50 values for SINT1 were 10.74 ± 1.55, 11.64 ± 0.78, and 13.22 ± 1.19 μm for PSA (6.1 kb)-Luc, PB-Luc, and ARR3-Luc, respectively. When SINT1 was combined with EPI-002, the curve shifted left with these two compounds having an additive effect on all three reporters at all concentrations except the lowest dose (C). The median combination index for PSA (6.1 kb)-Luc, PB-Luc, and ARR3-Luc reporter were 1.001, 1.016, and 1.012, confirming an additive effect. These data support that SINT1 and EPI-002 may have different binding sites on AR AF-1 and concomitantly different mechanisms of action.A standard approach to determine different mechanisms of two compounds is to examine whether combination treatment results in additive or synergistic responses compared with each monotherapy. Here we undertook these studies using three AR-driven reporters with SINT1 and EPI-002 used alone or in combination. The ICest dose C. The meA). Under these conditions, AR-V567es transcriptional activities were significantly inhibited by SINT1  and EPI-002 (positive control) but not by the inactive analog EPI-014  ether (BADGE·2H2O)) . Importantly, an AF-1 antagonist should block transcriptional activity of AR splice variants that lack LBD. Therefore, next we determined whether SINT1 could block endogenously expressed AR variants using LNCaP95 cells. These are androgen-independent cells that express both FL-AR and constitutively active AR splice variants that lack LBD , consistent with other reports that these cells are resistant to antiandrogens including enzalutamide . Androgen-induced levels of transcripts of FL-AR-regulated genes  were all significantly repressed by SINT1 with the exception of RHOU. These data support that SINT1 inhibits FL-AR transcriptional activity and that SINT1 blockade may be gene-specific, which may be due to its unique mechanism of action compared with antiandrogens.The ability of SINT1 to repress the expression of endogenous genes regulated by both FL-AR and AR-V7 was also evaluated using LNCaP95 cells. SINT1 did not lead to an increase in levels of FL-AR mRNA when compared with its relevant vehicle control, contrary to antiandrogens A. AndrogB). Evidence to support that SINT1 inhibits the transcriptional activity of AR splice variants, as shown in B and C, was provided by measurement of levels of endogenous expression of AR-V7 target genes. SINT1 significantly repressed levels of cyclin A2, CDC20, UBE2C, and CDK1 transcripts. AKT1 transcript levels were not repressed by any treatment. SINT1 blocks the expression of target genes regulated by FL-AR and AR splice variants, which is consistent with what would be expected from an inhibitor that binds to AR AF-1.SINT1 also had no significant effect on levels of AR-V7 transcript unlike bicalutamide and enzalutamide B. Evidenin vivo studies were carried out to determine dose and route of delivery. The maximum concentration (Cmax) in plasma following a single intravenous dose of 50 mg/kg of body weight was 4.15 μg/ml (∼8 μm), which was slightly lower than its IC50in vitro. SINT1 had a short elimination half-life (t½) of 1.16 h, the volume of distribution was 36.59 liter/kg, and the plasma clearance was 21.93 liter/h/kg  compared with DMSO-treated tumors  (A). SINT1 was also able to significantly reduce the growth of aggressive fast doubling LNCaP95 tumors that express both FL-AR and AR-Vs (B). Importantly, SINT1 caused tumor regression in four of seven CRPC LNCaP xenografts (C), and tumors had a less bloody appearance (E). In these animals, a serum PSA drop of ≥50% was achieved in six animals treated with SINT1 but in only one animal in the control group (D). Levels of FL-AR and AR-V7 transcripts were not significantly altered by SINT1 treatment in harvested LNCaP95 xenografts (F) nor was there support that SINT1 induced neuroendocrine differentiation as indicated by no significant changes in levels of expression of ENO2 and SYP genes (F)., n = 8) A. SINT1 nd AR-Vs B. Importnografts C, and tupearance E. In theol group D. Levelsnografts F nor wasYP genes F.A and B). Levels and cellular localization of AR protein were similar in xenografts from vehicle-treated and SINT1-treated animals (C). Importantly, there were marked decreases in the levels of PSA in xenografts treated with SINT1 compared with the vehicle control (D), which was consistent with the 50% drop in serum PSA levels in the majority of SINT1-treated animals. Together these in vivo data with the in vitro data support that SINT1 inhibits the transcriptional activities of FL-AR and AR-Vs.LNCaP xenografts harvested at the duration of the experiment were analyzed by immunohistochemistry staining for the proliferation marker Ki67 and for expression of