The inhibition of the androgen receptor (AR) is an established strategy in prostate cancer (PCa) treatment until drug resistance develops either through mutations in the ligand-binding domain (LBD) portion of the receptor or its deletion. We previously identified a druggable pocket on the DNA binding domain (DBD) dimerization surface of the AR and reported several potent inhibitors that effectively disrupted DBD-DBD interactions and consequently demonstrated certain antineoplastic activity. Here we describe further development of small molecule inhibitors of AR DBD dimerization and provide their broad biological characterization. The developed compounds demonstrate improved activity in the mammalian two-hybrid assay, enhanced inhibition of AR-V7 transcriptional activity, and improved microsomal stability. These findings position us for the development of AR inhibitors with entirely novel mechanisms of action that would bypass most forms of PCa treatment resistance, including the truncation of the LBD of the AR. Prostate cancer (PCa) is the most prevalent malignancy in men and the second most common cause of cancer-related death in Canada and the United States (US) among males . The andThe development of more recent antiandrogen drugs, such as enzalutamide and abiraterone, that competitively bind the ligand-binding domain (LBD) of AR helped decrease PCa mortality and morbidity significantly ,11,12,13In previous studies ,19,20, wIn a previous study , we idenHerein, we report the newly discovered series of chemically diverse small molecules that were characterized in numerous AR-relevant assays and shown to abrogate DBD dimerization effectively. In particular, two lead compounds, VPC-17160 and VPC-17281, demonstrated stronger inhibition of AR-V7 transcriptional activity and exhibited superior activity in mammalian two-hybrid assays compared to our previously identified lead (VPC-17005). Furthermore, compound VPC-17281 appeared to have significantly improved microsomal stability, which is critical for future hit-to-lead optimization. Overall, these findings provide insights on the inhibition of DBD dimerization and provide a foundation for further development of AR-targeting drugs that bypass most of the known mechanisms of PCa drug resistance.1/2 = 14 min). We utilized the structure of rat AR DBD dimer bound to the DR3 oligonucleotide (PDB (Protein Data Bank) code: 1R4I) to dock drug-like compounds from the ZINC15 database into the previously identified binding site [Our earlier lead VPC-17005 demonstrated good AR inhibition but sub-optimal pharmacokinetic properties with low microsomal stability .Compounds were first screened for their inhibition of AR-V7 transcriptional activity. To do so, we created a PC3 cell-based population that stably encodes (i) a doxycycline-inducible AR-V7 cDNA sequence , and (ii) the ARR3tk-nanoLuciferase reporter construct. Hence, nanoluciferase production was directly correlated with AR-V7 activity. This allowed the screening of the compounds without using the error-prone method of transiently transfecting V7 and reporter constructs. The inhibition efficacy of compounds was measured using doxycycline treatment along with an increasing dose of compounds. From this screen, a panel of compounds was identified with some anti-dimerization activity . Two hitompounds A. This d50 of 2 and 5 μM, respectively  AR transcriptional assay, where the expression of eGFP is under the direct control of an androgen-responsive probasin-derived promoter . Compounectively .50 values established at 2 μM and 6 μM, respectively  in PCa cell lines. PSA is an AR-regulated serine protease and is widely used as a biomarker for PCa . As expeectively C.50 of the compound in transcription assays. In contrast, parental compound VPC-17005 was displacing 75% of the androgen at 10 μM, suggesting that this compound was having some of its efficacy through the ligand-binding site. This would explain why VPC-17160 and VPC-17281 are more potent inhibitors than VPC-17005 in the AR-V7 transcription assay.To validate that this activity occurred via anti-homodimer inhibition rather than androgen displacement at the LBD, we measured the ability of VPC-17160 and VPC-17281 to displace a fluorescent-tagged androgenic ligand (FluormoneAL Green) from the AR-LBD. Even at high concentrations (50 μM), no significant hormone displacement was observed for compound VPC-17281 . We obseNext, the dimerization of full-length AR was assessed by a mammalian two-hybrid  assay, 50 of 25 μM in the LNCaP cell line. Interestingly, compound VPC-17281 caused a significant reduction in the growth of 22rv1 cells  and 22rv1 (AR-V7 dependent). As anticipated, all compounds effectively inhibited the growth of the AR-dependent LNCaP cell line. Compound VPC-17281 showed an IC1/2 > 90 min) compared to VPC-17005 (T1/2 = 14 min), whereas compound VPC-17160 showed poor stability with a half-life of 3 min. This could explain the lower potency of this compound in cell growth assays, as it is metabolized quickly in the media.We then tested the metabolic stability of these molecules using human microsomes. Additionally, the measured half-life in microsomes increased by approximately six-fold for compound VPC-17281  of the AR. Unfortunately, a large number of patients develop resistance to androgen displacement treatment. Thus, the development of drugs that target a different site on the surface of AR (such as the dimerization site) remains an attractive strategy to combat drug resistance.Previously we reported VPC-17005, a small molecule inhibitor that targets the dimerization interface on the AR DBD that demonstrated promising AR inhibition in androgen-sensitive LNCaP and enzalutamide-resistant MR49F cells in vitro . HoweverFurthermore, both lead compounds VPC-17160 and VPC-17281 inhibited the growth of 22rv1 and LNCaP cancer cell lines, proving the antineoplastic effects of the dimer inhibition. Importantly, the profound activity of VPC-17281 on the AR-V7 dependent 22rv1 cell line provides additional assurance as to the effectiveness of our lead inhibitor and its applicability for targeting castration-resistant prostate cancer cells. However, it is important to mention that VPC-17281 also demonstrated some activity in the PC3 cell line, which suggests off-target effects and possible toxicity. In contrast, VPC-17160 showed no effect on the PC3 cell lines suggesting its selectivity to AR and could represent the best starting template for further drug optimization.It should also be noted that microsomal stability represents one of the key pharmacokinetic properties of a candidate drug since a low microsomal half-life usually indicates poor bioavailability. VPC-17281 showed a greatly improved microsomal stability, while VPC-17160 was readily metabolized in microsomes. These results indicate that further optimization of the compounds is necessary to overcome the issues of toxicity and low microsomal stability.The crystal structure of rat AR DBD was downloaded from PDB database (1R4I) and prepared with Protein Preparation Wizard from Schrödinger ,32. The https://enamine.net/, Accessed 1 March 2021).All compounds were identified from the ZINC15 database and purchased from Enamine (Full-length human AR (hARWT) was encoded on pcDNA3.1 expression plasmid. Lentivirus plasmid pLIX402-ARV7  was a gift from Dr. N. Lack. pLIX402-Nanoluc was constructed by excising the ARV7 cDNA from pLIX402-ARV7  and ligating Nanoluciferase (Nanoluc or NLuc) cDNA amplified from pNL1.1(CMV) plasmid. Lentivirus plasmid encoding the AR-responsive reporter was created by replacing the Ubiquitin C promoter in pLB-U  with theLNCaP , 22rv1 , and PC3  cells were obtained in 2013 and authenticated by IDEXX Laboratories in 2014 every 6 months and tested for mycoplasma contamination every two weeks.Androgen displacement was assessed with the Polar Screen Androgen Receptor Competitor Green Assay Kit as per the instructions of the manufacturer.LNCaP cells incorporating an AR2PB-eGFP reporter construct (2 × probasin) are previously described . eGFP anwww.graphpad.com (accessed 1 March 2021).All statistical analysis and dose–response curves were generated using GraphPad Prism 6.07 software , In the current study, we discovered novel inhibitors of AR DBD dimerization through the synergetic application of virtual screening methods and experimental validation. The study was built upon a previously reported AR DBD inhibitor VPC-17005 that suffered from sub-optimal biostability and pharmacokinetics. The newly developed lead compounds, VPC-17281 and VPC-17160, belong to different chemotypes and exhibit significantly improved microsomal stability and anti-AR activity. This study broadens the repertoire of compounds targeting AR DBD dimerization and could provide useful insights for the further development of novel antiandrogens with unique and novel mechanisms of action. These drugs would be of immense value for CRPC patients that do not respond to conventional AR inhibitors.


