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S5: UT-155 prevents nuclear localization of AR and AR v567es. S6: UT-155 binds to AR AF-1 domain. S7: Results from microarray with SARDs.S8: UT-155 inhibits transactivation of AD1 and D567es AR transactivation. Metabolism properties of SARDs.

Antimitotics that target tubulin are among the most useful chemotherapeutic drugs, but their clinical activity is often limited by the development of multidrug resistance. We recently discovered the novel small-molecule DJ101 as a potent and metabolically stable tubulin inhibitor that can circumvent the drug efflux pumps responsible for multidrug resistance of existing tubulin inhibitors. In this study, we determined the mechanism of action of this drug. The basis for its activity was illuminated by solving the crystal structure of DJ101 in complex with tubulin at a resolution of 2.8Å. Investigations of the potency of DJ101 in a panel of human metastatic melanoma cell lines harboring major clinically relevant mutations defined IC50 values of 7–10 nmol/L. In cells, DJ101 disrupted microtubule networks, suppressed anchorage-dependent melanoma colony formation, and impaired cancer cell migration. In melanoma-bearing mice, DJ101 administration inhibited tumor growth and reduced lung metastasis in the absence of observable toxicity. DJ101 also completely inhibited tumor growth in a paclitaxel-resistant xenograft mouse model of human prostate cancer (PC-3/TxR), where paclitaxel was minimally effective. Our findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation tubulin inhibitor for cancer therapy.Significance: These findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation antitubulin drug for cancer therapy. Cancer Res; 78(1); 265–77. ©2017 AACR.

Androgen receptor (AR) mediates the growth of prostate cancer throughout its course of development, including in abnormal splice variants (AR-SV)-driven advanced stage castration-resistant disease. AR stabilization by androgens makes it distinct from other steroid receptors, which are typically ubiquitinated and degraded by proteasomes after ligand binding. Thus, targeting AR in advanced prostate cancer requires the development of agents that can sustainably degrade variant isoforms for effective therapy. Here we report the discovery and characterization of potent selective AR degraders (SARD) that markedly reduce the activity of wild-type and splice variant isoforms of AR at submicromolar doses. Three SARDs (UT-69, UT-155, and (R)-UT-155) bind the amino-terminal transcriptional activation domain AF-1, which has not been targeted for degradation previously, with two of these SARD (UT-69 and UT-155) also binding the carboxy-terminal ligand binding domain. Despite different mechanisms of action, all three SARDs degraded wild-type AR and inhibited AR function, exhibiting greater inhibitory potency than the approved AR antagonists. Collectively, our results introduce a new candidate class of next-generation therapeutics to manage advanced prostate cancer. Cancer Res; 77(22); 6282–98. ©2017 AACR.

Suppl. Methods

Captions for supporting data and detailed methods for chemical synthesis and standard biochemical assays

Supplementary Figures and Tables

NCI-60 screening data for DJ101

Resistance index values of DJ101 measured in drug sensitive PC-3 and DU-145 and their corresponding taxane resistant sublines.

Purpose:Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand-binding domain (LBD)-binding antagonists, are inactivated by common resistance mechanisms. It is important to develop next-generation mechanistically distinct drugs to treat castration- and drug-resistant prostate cancers.Experimental Design:Second-generation AR pan antagonist UT-34 was selected from a library of compounds and tested in competitive AR binding and transactivation assays. UT-34 was tested using biophysical methods for binding to the AR activation function-1 (AF-1) domain. Western blot, gene expression, and proliferation assays were performed in various AR-positive enzalutamide-sensitive and -resistant prostate cancer cell lines. Pharmacokinetic and xenograft studies were performed in immunocompromised rats and mice.Results:UT-34 inhibits the wild-type and LBD-mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and -resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses, when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist enzalutamide and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-protein–coupled receptor kinase and nuclear receptor family members.Conclusions:Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug.

Androgen receptor (AR) and its splice variants (AR-SVs) promote prostate cancer (PCa) growth by orchestrating transcriptional reprogramming. Mechanisms by which the low complexity and intrinsically disordered primary transactivation domain (AF-1) of AR and AR-SVs regulate transcriptional programming in PCa remains poorly defined. Using omics, live and fixed fluorescent microscopy of cells, and purified AF-1 and AR-V7 recombinant proteins we show here that AF-1 and the AR-V7 splice variant form molecular condensates by liquid–liquid phase separation (LLPS) that exhibit disorder characteristics such as rapid intracellular mobility, coactivator interaction, and euchromatin induction. The LLPS and other disorder characteristics were reversed by a class of small-molecule-selective AR-irreversible covalent antagonists (SARICA) represented herein by UT-143 that covalently and selectively bind to C406 and C327 in the AF-1 region. Interfering with LLPS formation with UT-143 or mutagenesis resulted in chromatin condensation and dissociation of AR-V7 interactome, all culminating in a transcriptionally incompetent complex. Biochemical studies suggest that C327 and C406 in the AF-1 region are critical for condensate formation, AR-V7 function, and UT-143’s irreversible AR inhibition. Therapeutically, UT-143 possesses drug-like pharmacokinetics and metabolism properties and inhibits PCa cell proliferation and tumor growth. Our work provides critical information suggesting that clinically important AR-V7 forms transcriptionally competent molecular condensates and covalently engaging C327 and C406 in AF-1, dissolves the condensates, and inhibits its function. The work also identifies a library of AF-1-binding AR and AR-SV-selective covalent inhibitors for the treatment of PCa.