AR and PSA. Consistent with SINT1 decreasing tumor volume, SINT1 also reduced the number of Ki67-positive cells in harvested xenografts , A and B animals C. Import control D, which Treatment of mCRPC with second generation inhibitors of the androgen axis such as abiraterone or enzalutamide has increased overall survival , 24. Unf25–Several mechanisms of resistance that could drive AR transcriptional activity despite application of enzalutamide or abiraterone include gain-of-function mutations of AR, breakthrough of androgen synthesis, and expression of constitutively active AR splice variants 1519, 29–3229–in vivo decreased the growth of CRPC tumors concomitant with decreased serum levels and expression of PSA, a clinically relevant AR-regulated gene. Changes in serum PSA are the earliest indication of tumor response and precede any changes in clinical symptoms (B) show a profound lack of protein bands detected by an antibody to biotin with the exception of AR. Second, SINT1 inhibited AR transcriptional activity but had no inhibitory effect on structurally related proteins PR and GR. AR DBD and LBD have ∼80 and 50% sequence homology, respectively, with PR and GR. AR NTD has less than 15% sequence homology with steroid hormone receptors, thereby making it an attractive drug target. However, the small regions of AR NTD that are conserved with other steroid hormone receptors are amino acid residues 1–30, 153–169 (47% with PR), and 236–247 (75% with GR). These regions are important for androgen-dependent transcriptional activity. Region 1–30 contains core residue sequence 23FQNLF27 required for optimal orientation and interaction of AR NTD with AR LBD for AR antiparallel dimer formation in response to ligand  directly binds AF-1, 2) inhibited forskolin-induced transactivation of AR NTD, 3) attenuated transcriptional activities of both FL-AR and AR splice variant, 4) decreased FL-AR- and AR-V7-dependent proliferation of prostate cancer cells, and 5) symptoms . Specifisymptoms B show a o ligand . The con-workers  may not Here the ability of SINT1 to inhibit constitutively active AR splice variant was shown directly in a transcriptional assay with AR-V567es and an AR-driven reporter gene construct as well as indirectly by blocking the growth of LNCaP95 cells that are reported to be driven by AR-V7 . EndogenC and B and C, and contributed to the preparation of the figures and manuscript. I. T. performed and analyzed the experiments shown in Figs. 1A and 4A. A. H. T. performed and analyzed the experiments shown in Fig. 2C. D. P. C. did the quantitative RT-PCR work in 6F. N. R. M. performed and analyzed the experiments shown in A (right), and B, and 3A. Z. L. performed histochemistry staining shown in Fig. 6C. J. W. performed the animal experiments in Fig. 1B. Y. I. performed and analyzed the experiments shown in Fig. 3B. L. Y. synthesized YLP19, -30 and -31. J. G. W. critically reviewed the intellectual content of the paper. R. J. A. extracted and purified SINT1 and synthesized YLP19, -30, and -31 as well as EPI-053. M. D. S. conceived and coordinated the study, wrote the paper, analyzed results, and designed C. A. B. performed and analyzed the experiments shown in Figs. 1


Diffusa Cyclotide-3 (DC3), isolated from Hedyotisdiffusa, has been experimentally demonstrated to inhibit the survival and growth of LNCap cells, which typically express T877A-mutated AR, the most frequently detected point mutation of AR in castration-resistant prostate cancer (CRPC). But the interaction mechanism between DC3 and AR is not clear. Here in this study we aim to explore the possible binding mode of DC3 to T877A-mutated AR from molecular perspective. Firstly, homology modeling was employed to construct the three-dimensional structure of the cyclopeptide DC3 using 2kux.1.A as the template. Then molecular docking, molecular dynamics (MD) simulations, and molecular mechanics/generalized Born surface area (MM-GBSA) methods were performed to determine the bind site and explore the detailed interaction mechanism of DC3-AR complex. The obtained results suggested that the site formed by H11, loop888-893, and H12 (site 2) was the most possible position of DC3 binding to AR. Besides, hydrogen bonds, hydrophobic, and electrostatic interactions play dominant roles in the recognition and combination of DC3-AR complex. The essential residues dominant in each interaction were specifically revealed. This work facilitates our understanding of the interaction mechanism of DC3 binding to AR at the molecular level and contributes to the rational cyclopeptide drug design for prostate cancer.Androgen receptor (AR) is a key target in the discovery of anti-PCa (Prostate Cancer) drugs. Recently, a