Renilla luciferase reporter gene, that is constitutively expressed under the control of an internal ribosome entry site (IRES) and is not regulated by steroid hormones. The corresponding SHR (wildtype or mutant/variant) is also expressed from the same construct. Using this improved reporter system, we revealed a large spectrum of transactivation activities within a set of previously identified mutations and variations of the androgen receptor (AR), the estrogen receptor α (ERα) and the glucocorticoid receptor (GR). This novel reporter system enables functional analysis of SHR mutants and variants in physiological and pathological settings, offering valuable preclinical, or diagnostic information for the understanding and treatment of associated diseases.Although the rapid development of high-throughput sequencing has led to the identification of a large number of truncated or mutated steroid hormone receptor (SHR) variants, their clinical relevance remains to be defined. A platform for functional analysis of these SHR variants in cells would be instrumental for better assessing their impact on normal physiology and SHR-associated diseases. Here we have developed a new reporter system that allows rapid and accurate assessment of the transcriptional activity of SHR variants in cells. The reporter is a single construct containing a firefly luciferase reporter gene, whose expression is under the control of a promoter with multiple steroid hormone responsive elements, and a  Many SHR variants have been identified in both common and rare diseases –3. All SRenilla luciferase reporter gene under the control of an internal ribosome entry site (IRES), an SHR-expressing cassette, and a firefly luciferase reporter gene driven by a promoter that is regulated by the corresponding SHR. Using these improved reporter systems, we undertook a comprehensive survey of a large number of AR, ERα and GR variants that were previously identified in clinical or preclinical studies. Our results reveal distinct transcriptional activities of these variants, providing insights into their roles in the pathogenesis of associated diseases.Luciferase reporter assays have been used for studying many cellular processes including intracellular signaling, gene expression, receptor activity, transcription factors, mRNA processing, and protein folding. Dual luciferase reporter systems have been used in the past to assay SHR activity in gene regulation with improved accuracy by using one luciferase as an internal control. These systems usually involve co-transfection of three expression vectors  into a cell line, which could introduce additional variables and thus impact assay accuracy. In this study we engineered multiple single-vectored, dual-luciferase reporters that can rapidly and accurately measure the activity of individual SHRs in cells. These reporters contain a constitutively expressed 2. Huh-7, COS-7, Hela, HEK293T, and HepG2 cells were cultured in phenol DMEM(H) medium  supplemented with 10% FBS (Gibco) at 37°C in a humidified atmosphere with 5% CO2.The Huh-7 and COS-7 cells were obtained from Peking Union Medical College . PC3 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco) in a humidified atmosphere with 5% COPSA61-Luc, pcDNA3.1(+)-AR and pSG5-hERα were kindly provided by Jörg Klug, JLU Giessen, Germany. 4×ARE-Luc and 2×GRE-Luc plasmids were kindly provided by Prof. Michael Carey at UCLA and Iain J. McEwan at University of Aberdeen, respectively. The ARE-I-II-III-Luc vector was generated by replacing the PSA61 region in PSA61-Luc (between the BamHI and EcoRI sites) with an ARE-I-II-III fragment (amplified through overlapping PCR).Renilla luciferase reporter gene; (d) the full length wild type AR fragment was then inserted upstream of the IRES sequence in pcDNA3.1(-)-4×ARE-Fluc-Rluc between the XhoI and XbaI sites.The pcDNA3.1(-)-4×ARE-Fluc-AR-Rluc was generated through the following steps: (a) the CMV promoter in pcDNA3.1(-) vector was replaced with the 4×ARE and minimal promoter, amplified from the 4×ARE-Luc plasmid; (b) the firefly luciferase gene was inserted into the pcDNA3.1(-)-4xARE vector downstream of the minimal promoter; (c) the neomycin gene in the pcDNA3.1(-)-4×ARE-Fluc was replaced with IRES and the To generate pcDNA3.1-4×ARE-Fluc-AR-Vs-Rluc constructs, various AR variant sequences (AR-Vs) were amplified by PCR from pcDNA3.1(+)-AR based on previous studies –10, whicTo generate pcDNA3.1-4×ERE-Fluc-ERα-Vs-Rluc plasmids, a 4xERE promoter sequence containing four copies of ERE was synthesized by Qingke Biotech  a. ERα wildtype cDNA was amplified from pSG-hERα and used to replace the AR cDNA in pcDNA3.1(-)-4×ARE-Fluc-AR-Rluc. ERα variant and mutant sequences were generated by overlapping PCR and used to replace ERα-wt cDNA in the vector to generate pcDNA3.1-4×ERE-Fluc-ERα-Vs-Rluc expression constructs.To generate pcDNA3.1(-)-4×GRE-Fluc-GR-Vs-Rluc, the 4xGRE promoter sequence was amplified from the 2×GRE-Luc vector by overlapping PCR, and the wildtype GR cDNA was amplified from cDNA isolated from HeLa cell, as described . 4×GRE aSequences of all the primers used to create AR-wt, ERα-wt, and GR-wt cDNA, as well as their corresponding variants and mutations, are described in Renilla luciferase. Transfection efficiency was normalized by Renilla luminescence signals. The results are presented as the mean ± SD of triplicate samples.HEK293T cells were seeded in 6-well plates and cultured in complete medium containing 10% charcoal-stripped fetal bovine serum  for 24h. Subsequently, they were transfected with different constructs using the Lipofectamine 3000 transfection reagent  according to the manufacturer's protocol. Six hours after transfection, the medium was replaced with fresh charcoal-stripped medium containing either corresponding hormones , or DMSO solvent . After 48 h of incubation, cells were then washed, lysed, and harvested. Dual-Luciferase Reporter Assay  was then performed in duplicates to measure the activities of firefly luciferase and t-test and P < 0.05 is considered significant. Data are presented as mean ± SD. Analysis was conducted using Prism v6.0c (GraphPad Software).All reported values represent the average of triplicates. Statistical analysis of differences between samples was performed using two-tailed student's In general, activation of receptors requires not only ligands binding but also recruitment of various coactivators to the transcriptional complex. Thus, an appropriate cellular context and a sensitive and effective promoter are important factors for consideration for assessing AR transcriptional activity. After surveying androgen regulation of PSA61-Luc in six AR-negative cell lines, we found the promoter displayed the highest fold stimulation activity (16.6-fold change) in HEK293T compared to other cell lines . WhereasIn order to assess the effects of AR alterations in low androgen concentrations in castration environments, we constructed pcDNA3.1-4×ARE-Fluc-AR-wt-Rluc reporter  and tranAfter surveying six different cell lines and three promoters displaying different efficiency of activation, we chose HEK293T and 4×ARE-Luc to compare the transcriptional activity of AR-wt and its mutants or variants , S2A,B. As shown in Among the 22 AR variants examined, 13 displayed a complete loss of function in the presence or in the absence of R1881. Some of them, including AR-V1, AR-V2, AR23, AR45, and AR8, have been characterized before , 25, 26.AR mutations are rare in the early stages of untreated PC in patients, but occur much more frequently in castration-resistant prostate cancer (CRPC) . Gain-ofSince discovered in early 1990s, many naturally occurring splice variants of ERα with different coding sequences have been identified in cell lines and clinical samples , 31. To In addition to splicing variants, we also examined the transcriptional activity of ERα mutants which have been identified in 10 to 54.5% metastatic, ERα-positive breast cancers , 19, 34.Previous structural studies indicated that Y537S and D538G mutations cause increased interaction of ERα with its coactivators such as SRC-1, due to the shift of helix 12 to a conformation similar to that of estrogen-bound ERα-wt -4×GRE-Fluc-GR-Rluc reporters expressing a serial of GR mutants and splice variants . The 10 Increased expression of GRβ (which does not contain helix 12 of LBD but has a different amino acid sequence in helix 11 compared to GR-wt) was observed in hormone-insensitive autoimmune diseases , blood sThe mRNA of GR-P variant accounts for 10–20% of total GR RNA in normal cells, which is much higher than GR-β. Certain blood cancer  and solid tumors  have increased GR-P, up to 50% of total GR-mRNA. However, it exhibited virtually no transactivation activity in our assay.GR-A, which lost exons 5–7 during alternative splicing, lacks trans-acting regions and nuclear localization regions. Consistently, the observed transactivation activity of GR-A was minimal. The increase of GR-A expression may contribute to glucocorticoid insensitivity.GR-γ, which is associated with hormone insensitivity in leukemia and non-small cell lung cancer , containIn addition to splicing variants, we also examined the transcriptional activity of multiple translation variants of GR. Expression of these translational variants is tissue- and cell- specific and might be one of the mechanisms underlying their different varying sensitivity. These translational variants might also contribute to glucocorticoid-related side effects. For instance, higher levels of translational variants C2 and C3 in some patients may result in higher glucocorticoids sensitivity and more frequent side effects such as glucocorticoid-induced osteoporosis. GR-C1, GR-D1, GR-D2, and GR-D3 exhibited clear loss of function compared with GR-wt in the presence of DEX. Both GR-C2 and GR-C3 had slightly higher transcriptional transactivation than GR-wt when treated with 10 nM DEX, consistent with a previous report . The traPrimary glucocorticoid resistance due to mutations in the GR gene was found in 23 cases, most of which were single nucleotide mutations , 44, insWe also explored the relationship between two common SNPs and glucocorticoid sensitivity. N363S had been reported to be associated with increased glucocorticoid sensitivity, decreased bone density , and freRenilla reporter gene downstream SHR mRNA under the control of IRES for expression normalization ensures that the firefly luciferase reporter activity accurately reflects the transcriptional activity of SHRs. Our reporter systems also provide a platform for direct assessing and comparing different SHR mutants and variants in their transcriptional activity, which allows for the linking of specific SHR alterations with clinical phenotypes and treatment outcome. Furthermore, our reporter systems are versatile and can be adapted to report the activity of other hormone receptors such as mineralocorticoid receptor (MR), progesterone receptor (PR), and thyroid hormone receptor (THR), by replacing the hormone responsive promoter in the pcDNA3.1(-)-4×ARE-Fluc-AR-Rluc vector with mineralocorticoid receptor elements (MREs), progesterone receptor elements (PREs), or thyroid hormone receptor elements (THRs), and replacing the AR gene with MR, PR, or THR.The frequent alterations of AR, ERα, and GR genes are clinically important due to their specific functional differences . The estOur sensitive assay systems have revealed functional alterations in AR, GR or ERα caused by genetic mutations or splicing variations, with many results being consistent with previous studies , 32, 43.The datasets generated for this study are available on request to the corresponding author.FX, ML, and LW conceived the study and designed the experiments. HJ, YL, MT, ZL, and LZ performed the experiments. FS, YZ, JZ, and HL made clinical validation. LL, BA, and JM collected clinical interpterion. HJ and FX wrote the manuscript. All authors read and approved the final 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.


A) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A2B adenosine receptor (A2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABA We chose indole as a privileged scaffold owing to its recognized ability of being exploited to obtain drug-like molecules flunitrazepam) from neuronal membranes obtained by the cortex of bovine brain. In the light of the knowledge about the GABAA isoforms acquired several years after our initial studies, such binding data correlated mainly with the α1 BzR subtype.At that time, the endpoint in our biological experiments was the ability of the tested compound to displace a radioligand aminoacids of series 1. Expectedly, the esters performed better than the corresponding acids. The two compounds provided with the highest affinity as racemic mixtures in the previous work flumazenil from recombinant rat α1, α3 and α5 BzR subtypes. All of them displayed high affinity for α1β3γ2 receptors and moderate to good selectivity for α3 and α5 subtypes. In series N-(heteroarylmethyl)indol-3-ylglyoxylamides 16 were subsequently investigated flumazenil from recombinant rat α1, α2 and α5 GABAA/BzR pure isoforms. All of them showed binding selectivity for the α1 subtype over the α2 and α5 subtypes.Four of the most potent compounds of series 6 by inserting lipophilic substituents at the 4′-position of the side phenyl ring, some of which were characterized by considerable steric bulk 3 and C≡C-CH2Si(CH3)3) methyl-2-(4′-nitrophenyl)indol-3-yl) glyoxylamide (18e) as a high affinity radiolabelled probe of TSPO PK11195. Our experiments showed a direct correlation between the efficacy of TSPO ligands and their RT values -PK11195 and expressed as Ki values. The strategy resulted successful as all the new compounds revealed to disrupt the p53-MDM2 complex and bind to TSPO at nanomolar concentrations.The tumour suppressor protein p53 promotes apoptosis by interacting with members of the protective Bcl2-family proteins which in turn mediate the release of cytochrome c. P53 is a transcription factor that controls cellular response to stress by inducing cell cycle arrest or apoptosis ,91. The l esters ,94. An i20 2, R3 = CH3), showing the highest ability to dissociate the p53-MDM2 complex (IC50 = 4.3 nM) and the highest affinity for TSPO (Ki = 87 nM), was selected for further biological studies, giving the following results: (i) reactivation of the p53 function and inhibition of the GBM cell growth, triggering subsequent apoptosis; (ii) no efficacy on a GBM cell line expressing mutant p53, supporting the involvement of this protein in the observed effect; (iii) reduction of viability of glioma cancer stem cells (CSCs), which are less sensitive to anticancer agents and responsible for GBM recurrence benzimidazoles  as geometrically constrained analogues of indole derivatives (23) triazino [4,3-ves (23) ,104.6H5, C6H4-p-OCH3, 2-furyl, 2-thienyl) displayed Ki values at the BzR  ranging from 13 nM to 56 nM. The most potent TBIs  [25) identified as the first non-xantine antagonist of the A1, A2A and A3 ARs [It is worth noting that the structures of certain BzR ligands are similar to those of several antagonists of the A895 (24)  with thed A3 ARs .26) purposely designed as potential A1 AR antagonists through insertion of substituents not only at position 3 (R) but also at position 10 (R’) of the tricyclic system [In the light of the above consideration, we prepared some novel TBIs (c system .i 83 nM) and selective one at the human A1 AR had R = R’ = phenyl [Among the new TBI derivatives, the most potent (K= phenyl .26a provided with high potency (IC50 of 3 nM) and selectivity for the human A2B AR [In 2012 we disclosed a TBI derivative n A2B AR . For a ln A2B AR .2B AR is remarkable. Particularly, selective agonists of this receptor have been reported to reduce inflammation after ventilator-induced lung injury [2B AR antagonists have been regarded as candidates for the treatment of cancer [The therapeutic potential of agonists and antagonists of the Ag injury  and to mg injury . Selectif cancer ,114, colf cancer ,116 and f cancer ,118,119.27a–c, 28a,b featuring a diketo moiety as a linker designed as open chain analogues of the TBI 26a. Additionally, we purchased two indole derivatives 29a,b characterized by an amide linker [Continuing our searches of novel lead compounds binding to ARs, we synthesized five indole derivatives e linker .27–29 for the human A1, A2A, and A3 AR expressed in CHO cells was determined by measuring their ability to displace specific radioligands from the above receptors. Compounds 27a and 27b exhibited submicromolar affinities for the A1 AR , whereas the remaining five indoles showed no appreciable affinity for the three AR subtypes. Functional experiments showed that 27a and 27b behaved at the A1 AR as antagonists. The affinity of indoles 27–29 and the human A2B AR, we measured to what extent our compounds modified the levels of cAMP in CHO expressing only this receptor. None of the compounds increased the cAMP levels at the concentration of 10 μM, clearly showing a lack of A2B AR agonist activity. However, when the experiments were repeated in the presence of 5′-(N-ethylcarboxamido)adenosine (NECA), which acts as unspecific agonist of the ARs, 27a and 28a,b potentiated its agonist effects, suggesting that these three compounds interact with the A2B AR as positive modulators. Conversely, 27b,c and 29a,b potently counteracted the NECA-mediated increase in cAMP, indicating that they act as negative modulators of the A2B AR. To evaluate the pharmacological effect resulting from interaction between 27–29 are so far the only A2B AR allosteric modulators reported in literature.We were very satisfied by these unexpected preliminary results  as our indole derivatives 2B AR was characterized in more detail [27–29 in modulating the activity of A2B AR agonists were determined by assessing the effects of different concentrations of each of them on cAMP accumulation induced by an EC50 concentration of NECA (100 nM). The resulting concentration-response curves indicated that 27a and 28a,b exhibit similar submicromolar potencies at A2B AR, with EC50 values between 250 nM and 446 nM.In a subsequent paper, the pharmacology of the new indole derivatives at the Ae detail . The pot27b and 29a,b, obtained under the same conditions, fitted a two-site equation model, suggesting that these compounds recognize two sites of the A2B AR with different affinities. The potency values obtained for the high and low affinity states of the receptor were in the subnanomolar/nanomolar and micromolar range, respectively. Conversely, the concentration-response curve of 27c fitted a one-site equation model, revealing that this compound recognizes a unique site of the A2B AR with nanomolar affinity.The concentration-response curves of 27a and 28a,b enhance the efficacy of the agonist without affecting its potency, while 27b,c and 29a,b decrease either the efficacy and potency of the agonist. Several studies report that agonist efficacy and potency are not necessarily both modified by allosteric modulators [27a and 28a,b affects specific conformational states of A2B AR so as to improve the functional coupling to the intracellular signaling system without altering the conformation of the orthosteric site. Probably, 27b,c and 29a,b affects the agonist potency by shifting the receptor conformational states toward the resting ones; at the same time, they decrease the agonist affinity by deforming the conformation of the orthosteric site.Concentration-response curves in which the cAMP was measured by varying the concentration of the tested compound as well as the concentration of NECA gave us further information about their mechanisms of action. Particularly, from these curves we could infer that dulators ,123. A p2B AR in several physiological and pathological processes, discussed previously in this chapter, the positive and negative allosteric modulators of this receptor represent promising tools to identify novel druggable compounds.In virtue of their indirect mechanism of receptor modulation, allosteric modulators of G-protein-coupled receptors offer therapeutic advantages compared to agonists and antagonists. Particularly, they tune pharmacological responses only when and where the endogenous agonist is present in the specific tissue. Given the role played by A2B AR agonists in the treatment of bone-related diseases  [28b potentiates the effects of either adenosine and synthetic A2B AR agonists in mediating osteoblast differentiation in vitro. In detail, by treating the MCSs with 28b we observed an increase in the expression of osteoblast-related genes (runx2 and osterix) and osteoblast marker proteins  associated with a stimulation of osteoblast mineralization.Shortly after we reported the therapeutic potential of the indole derivatives acting as allosteric enhancers of Al-union) . Particu2−·), hydrogen peroxide (H2O2), hydroxyl radical (OH·). ROS are potentially cytotoxic as they damage DNA, RNA, enzymes and cellular membranes; they are generated from molecular oxygen during physiological processes  or pathological events . Very recently, we have published a paper  disclosiThe cells reduce oxidative stress through radical scavengers  and antioxidant enzymes, both inactivating the ROS. The antioxidant enzymes include superoxide dismutase, catalase, heme oxygenase-1, glutathione S-transferase, NADPH:quinone oxidoreductase 1 and transketolase ,129,130.The expression of the above enzymes is regulated by the so called Keap1-Nrf2-ARE system , whose mInhibitors of the Keap1-Nrf2 interaction (KNI) are considered a promising new class of anti-inflammatory agents to treat diseases involving chronic oxidative stress, such as diabetes, cancer and neurodegenerative disorders . The Keap1 binding cavity hosting the KNI inhibitors can be divided into six subpockets (P1-P6) . P1 and Most of the KNI inhibitors reported in literature are chemically heterogeneous small molecules featuring a planar or quasi-planar scaffold which bear at least one aromatic ring and/or a weak acidic group involved in the abovementioned electrostatic interactions . 30a–i, among which 30a–d were synthesized, whereas 30e–i were purchased.Our experience with indole as scaffold, led us to believe that it would be feasible to design indole derivatives acting as KNI inhibitors. The design of the compounds to be tested was mainly guided by the 3D structures of some KNI inhibitors co-crystallized with Keap1 ,136,137.30a–d); (b) acidic ; ampholytic (30g–i). The presence of methoxy group(s) or a methylendioxy moiety in the structures of 30a–i was regarded as a chance for our compounds to strength potential cation-π interactions and/or engage H-bonds with the target protein. The thiophene ring featured by 30e–g confers conformational rigidity and represents an electron-rich ring potentially able to interact with arginine residues. Compounds 30a–i were evaluated for their ability to inhibit the Keap1-Nrf2 interaction through a cell-based luciferase reporter assay [30g was tested at the concentration of 5 μM owing to its limited solubility in phosphate buffer. t-Butylhydroxyquinone (t-BHQ) was employed as a positive control [Based on their acid-basic properties, these compounds can be divided into three groups: (a) non-ionizable . The remaining compounds displayed activities below 50%. A western blot analysis confirmed that 30e,f and 30g increase the expression of Nrf2 and of two enzymes encoded by its downstream target ARE genes, namely NADPH:quinone oxidoreductase 1 and transketolase. The same three best performing compounds showed to be non-cytotoxic when tested on human peripheral blood lymphocytes.Compounds 30g and Keap1, using available 3D structures of this protein [30g makes a salt bridge with the Arg483 protonated side chain and a charge-reinforced H-bond with the Ser508 hydroxy group; the thiophene ring establishes a cation-π interaction with the Arg415 positively charged side chain. Such a cation-π interaction cannot be established by the less active compounds 30h,i which bear a carboxylic group but lack an aromatic ring attached to the indole nitrogen. Furthermore, 30h,i are much more flexible than 30e,f and 30g.Docking simulations of the interaction between  protein , allowedm-methoxy)benzylaminomethyl substituent of 30g establishes hydrophobic interactions with Val512 and Leu472 side chains and a H-bond between the m-methoxy oxygen and the Leu472 backbone NH. The protonated nitrogen of 30g is not involved in any type of electrostatic interaction. This suggests that the higher activity of 30g with respect to those of 30e,f may be ascribed to the different length of the linker between the indole nucleus and the m-methoxy moiety. The indole ring of 30g contributes to the binding affinity through weak hydrophobic interactions with the Ala556 methyl group and the Arg415 dimethylene fragment.The (The works described in this review confirm how useful and versatile indole can be as a molecular scaffold in designing drug-like molecules.


Guess Who, which is a form of zero-sum symmetric game with perfect information. We discuss some preliminary strategic insights and formally derive an optimal strategy and win-probabilities for the game. We discuss the first-mover advantage in the game and other strategic aspects coming out of the optimal strategy. While the paper is based on the popular children’s game, our analysis generalises the actual game by allowing any initial game state with an arbitrarily large number of starting characters. With the aid of these mathematical results you can now comprehensively thrash your young children and be a terrible parent!Are you Richard? Are you Anne? We look at the strategic problem in the children’s guessing game  Guess Who which forms the basis for this article, either through having played the game during childhood, or having had children who have done so, or perhaps both. The game was designed by Theora Designs and was first manufactured by Milton Bradley in 1979. At the time of publication of this paper it is presently manufactured and distributed by Hasbro. The game has been redesigned several times and re-released in many forms. The structure of the game has remained the same since its initial design and has been a staple in toy stores for over forty years.Most readers will already be familiar with the children’s game Guess Who is a two-player guessing game teaching children rudimentary skills in logic. For readers unfamiliar with the game, we set out a brief explanation. Each player has an identical game-board showing the faces of twenty-four named characters. These characters have various different characteristics—some are men, some are women, some have facial hair, some are wearing hats, some have glasses, and so on.  Each player randomly selects a character from a set of cards that matches the characters on the game-boards. Each player has a different character from the game-board and keeps this character secret from the other player. Taking it in turns, each player asks a question about the other player’s character that can be answered with a simple yes or no—e.g., “Are you wearing glasses?”  After receiving a yes or no answer the player asking the question is then able to eliminate incompatible characters on his game-board by placing these face down, so that only the remaining face-up characters are possible characters that might be held by the other player. By guessing the characteristics of characters, player narrows down the search through this process of elimination. When one player is able to correctly guess the other player’s character as his question, he wins the game; specific variations in rules for the final guess are discussed soon.Guess Who it is their first exposure to the basic logical idea of eliminating a false hypothesis through contradictory information. As children progress in their understanding of the game they can also learn some basic probabilistic intuition which gives them a higher chance of winning the game. These features make the game a good introduction to logical reasoning. As an object of academic study, the game is useful because it is a simple discrete “search race” that bears resemblances to more complex problems in sports, industrial competition, and other competitive ventures. In this regard, the game has two interesting strategic aspects that occur in broader competitive problems: (1) the agents are able to choose between more “conservative” and more “risky” strategies in their moves; and (2) the agents are able to observe the progress of their competitor and adjust their own strategy accordingly.For many young children, Guess Who is a “search race” in which each player is searching for an unknown item which is known to be equiprobable from a finite group of objects. Search algorithms for binary search are studied extensively in computer science and have led to the use of the “split-in-half search” which operates by splitting the remaining objects evenly (or as close as possible if there are an odd number of objects) on each search iteration . . ThiGuess Who is advertised as being suitable for children aged six and up, but in the experience of the present author it is also suitable (and fun) for younger children—essentially any child who is old enough to ask and answer basic yes-no visual questions about faces. The game is useful in teaching young children rudimentary logic and efficient searching. Psychological research has analysed the types of questions asked by people of different age groups in this kind of search game and in similar search games .  [et al.  find thaGuess Who with a game-board that has an arbitrary number of characters. We will look at the strategy of this game starting from any given game-state, which is a specification of the number of remaining characters on the game-board of each player. We assume that the only information available to each player is the state of their own game-board and the series of questions asked during the game. This is sufficient to allow each player to determine the state of the opponent’s game board other than determining which character they have eliminated due to holding that character. Each player is aware of the game-state at each point and their decision is determined entirely by this game-state. To be clear, we assume that there is no sneaky business insofar as using other information from the opponent’s behaviour or anything else which would give a strategic advantage from matters outside of the game. This assumption excludes strategies that infer opponent’s likely actions from exogenous behavioural clues.In this paper we will consider a generalised variant of how many characters to test with his guess.Each player is able to ask questions that will effectively seek to confirm or eliminate a given subset of the characters remaining on his game-board. Although it is common for questions to refer to characteristics like hair colour, facial hair, etc., it is always possible to refer to any chosen subset by specification of the logical disjunction of the characters by name—e.g., “Are you Max or Claire or Anne or Tom or … or John?” It is also possible to ask a simple ranking question to refer to a subset of any chosen size—e.g., “Does your name come before the word ‘Milk’ in the alphabet?”  Under such an approach the names and characteristics of the characters are arbitrary, and confer no useful information for the strategic decision. The strategic choice for a player is to decide n, m) where he has s<n be the integer number of characters the player chooses as a subset to confirm or eliminate with his guess, disallowing the empty or full sets. It is notable that there is a partial symmetry to our problem—if the player chooses a subset of s>1 characters, this is equivalent to choosing the other subset of n−s characters.Suppose that a player is in the game-state (https://www.hasbro.com/common/instruct/GuessWho.PDF). Correspondingly, the player does not win the game merely by narrowing down to a single character until they make a “final guess” of this character. We will denote this “final guess” as the move s = 0, and we note that this move is distinct from the move s = 1, where the player also attempts to narrow down to a single character, but does not make this character his “final guess” (and therefore does not win or lose at the end of the move). We will also examine a rules variant where a player can guess a single character to win, but does not lose if they guess incorrectly.  In this latter case we take the move s = 1 to be a winning move if it succeeds in narrowing the characters to a single remaining character . In this variant the move s = 1 is not equivalent to s = n−1 since the former guess allows the player to win on the present turn while the latter does not.) These two rules variations are summarized in Our analysis will examine two variants of the game, using different rules for the “final guess” to win the game. The official game rules provide that a player may use their move to make a guess of the opponent’s character—if the guess is correct they win the game but if the guess is wrong they lose the game  with We consider the case where there is a fixed positive payoff for winning and an opposing loss for losing leading to a zero-sum game (we refer to this particular type as a “win-loss” game). Under this system of payoffs, each player seeks to maximise the probability of winning the game, and adopts a strategy with a view to this optimisation criterion. Since the probability of winning depends on the strategy of the other player, this is a game-theory problem. Ours is a two-person win-loss game with perfect information, which is presented in extensive form. Our generalised problem is a specification of the set of games corresponding to every possible starting game-state . In parGuess Who allows any game-state  with s1 and the other player (the “waiting player”) adopts strategy s2. For brevity we let s≡s1 denote the move chosen by the moving player in the current game state. The game rules provide that each character is selected at random; we take this as simple random sampling without replacement, so that each character is marginally uniformly distributed over all characters not already eliminated.Our strategic analysis of s = 0, in which case he wins if he guesses correctly (with probability 1/n) and loses if he guesses incorrectly. Alternatively, he can guess s≥1 in which case he cannot win with his present guess, but he still reduces the characters on his game-board and passes the opportunity to move to his opponent. From these options, we see that the probability that the moving player wins is given recursively by:Under the official rules (I) the moving player can guess vice versa.) but does not lose if he guesses incorrectly.  