novel cyclopeptide  Prostate cancer (PCa) has become the second frequently diagnosed cancer in men throughout the world  , a member of steroid hormone group of nuclear receptor superfamily, plays an essential role in the development and proliferation of prostate cancer  analogs] means  is a novel cyclopeptide isolated from the traditional Chinese Medicine (TCM) Hedyotisdiffusa  simulations and molecular mechanics/generalized Born surface area (MM/GBSA) methods and various MD trajectory analysis methods were combined to explore the most possible binding site of DC3 to AR, investigate the key residues dominant in the binding process, and elucidate the detailed interaction mechanism. The results are expected to reveal the interaction mechanism of DC3-AR complex, promote the development of DC3 and correlative cyclopeptide AR antagonist, which will contribute to the rational drug design for prostate cancer.Homology modeling is a common technique to construct three-dimensional structure from amino acid sequence using homologous proteins with known structure as templates . The missing loop regions were refined by Discovery Studio 2.5. , the latter 700 ps were carried out without any restraint. Minimization and heat, as well as equilibration were executed in the Sander program. Finally, a 150 ns production of MD simulations of each system was performed in the PMEMD program at 310.0 K, 1 atm in the NPT ensemble without any restraint. The Langevin thermostat was used to control the temperature and the Berendsen barostat was used for constant pressure simulation. The time step was set as 2fs, and the coordinates of trajectories were recorded every 2 ps. During this simulation, the SHAKE algorithm  simulations were operated through Amber12 package  and solvent-phase (ΔGsol). –TΔS represents entropy contribution. Entropic calculation is time-consuming, and its value will fluctuate if a small quantity of snapshots were adopted  from bonds, angles, and torsions, van der Waals (ΔEvdw) and electrostatic energies (ΔEele) as follow:where ΔH represents enthalpy contribution, which is composed of enthalpy changes in gas-phase , electrostatic contribution (ΔEele), polar solvation contribution (ΔGGB), and nonpolar solvation contribution (ΔGSA):To further explore the detailed interaction information of DC3-AR complex, free energy decomposition was performed by using MM-GBSA method to identify the key residues responsible for binding energy. The contribution of each residue was calculated without considering the contribution of entropies. The contribution is defined as the sum of van der Waals contribution  analysis of the Cα atoms during the last 50 ns of the first parallel trajectory was performed to explore the correlated motion between residues of DC3-AR complex. The cross correlation matrix Ci and ΔRj represent displacements of atom i and j, respectively. The value of Cij fluctuated from −1 to 1, the positive value indicates a correlated motion between the residue i and residue j, while negative values indicates an anti-correlated motion.Where ΔRhttp://www.cgl.ucsf.edu/chimera).Clustering analysis was conducted by using the MMTSB toolset in Amber 12 to determine the representative structure of DC3-AR complex during the last 50 ns MD simulations. Firstly, the similar conformations of DC3-AR complex generated from the trajectory were classified into one cluster, and the most populated cluster has maximum number of conformations. Centroids of the generated clusters were then calculated and generated. Subsequently, RMSD of each structure was calculated with respect to specific centroid. Ultimately, the structure with lowest RMSD to cluster centroid from the most populated cluster was defined as the representative structure of DC3-AR. After that, the representative structure was adopted to generate the hydrophobic and electrostatic interaction surface of DC3-AR complex by using UCSF Chimera package   values of DC3-AR complexes backbone atoms were calculated relative to the initial structures to monitor the stability and overall convergence of each system during the simulation process. As shown in Figure The root mean square fluctuation (RMSF) reveals the fluctuation of certain residues during simulation process around its average position, which is also a tool to assess the dynamics stability of system. Here, RMSF values of Cα atoms in the last 50 ns were calculated by employing the first parallel trajectory of each system. RMSF of DC3 residues were shown in Figure ele) in system 2  can be found to make a great contribution to the low binding free energy of the whole system, which reflects that significant electrostatic interactions may exist between DC3-AR complex and contribute