If he does not win with his present guess he reduces the number of characters on his game-board and passes the move to his opponent (who now becomes the moving player). Alternatively, he can guess s>1 in which case he cannot win with his present guess, but he still reduces the characters on his game-board and passes the opportunity to move to his opponent. From these options, we see that the probability that the moving player wins is given recursively by:Under the variant rules (II) the moving player can guess Since our generalised “game” is symmetric, it follows that both players must have the same set of optimal strategies, and each optimal strategy pair is a subgame-perfect Nash equilibrium. Hence, we are able to identify the optimal strategies using the recursive equations that come from this equilibrium. Let n and m accordingly. However, it is just as easy to proceed for the general case, allowing an arbitrary number of characters. This gives the optimal strategies for specific game-states as a consequence.) = p. It is also helpful to conduct our analysis in terms of a = nm∙p. The optimization criterion leads to the following simple recursive equations for the two variants. Under the official rules (I) we have the recursion:The above optimisation criterion leads us to a recursive equation for the optimal strategy. Since both players are using the same strategy we drop the reference to the strategy Under the variant rules (II) we have the recursion:Any pure strategy satisfying these recursive equations for all game-states is an optimal pure strategy of the infinite generalised problem. Duersch, Oechssler and Schipper  give soms = 1 with corresponding win-probability p = 1 giving a = m. (This applies under both variants of the game.)We can obtain the optimal strategy by solving the above recursive equations using backwards induction, starting with the case where the moving player has a single remaining character. In this case we know that the player will guess this remaining character and will be correct, thereby winning the game. For all aI and aII for values 1≤n, m<24  are shown at the end of this paper.Starting with this baseline case and using the technique of backwards induction we can obtain the win-probability function under optimal strategy. Matrices showing the resulting functions The recursive formulae in the previous section allow computation of the win-probabilities for both rules variants under optimal play. Using these win-probabilities it is possible to quantify the first-mover advantage and game state advantage that accrue to the moving player. These can be defined as the symmetric and anti-symmetric parts of the win-probability function:The first-mover advantage and game-state advantage under both rules variants is shown below in the heatmaps in Guess Who provide that the youngest player should always move first. This rule implicitly recognises the first-mover advantage in the game and attempts to ameliorate this advantage by giving the first move to the player expected to use the most sub-optimal play.) It is also interesting to observer that, aside from cases of low values for n and m, the first-mover advantage under the official rules is higher in the bifurcating regions where the game-state is uneven, whereas the first-mover advantage under the variant rules is higher where the game-state is even. The win-probability is the combination of these two effects—it is shown in As can be seen from Guess Who has twenty-four characters and each player eliminates his own character from consideration before guessing. This means that the game starts at game state  . If both players play an optimal strategy then the win probabilities for the first player under the two rules variants are:A standard game of Guess Who has a first-mover advantage, it is possible to correct this to create a “fair” game by altering the starting state of the game to one where there is a game-state advantage to the waiting player that is equivalent to the first-mover advantage of the moving player. The fair game-states where this occurs are shown in the Although aI and aII for any range 1≤n, m<U, which then gives the corresponding values for the win probabilities, first-mover advantage and game-state advantage, under either rules variant. In the next section we derive a non-recursive formula for the win probability and optimal pure strategies for the variant rules.The recursive formulae above allow easy derivation of matrices for We will conduct an analysis of the optimal strategy only for the variant rules (II). This is the more interesting of the two cases, since it holds a closer resemblance to other “search race” game theoretic problems in broader fields such as industrial competition. It also has smoother properties for its win-probability function and a relatively simple form for its strategic optima, which give some intuitive insight into the nature of a “search race” problem.aII−aII have a simple form that can be expressed using a number series introduced in Theorem 1 below.We present our optimality findings as a series of theorems, with all proofs in the Theorem1: The win-probabilities when both players use optimal strategies are:k are taken from the following series:Theorem 2: We can rewrite the function aII in simplified form as:Theorem3: Any pure strategy s* is an optimal pure strategy (under the variant rules) if and only if its guesses all fall within the range k are taken from the following series:Corollary 1: The following strategy s* is an optimal pure strategy (under the variant rules):Corollary 2: The following strategy s* is an optimal pure strategy (under the variant rules):Remark: Another way of expressing this solution is as follows. For simplicity, consider the case where n≥4 and m≥4. If 3∙2k<n≤3∙2k+1 and m>3∙2k for some integer k≥0 then we have ⋆n = 3∙2k+1≤⋆m. In this case we have:n>3∙2k+1 and 3∙2k<m≤3∙2k+1 for some integer k≥0 then we have ⋆n>3∙2k+1 = ⋆m. In this case we have:Alternatively, if Guess Who under the variant rules, and the win-probabilities that result when both players use an optimal strategy. Theorems 1–2 give the win-probability from any game-state in the case where both players play an optimal strategy. Theorem 3 shows us that there is a wide scope for the optimal strategy, with a range of allowable guesses in each game-state. It is notable that each optimal strategy is in accordance with our preliminary intuition regarding the strategies for a player who is ahead or behind in the game. In particular, we can see from Corollary 1 that one optimal strategy is for the player to use the ‘split-in-half search’ as his guess, taking s = max for all his moves unless his opponent has only one or two characters left. In this latter case the opponent is sufficiently close to winning the game that the moving player should make a guess s = 1 of a single character, even if this is not the split-in-half strategy from his position. This optimal strategy shows us that the player who is behind in the game may delay his ‘risky search’ until quite late, when his opponent has only one or two characters remaining on the game board.These theorems set out the optimal strategies for n≤⋆m). If he does not have a sufficiently small number of characters then it is optimal for the moving player to take a ‘risky search’ where he takes s = max/2⌋) as his guess. Unlike the split-in-half search, this guess does not minimize the expected number of characters eliminated, but it is equally optimal due to the fact that the moving player is sufficiently far behind in the search race to warrant a risk being taken to overtake his opponent. Even in this case we see that the risky search bears a resemblance in form to the split-in-half search—it is made based on a number of characters related to the number the opponent has remaining.On the other hand, it is possible to undertake risky play earlier in the game while still using an optimal strategy. In particular, we can see from Corollary 2 that another optimal strategy is for the moving player to use a ‘split-in-half search’ if he has a sufficiently small number of remaining characters relative to his opponent ≡(3x−1)/(3x2). It is easy to show that ½<dn≤1 and ½<dm≤1 so that 2/3≤A≤3/4 in both parts of the expression.  This is a narrow range, which means the main thing that affects the win-probability is the ratio r, as we expect.For n≥3 and m≥3, if the ratio r is held constant and the magnitude of the values of n and m are increased (in proportion to each other), the function oscillates slightly due to the changes in the function A, but it holds its value approximately. If the magnitude of the values of n and m is increased by an integer power of two then the win-probability remains unchanged  = pII for all n≥3 and m≥3) but if a different multiple is applied then there can be a slight change in the function, owing to the oscillation in A that we mentioned. Fair game-states, where each player has an equal chance of winning, occur in cases when r = 3/2 (dn = 1) or r = 4/3 (dm = 1). The win-probability for the game has already been shown in The pattern of values in the win-probability function should now be a bit more discernible. In the case when n = ⋆n = ⋆m = m . At these points we have GII = 0 and FII = 1/6 so that pII = 2/3 . The game-state advantage accrues most intensely when the player has substantially fewer characters than his opponent. Since we have shown this advantage for the moving player, this occurs in game states where n is substantially less than m .Our present results also allow us to further elucidate the first-mover advantage and game-state advantage for the variant rules shown in Guess Who, and we have derived corresponding results for the first-mover advantage and game-state advantage for the players.  We have also derived the set of pure optimal strategies for the rules variant where an incorrect character guess does not lead to loss of the game.In this paper we have derived win probabilities under optimal strategy for play for two variants of the children’s game Guess Who is a type of ‘search race’ in which players seek to conclude their search successfully before their opponent does the same. The game using the variant rules is a discrete turn-based game, but its structure bears a resemblance to other forms of search race occurring in fields such as sporting competitions and industrial competitions. In particular, the game allows players to vary their strategy between “conservative” and “risky” strategies, and adapt this selection to the known state of progress of themselves and their opponent towards the goal. As with many search problems, the optimal strategy involves using a ‘split-in-half search’ to minimise the number of search steps. However, in game states where the other player is sufficiently close to winning it can be optimal to depart from this method and instead engage in ‘risky play’ which seeks to overtake the opponent in the search race.Guess Who. The probability of winning from any game state is easily calculated from the formulae in this paper, and has also been shown graphically in a heat map.The optimal strategy we have derived here is actually a set of pure strategies that are subgame perfect Nash equilibria of the relevant problem, and moreover, these form a single effective strategy set, insofar as all strategies in the set are best-responses to each other. We have also shown that under optimal play there is a first-mover advantage, such that first player has an advantage in the standard game of Guess Who. Under optimal play there is an advantage to the first-mover, though the advantage is larger in the variant rules than in the official rules. If both players use an optimal strategy from the starting game state  then the probability that the first player will win the game is pI = 0.5595 under the official rules or pII = 0.6597 under the variant rules. Whilst the game is “unfair” it is possible to alter the starting game-state to obtain a fair game. There are a number of fair game states in the game where the waiting player has a game-state advantage that is of the same magnitude as the first-mover advantage of the moving player, leading to a fair game.We conclude this analysis by summarising some basic facts about the optimal strategy and consequent win-probability in the two variants of S1 Appendix(DOCX)Click here for additional data file.


Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients’ survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked. Prostate cancer is the most commonly diagnosed malignancy in men and a major health issue, as it represents the third highest cause of death by cancer in industrialized countries . The proInhibition of the androgen axis by androgen deprivation therapy (ADT) is the predominant strategy for the treatment of metastatic prostate cancer. It can be achieved surgically (orchiectomy) or pharmacologically, using agonists or antagonists of GnRH to block the testicular synthesis of androgens. However, despite low levels of circulating testosterone, disease progression is invariably observed, leading to a castration-resistant state, in which tumor growth relies on the intracrine production of androgens. This justified the development of AR antagonists that prevent AR signaling by inhibiting the ligand binding to the receptor, or by inhibiting the translocation of AR to the nucleus, and its subsequent binding to ARE sequences on the DNA. Though a significant improvement of survival has been observed with the approval of first  and second generation non-steroidal AR antagonists , resistance to these agents is also observed. Several AR-dependent mechanisms have been described to explain this resistance . U2OS, DU145, and VCaP cells were cultured in Dulbecco’s Modified Eagle (DMEM) medium, and PC3 and LNCaP-FGC cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum without antibiotics. HG5LN (HeLa GAL4REx5-luciferase) and HELN (HeLa ERE-luciferase) cells were cultured in DMEM/F-12 medium supplemented with 5% fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL of streptomycin and 1 mg/mL geneticin. HELN and HGLN5 cells stably expressing MR, PR, GR, and PXR were cultured in the same medium, supplemented with 0.5 µg/mL puromycin. All cell lines were cultured at 37 °C under 5% COHigh throughput screens and antagonism tests were performed in DMEM, supplemented with 5% charcoal-stripped serum medium in the presence of 1% penicillin and streptomycin.High-content screening was performed using the Prestwick Phytochemical Library , a collection of 320 pure natural products, mostly derived from plants. Harmol hydrochloride was obtained from Santa Cruz Biotechnology, Inc. Harmine, harmane, R1881, spironolactone, RU486, R5020, dexamethasone, and aldosterone were purchased from Sigma Aldrich . Enzalutamide was purchased from Selleckchem , SR12813 from Bio-Techne . All reagents were dissolved in dimethyl sulfoxide (DMSO) at 10 mM before use.GGCTCTGGTAGTTCTTGGA) or ARE(PB) (AATAGGTTCTTGGAGTACTTTAC) inserted in the collagenase-luciferase-hygromycine plasmid   cells, as previously described ,33,34. HU2OS reporter cells stably expressing hAR (U2OS-hAR-ARE-Luc), or hARv7 (U2OS-hARv7-ARE-Luc), and U2OS-ARE-Luc control cells were plated in clear-bottomed 96-well plates in DMEM, supplemented with 10% FBS at 80% of confluence. The day after, the medium was replaced by DMEM without phenol red, supplemented with 5% charcoal-stripped serum in the presence of 100 units/mL of penicillin and 100 μg/mL of streptomycin. Each compound from the library was then added to U2OS-hAR-ARE-Luc and U2OS-ARE-Luc cells at 4 concentrations  for an additional 16 h at 37 °C, in the presence of 1 nM R1881 that corresponds to a suboptimal concentration, inducing 80% agonistic activity. We used R1881 because it is less subject to metabolism compared to dihydroxytestosterone (DHT). The medium was then replaced with a test medium containing 0.3 mM luciferin, and luminescence was measured using a MicroBeta Wallac luminometer . Screening were performed in duplicate in two separate experiments, and data were expressed as % of the maximal activity obtained with 100 nM R1881 alone. Enzalutamide (1 µM) was used as a positive control. The antagonistic activity of the positive hits towards hAR was validated using the same protocol in the absence of R1881, or in the presence of 1 or 100 nM of the agonist, corresponding to approximately 80% and maximal luciferase activity, respectively. Tests were performed in triplicates using 6 concentrations (0.01 to 3 µM) of each compound, and the results were expressed as percentages ± SEM of the luciferase activity obtained in the presence of 10 nM R1881. Screening for ARv7 inhibitors was performed using U2OS-hARv7-ARE-Luc and U2OS-ARE-Luc control cells with the same protocol in the absence of R1881.Transactivation assays to evaluate the activities of harmol and enzalutamide on other nuclear receptors were performed using the same protocol as for U2OS cells. Agonistic activities were evaluated in HMLN GR, HELN PR, HG5LN MR, and HG5LN PXR cells, using dexamethasone 100 nM, R5020 100 nM, aldosterone 100 nM, and SR12813 3 μM as a positive control, respectively. Antagonistic activity towards GR, PR, MR, and PXR were evaluated by co-incubating increasing concentrations of the tested compound with a concentration of agonist that induced 80% of maximal luciferase activity, i.e., 1 nM R5020 (for PR), 5 nM dexamethasone (for GR), 1 nM aldosterone (for MR), 300 nM SR12813 (for PXR). RU486 was used as a control antagonist for GR and PR, and spironolactone for MR.® imaging cytometer (Nexcelom Bioscience). The results (n = 3 ± SEM) were expressed as mortality ratios (number of dead cells in treated samples vs. untreated controls) as a function of drug concentrations. Then, proliferation was assessed following cell lysis with the CellTiter-Glo® Luminescent Cell Viability Assay reagent , according to the manufacturer protocol. Quantification of the luminescence was performed using a MicroBeta Wallac luminometer (PerkinElmer), and percent growth was calculated relative to DMSO treated cells. IC50 values were determined using the Prism® software, and are the results of 3 independent experiments.Spheroids were generated by seeding prostate cancer cells in non-adherent U-bottom 96-well plates in medium supplemented with 5% of charcoal stripped FBS. VCaP, LNCaP-FGC, DU145, or PC3 cells were seeded in duplicate at a density of 2000, 500, 600, and 600 cells per well, respectively. Three days after, spheroids were treated with indicated concentrations of the drugs in the presence of 1 nM R1881, and both cell death and proliferation were assessed 7 days later. Cell death was first evaluated by the incubation of the spheroids with propidium iodide (1 mg/mL) for 30 min, and quantitation of red fluorescence was performed using a Celigo6 and 2 × 106 cells per well, respectively) and, 48 h later, complete medium was replaced by charcoal-stripped medium with 5% FBS. Cells were then treated for 24 h with the tested compounds, and RNA extraction was performed using the Quick RNA Miniprep kit , according to the manufacturer’s protocol. Reverse transcription was performed with 0.5 µg total RNA using the SuperScript III reverse transcriptase kit , according to the manufacturer’s protocol, in a final volume of 20 µL. The levels of genes transcripts were measured by real time PCR using a LightCycler 480 system  in the presence of SYBRGreen (SYBR® Premix Ex Taqtm) and the following primers: FKBP5, sense 5′-GCGGCGACAGGTTCTCTACTT-3′; antisense 5′-TCATCGGCGTTTCCTCACCA-3′; PSA, sense 5′-CCCTGTCCGTGACGTGGATT-3′; antisense 5′-CAGCAAGATCACGCTTTTGTTCC-3′; TMPRSS2, sense 5′-GGACAGTGTGCACCTCAAAGAC-3′, antisense 5′-TCCCACGAGGAAGGTCCC-3′; GR, sense 5′-ACAGCATCCCTTTCTCAACAG-3′, antisense 5′-AGATCCTTGGCACCTATTCCAAT-3′; HPRT, sense 5′-CTGACCTGCTGGATTACA-3′; antisense 5′-GCGACCTTGACCATCTTT-3′; GAPDH, sense 5′-AATTGAGCCCGCAGCCTCCC-3′; antisense 5′-CCAGGCGCCCAATACGACCA-3′. Expression levels were evaluated with the ΔΔCt method and were normalized to GAPDH. Results are expressed as mRNA expression, relative to control (untreated cells), and are the mean ± SEM of 3 independent experiments.LNCaP and VCaP cells were seeded in 6-well plates  for 30 min at 4 °C and centrifuged for 15 min at 15,000 g and 20 µg of proteins from the supernatants were electrophoresed on a 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Immunoblotting was performed using the following antibodies: AR , GR , and GAPDH , and proteins were visualized by chemiluminescence detection using the ECL RevelBlot Plus or RevelBlot Intense .3H]-R1881 (41.3 Ci/mmol specific activity) in the absence or presence of enzalutamide, harmol (0.01–10 μM), or unlabeled R1881 (0.01–300 nM) for 3 h at 37 °C, and washed three times with 200 μL of cold PBS, to eliminate unbound material. Cells were then lysed with 50 μL of LSC-cocktail , and [3H]-bound radioactivity was counted in scintillation liquid with a microbeta trilux . Non-specific binding was determined in the presence of 300 nM unlabeled R1881. Specific binding was calculated by subtracting non-specific binding from the total binding, and expressed as disintegrations per minute (dpm). The results were plotted as % of maximal [3H]-R1881 specific binding, which was obtained in the absence of a competitor (250–300 dpm) and set at 100%. IC50 values were defined as the concentrations required to decrease maximal [3H]-R1881 binding by 50%. Relative binding affinities (RBA) were calculated as IC50 ratios between each competitor and R1881. The RBA value for R1881 was arbitrarily set at 100.U2OS hAR-AREluc cells were seeded at a density of 80,000 cells per well in clear-bottomed 96-well plates . The day after, cells were labeled with 0.3 nM -R1881, and increasing concentrations of unlabeled R1881 or of the tested drugs  in its LBD, which is known to affect steroid binding characteristics and response to antiandrogens. VCaP cells express both the wild-type form of AR and its ARv7 variant, though the wild-type form is predominantly expressed at the basal level . The resA similar effect was observed for harmol hydrochloride, though higher concentrations of the drug were required, which is in accordance with its lower affinity for AR. Conversely, harmol hydrochloride had no effect on the growth of DU145 or PC3 cancer cell lines that do not express AR B. We furAs it was already demonstrated for enzalutamide, we found that growth inhibition of VCaP and LNCaP spheroids induced by harmol hydrochloride was accompanied by a significant decrease of R1881-mediated expression of well-known AR target genes, such as TMPRSS2, FKBP5, and PSA, whereas the expression of the AR non-target gene HPRT was unaffected C. In accWe then tested the effect of harmol hydrochloride and enzalutamide for their potential activities towards the progesterone (PR), glucocorticoid (GR), mineralocorticoid receptors (MR) and PXR (NR1I2), using dedicated luciferase reporter cell lines  . In contrast, enzalutamide displayed antagonistic activity towards PR for concentrations ranging from 0.5 to 3 µM C, and coAltogether, our data validate the