greatly to the system stability.In order to explore the interaction energetic features of DC3-AR complexes, MM-GBSA method was employed to calculate the binding free energies of each system. The average binding free energies and detailed energetic contribution components of the last 50 ns of parallel trajectories were calculated and shown in Table The dynamic cross-correlation matrices (DCCM) analysis was further analyzed Figure  to invesThe stronger cross-correlation between DC3 and AR residues found in system 2 might also due to the formation of hydrogen bonds during MD simulations. Hydrogen bonds, as critical indicators of nonbonding interactions, play vital roles in the protein-ligand recognition process  is the most possible site of DC3 binding to AR complex.To fully explore the binding modes and interaction mechanisms of DC3 and AR, system 2 was further studied to reveal the complicated binding mechanism of DC3-AR complex.According to the RMSF values of DC3 residues and AR residues shown in Figures The representative conformation of DC3-AR complex during MD simulation was extracted by clustering analysis. The first parallel trajectory of system 2 was grouped into 4 clusters based on the conformational similarity. The most populated cluster contained14068 frames, which accounted for 56.27% of all frames extracted from the last 50 ns MD simulation. Then the conformation with least RMSD value in most populated cluster was defined as the representative structure. Here, the representative structure of DC3-AR complex extracted from MD simulations trajectory and the initial structure acquired from molecular docking were plotted in Figures To figure out the binding mechanism between the key residues in the binding process of DC3-AR complex, hydrophobic and electrostatic interaction surfaces of representative structure were generated. The hydrophobic interaction surface was plotted in Figure The electrostatic interaction surface was shown in Figure To further validate the interaction formed between key residues and investigate the formation process, the distances of key residue pairs mentioned above vs. simulation time were calculated and plotted in Figure To characterize and intuitively exhibit the underlying dynamical cross-correlations among different parts of DC3-AR complex, the overall cross-correlation networks of system 2 and apo-AR system were constructed. As shown in Figure In this work, the three-dimensional structure of cyclopeptide DC3 was firstly constructed by homology modeling technology using 2kux.1.A as template. Then molecular docking was carried out to predict possible binding site and preferred orientation of DC3 into AR. Finally, four systems with best docking score from top four clusters were selected to perform 150 ns all-atom molecular dynamics (MD) simulations. The MM/GBSA method and a series of MD trajectory analyses were subsequently conducted. The analyses of RMSF, binding free energy, DCCM and hydrogen bonds indicated that DC3 showed a higher binding affinity to AR in site 2  and this system showed obvious superiority in stability comparing to other systems. Besides, much more intermolecular hydrogen bonds were constantly formed in system 2 with high occupation. Stronger cross-correlation among DC3 residues and stronger anti-correlation between DC3 and AR residues also exited here. These results suggest that DC3 is most likely to bind to AR in site 2 encompassed by H10, H11, loop888-893, and H12. Subsequently, combining further analysis of free-energy decomposition, interaction surface, distance, and cross-correlation network, it can be observed that hydrogen bonds, hydrophobic, and electrostatic interactions play dominant roles in the recognition and combination of DC3-AR complex. For hydrogen bonds, it frequently existed between K669-H885, D890-R666, S900-T647, and D890-E643. Besides, K665, R666 of DC3, and E706, E709, D890, E893, E897 of AR made great contributions to electrostatic interaction values. V649, V650, V651-V801, and L654, V657, L658-L880, L881 play essential parts of hydrophobic interaction. These results elucidated the detail interaction mechanism of DC3-AR complex and the key residues dominated in specific interaction. These findings will significantly facilitate our understanding of action mode of DC3 to AR at the molecular level, and contribute to the future rational cyclopeptide drug design for prostate cancer.JL: conceived and coordinated the study; HZ and TS: did the homology modeling, molecular docking, molecular dynamics simulations, and they wrote the paper; YY and CF: helped to do molecular simulations. All authors analyzed the results and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.