use of U2OS cell model for the screening of compounds that could alter the transcriptional activity of nuclear receptors, including the androgen receptor. They further identified harmol hydrochloride as a competitive and selective antagonist of the androgen receptor.Despite the approval of new generation AR antagonists, resistance to castration remains one of the major causes of prostate cancer treatment failures, emphasizing the need to identify more potent inhibitors of the androgen axis signaling. In the search for new derivatives, numerous studies using cell-based high content functional screens of chemical libraries from various origins have been performed. In these studies, the inhibition of AR activity was quantified using either the transcription of AR reporter genes ,26, AR lThe object of our study was to emphasize the importance of the choice of the cell model to screen for inhibitors of the AR transcriptional activity. For this purpose, we considered the previously validated U2OS osteosarcoma cell model stably transfected with AR or ARv7, and a luciferase reporter system. We confirmed that this model is highly selective to low levels of androgens, and is relevant for the absence of response to other nuclear receptors ligands and a high fold induction by AR ligands, which facilitates the identification of potential inhibitors  and Figu. We thenOur study also reveals, for the first time, the high selectivity of harmol hydrochloride towards AR, as it could not inhibit or activate the other nuclear receptors of the same family. Moreover, we found that harmol hydrochloride had no effect on the activity of PXR (NR1I2), a master regulator of the expression of genes involved in drug metabolism, such as CYP3A4, a well-known cytochrome implicated in the metabolism of numerous xenobiotics, including anticancer drugs . This isAltogether, our data confirm that U2OS osteosarcoma cells represent a pertinent model for HCS-based functional screening of potential inhibitors of the transcriptional activity of the androgen receptor. Indeed, prostate cancer cells with heterogenous expression of AR and other nuclear receptors, as well as their associated cofactors that may differ drastically between each other, could eventually mask the activity of potential hits, and may not be the most pertinent models. Our results not only identified harmol as a competitive inhibitor of AR, but also show, for the first time, the selectivity of this derivative towards AR. This finding has potential implications with respect to the use of second-generation AR antagonists, such as enzalutamide. Indeed, conversely to harmol, enzalutamide can activate PXR and lead to potential drug–drug interactions that are associated with adverse events. Moreover, the fact that harmol antagonizes AR through a mechanism that differs from enzalutamide, one can hypothesize that it could exert a different biological response. The active conformation of the AF2 helix is not sterically precluded by harmol, so it might be more dependent on the expression level of coregulators, thereby generating some cell- or tissue-specificity, and may therefore represent an interesting therapeutic alternative to currently approved AR antagonists used in the clinic.


Rationale: People living with both chronic obstructive pulmonary disease (COPD) and frailty have high potential to benefit from pulmonary rehabilitation but face challenges completing programs. However, research to understand ways to optimize participation in this group is lacking.Objectives: To explore the experiences, needs, and preferences of people with COPD and frailty referred for outpatient pulmonary rehabilitation.Methods: Semistructured interviews with people with COPD and physical frailty, purposively sampled by age, living status, level of frailty, and completion of pulmonary rehabilitation. Thematic analysis with a critical realist perspective was used, involving relevant stakeholders with clinical, academic, and lived experience for interpretive rigor.Results: Nineteen people with COPD and frailty were interviewed, with a median age of 78 years . Nine did not complete their pulmonary rehabilitation program. Four themes were identified: striving to adapt to multidimensional loss, tensions of balancing support with independence, pulmonary rehabilitation as a challenge worth facing, and overcoming unpredictable disruptions to participation. Participants described constantly adapting to their changing health and resulting multidimensional losses . This involved traversing between independence and seeking support, set against a mismatch between their needs and what support is available. People with COPD and frailty can be highly motivated to participate in pulmonary rehabilitation, despite the physical and mental demands it entails, and report a range of benefits. Yet in the context of changeable health, they must often overcome multiple unpredictable disruptions to completing rehabilitation programs. Participant determination and flexibility of services can facilitate ongoing attendance, but for some, these unpredictable disruptions erode their motivation to attend.Conclusions: People with COPD and frailty experience accumulating, multidimensional loss. This group are motivated to complete pulmonary rehabilitation but often require additional support and flexibility owing to fluctuating and unpredictable health. Person-centered approaches should be considered to minimize disruptive health events and support pulmonary rehabilitation participation and completion. Service adaptations could allow more flexibility to meet the changing needs of this group and enable communication around how pulmonary rehabilitation might align with their priorities. Chronic obstructive pulmonary disease (COPD) affects multiple body systems and has been described as reflecting an “accelerated aging” . COPD frFrailty is a multidimensional syndrome characterized by decreased reserve and diminished resistance to stressors . PhysicaParticipating in exercise improves outcomes for people with COPD , 14 or fPeople with COPD report multiple challenges to participation in exercise-based interventions, including lack of perceived benefit, concurrent burden of comorbid conditions, conflicts with other priorities, difficulties with mobility and travel, fear of worsening symptoms, low energy and motivation, and exacerbations of their COPD –23. Simi1) understand the experiences and preferences of people living with COPD and frailty; 2) identify current support and areas of unmet need; and 3) explore motivation for, and barriers to, continued participation in pulmonary rehabilitation.Understanding (non-)participation and identifying optimal ways of supporting people with COPD and frailty is a priority for improving outcomes for this population . People We conducted a qualitative interview study within a critical realist paradigm . This meWe recruited participants from two London hospitals providing outpatient pulmonary rehabilitation. Clinical staff identified potential participants during their initial assessments for pulmonary rehabilitation. A researcher then periodically followed up with those interested in participating, so that they could be potentially sampled when they stopped or completed their pulmonary rehabilitation.People referred for pulmonary rehabilitation with a physician diagnosis of COPD, who at initial assessment were identified as physically frail using the Short Physical Performance Battery  SPPB; s. PatientWe purposively sampled participants by age (> or ≤80 yr), living status , level of physical frailty (SPPB scores of > or ≤7), and completion of pulmonary rehabilitation (did or did not complete). Within the group who did not complete pulmonary rehabilitation, we attempted to sample those who were and were not admitted to hospital.A female researcher (L.B.) with a background in psychology and palliative care research  conducted the interviews in participants’ preferred locations, between October 2018 and April 2019. L.B. had previous training in qualitative research and experience in conducting interviews with people with serious illness and their families. L.B. was not known to participants before the interviews.The interviews followed a semistructured interview topic guide (online supplement E1) developed with input from people with lived experience relevant to both COPD and frailty, and their informal carers (service user representatives). The topic guide explored participants’ current health and priorities, support and unmet needs, and expectations and experiences of pulmonary rehabilitation. On the advice of the service user representatives, the researcher identified and used participants’ own language in relation to frailty, for example, slowing down, difficulties walking, lack of strength or energy. Service user representatives also prompted the researcher to consider the participant’s assets and resilience in addition to limitations. Interviews were audio-recorded and transcribed verbatim. The researcher completed detailed field notes to describe interview flow, contextual factors, participant responses, and initial reflections immediately after each interview.Data collection continued until the data set was deemed to be approaching thematic saturation  i.e., r dimensioWe conducted a reflexive thematic analysis to identify patterns of meaning within the data . First, The London Camberwell St Giles Research Ethics Committee (ref. 18/LO/1197) approved this study. We obtained written informed consent prior to interviews.n = 17), but two took place at the researcher’s university. In three interviews, participants were accompanied by a family member: two who consented for their contributions to be included and one who was present but did not participate.Of 49 eligible people introduced to the study, 19 were interviewed . SixteenFour themes were identified: striving to adapt to multidimensional loss, tensions of balancing support with independence, pulmonary rehabilitation as a challenge worth facing, and overcoming unpredictable disruptions to participation. Subthemes and illustrative quotes are shown in Participants reported an accumulation of health events and symptoms, describing their health as “quite up-and-down” over the preceding years. Overall, they described feeling not as well as they used to be and were accustomed to facing multiple health concerns. Participants described multidimensional loss across different areas of their lives, from loss of mobility and usual activities, to loss of relationships and life-space (mobility within their community), and loss of confidence and motivation. These primary concerns were often a result of their persistent breathlessness and reduced mobility but were also influenced by low energy, pain, throat-related symptoms, decreasing memory and cognition, anxiety, and poorer strength and balance.In the face of multidimensional loss, participants described resilience and capacity to keep adapting to a changing self. They were driven by their priorities of maintaining a sense of normality, remaining independent, and staying connected with others. This included changing how they did something  and/or changing how they thought about it . Whereas some adaptations were automatic and straightforward, others were emotionally challenging, particularly those involving accepting limitations. However, where losses kept building, some found it harder to keep up, and they could begin to experience a loss of purpose in their existence. This appeared more common in those living alone.As part of adapting, participants experienced tensions of balancing support and independence. Health and social care professionals, plus families and friends where present, often helped with filling the gaps and supporting their adaptation as it became more difficult to do things without assistance. However, this required negotiating the right balance between persevering alone and asking for help. This was an ongoing process of figuring out, by themselves and with others, how to adapt in a way that still maintained some sense of independence and did not make them feel like a burden. Against a background discourse about underresourced and overstretched services, some found this difficult.Achieving the right balance was also made harder by instances of mismatches and mistrust. Mismatches occurred when people received conflicting advice  or when services were offered routinely or reactively, rather than responding flexibly and proactively to fluctuating needs. For example, one participant described her difficulty getting a walk-in shower from local services, who presumed she did not need one because she had turned down their offer of a chairlift. She also noted the challenges of expressing her needs when her health can be so varied, for example, needing more support when she had a chest infection. Mistrust resulted from confusion or uncertainty around care, such as juggling multiple appointments, and not being clear on the purpose of the appointments. Mistrust could also stem from disagreements about appropriate support , and poor communication with, or not feeling listened to by, those providing support. These experiences could fracture relationships and create feelings of abandonment.The final influence on this balancing act was the compounding effects of inaccessibility. People mentioned disabling systemic barriers to getting support to suit their needs, such as things being too costly and/or physically inaccessible. When already concerned about being a burden, feeling frustrated by mismatches, and/or having lost trust in services, people were less likely to feel accessibility issues could be overcome and would disengage.Against this background of adapting to multidimensional loss and negotiating support and independence, participants had agreed to an assessment for pulmonary rehabilitation. All were motivated by a desire for change, whether to improve their health and symptoms or looking for an opportunity to get out of the house.Those who attended pulmonary rehabilitation described it as physically and mentally challenging. As participants were often experiencing high levels of breathlessness and low energy, it was physically demanding to travel there and complete the exercises, and psychologically challenging to overcome their fears  and stay motivated. Yet at the same time, most participants, including some who stopped attending pulmonary rehabilitation part-way through their program, emphasized the benefits of experiencing a safe and encouraging atmosphere at pulmonary rehabilitation. This resulted from skilled and supportive staff, appropriate tailoring of exercises and monitoring throughout, and being in a group with people like themselves. This type of atmosphere helped to address their fears around the safety of exercising “at their age” and with their particular health experiences .When people then perceived benefits from participating, this also motivated ongoing engagement with pulmonary rehabilitation. People also described regaining strength, energy, control over their breath, increased motivation, and confidence—often reflecting the areas where they had previously described losses. A few participants were less sure about physical benefits, particularly when comparing with previous attendance or function. However, they often still described social and psychological benefits of participating in a group activity outside of their homes, particularly when living alone. Consequently, attendees felt participation was worth it, despite the physical and psychological demands.For participants who did not start, stopped attending, or missed sessions within their pulmonary rehabilitation, the unpredictable causes of these disruptions were often apparent. Common challenges included periods of illness  or conflicting priorities .In many cases, these participants remained motivated and keen to return when they were able. Some were determined despite these disruptions: they still saw pulmonary rehabilitation as a challenge worth facing and as a good fit to support their ongoing adaptation and address their priorities. For others, it was also the rapport and flexibility of services that helped overcome disruptions. This included feeling able to discuss canceling or moving single sessions when unexpected events arose, and for two participants, negotiating less frequent attendance  when twice per week felt like too much.However, there were circumstances when people felt unable to overcome a disruption and pulmonary rehabilitation was no longer seen as a good fit. In some cases, disruptions were simple yet crucial issues around cost or physical accessibility. For others, a deterioration in health meant they now felt it was not going to address their needs or that it was physically beyond their capabilities. This type of disruption eroded their motivation to attend, and they stopped engaging with the service.People with COPD and frailty experience accumulating, multidimensional loss. In striving to adapt, participants report having to negotiate the right balance for them between independence and support. Often pulmonary rehabilitation contributed to this balance: as an opportunity to actively improve their own health, with encouragement and support from others. These positive experiences and outcomes motivated participants to attend pulmonary rehabilitation, despite its physical and mental challenges. However, owing to fluctuating health and multimorbidity, several participants experienced multiple unpredictable disruptions to completing their program. Rapport with and flexibility from service providers helped overcome these disruptions for some. For others, this disruption meant they no longer felt able to engage with pulmonary rehabilitation.The multidimensional impact of living with COPD –42 is weInterventions with potential to build resilience around multidimensional losses, and reduce the impact of unpredictable disruptions to health, may have greatest benefit for people with both COPD and frailty. Person-centered approaches suited to heterogeneity, complexity, and multimorbidity are required. One strategy potentially suited to this population might be integration with geriatric specialists to address reversible frailty causes, polypharmacy, and malnutrition . ComprehAdjusting pulmonary rehabilitation services to be more flexible and responsive may also be appropriate for people with both COPD and frailty. Participants’ descriptions of balancing independent adaptation and support-seeking reflected how, as stressors on their health increase and decreased, the amount of personal and professional resource required to adapt was equally variable . HoweverService adaptations that foster greater rapport with, and flexibility for, those identified as also living with frailty might facilitate participation. For example, services could offer closer supervision, preempt potential disruptions, and create shared plans for when this might occur. They could also make additional follow-up contacts before discharging people missing sessions, and/or prioritize reentry of those with frailty into services. This might be helped by more nuanced criteria for completion than the current binary approach. Instead, services could consider the number of sessions attended, note achievement of personalized goals, and acknowledge when sessions are missed owing to uncontrollable events. Such strategies to work flexibly with people with both COPD and frailty should be codeveloped to maximize acceptability, uptake, and potential effectiveness. Incorporation of home-based rehabilitation may also be helpful and can be an effective way of engaging people who cannot or prefer not to attend center-based pulmonary rehabilitation , 58. HowFinally, we must consider carefully how we communicate around pulmonary rehabilitation. As people with both COPD and frailty experience more unpredictability, they may increasingly use emotion-focused adaptations. Emotion-focused adaptations may include reframing perceptions of themselves and/or pulmonary rehabilitation such that it is not seen as a good fit  . To avoiHelpful approaches might include empathetic communication suggesting problem-focused strategies to support engagement , but also importantly addressing the emotional aspects of their experience . This could include learning from the success of motivational interviewing-based health coaching . If pulmPurposive sampling facilitated inclusion of diverse experiences, including those who stopped attending, and those who never commenced, pulmonary rehabilitation. However, our sample was limited to two urban sites, mainly to people with white ethnicities, and to people who attended their initial assessment for pulmonary rehabilitation. Approximately one in three people referred to pulmonary rehabilitation in the United Kingdom do not attend their initial assessment . This liPeople living with both COPD and frailty experience accumulating, multidimensional loss. This group are motivated to complete pulmonary rehabilitation but often require additional support and flexible services owing to their unpredictable health. Person-centered approaches suited to people with multiple conditions should be considered to minimize disruptive health events and support pulmonary rehabilitation attendance. Alongside this, services need to prioritize supportive communication around how pulmonary rehabilitation may align with participant’s priorities and consider more flexible delivery models to meet the fluctuating needs of this group.