A series of propanamide derivatives were designed, synthesized, and pharmacologically characterized as selective androgen receptor degraders (SARDs) and pan-antagonists that exert a broad-scope androgen receptor (AR) antagonism. Incorporating different basic heteromonocyclic B-ring structural elements in the common A-ring–linkage–B-ring nonsteroidal antiandrogen general pharmacophore contributed to a novel scaffold of small molecules with SARD and pan-antagonist activities even compared to our recently published AF-1 binding SARDs such as UT-69 (11), UT-155 (12), and UT-34 (13). Compound 26f exhibited inhibitory and degradation effects in vitro in a wide array of wtAR, point mutant, and truncation mutant-driven prostate cancers (PCs). Further, 26f inhibited tumor cell growth in a xenograft model composed of enzalutamide-resistant (EnzR) LNCaP cells. These results demonstrate an advancement toward the development of novel SARDs and pan-antagonists with efficacy against EnzR prostate cancers.

Traditional endocrine therapy for prostate cancer (PCa) has been directed at suppression of the androgen receptor (AR) signaling axis since Huggins et al. discovered that diethylstilbestrol (DES; an estrogen) produced chemical castration and PCa tumor regression. Androgen deprivation therapy (ADT) still remains the first-line PCa therapy. Insufficiency of ADT over time leads to castration-resistant PCa (CRPC) in which the AR axis is still active, despite castrate levels of circulating androgens. Despite the approval and use of multiple generations of competitive AR antagonists (antiandrogens), antiandrogen resistance emerges rapidly in CRPC due to several mechanisms, mostly converging in the AR axis. Recent evidence from multiple groups have defined noncompetitive or noncanonical direct binding sites on AR that can be targeted to inhibit the AR axis. This review discusses new developments in the PCa treatment paradigm that includes the next-generation molecules to noncanonical sites, proteolysis targeting chimera (PROTAC), or noncanonical N-terminal domain (NTD)-binding of selective AR degraders (SARDs). A few lead compounds targeting each of these novel noncanonical sites or with SARD activity are discussed. Many of these ligands are still in preclinical development, and a few early clinical leads have emerged, but successful late-stage clinical data are still lacking. The breadth and diversity of targets provide hope that optimized noncanonical inhibitors and/or SARDs will be able to overcome antiandrogen-resistant CRPC.

We report herein the design, synthesis, and pharmacological characterization of a library of novel aryl pyrazol-1-yl-propanamides as selective androgen receptor degraders (SARDs) and pan-antagonists that exert broad-scope AR antagonism. Pharmacological evaluation demonstrated that introducing a pyrazole moiety as the B-ring structural element in the common A-ring-linkage-B-ring nonsteroidal antiandrogens' general pharmacophore allowed the development of a new scaffold of small molecules with unique SARD and pan-antagonist activities even compared to our recently published AF-1 binding SARDs such as UT-155 (9) and UT-34 (10). Novel B-ring pyrazoles exhibited potent AR antagonist activities, including promising distribution, metabolism, and pharmacokinetic properties, and broad-spectrum AR antagonist properties, including potent in vivo antitumor activity. 26a was able to induce an 80% tumor growth inhibition of xenografts derived from the enzalutamide-resistant (Enz-R) VCaP cell line. These results represent an advancement toward the development of novel AR antagonists for the treatment of Enz-R prostate cancer.

Interfering with microtubule dynamics is a well-established strategy in cancer treatment; however, many microtubule-targeting agents are associated with drug resistance and adverse effects. Substantial evidence points to ATP-binding cassette (ABC) transporters as critical players in the development of resistance. Herein, we demonstrate the efficacy of DJ95 (2-(1H-indol-6-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine), a novel tubulin inhibitor, in a variety of cancer cell lines, including malignant melanomas, drug-selected resistant cell lines, specific ABC transporter-overexpressing cell lines, and the National Cancer Institute 60 cell line panel. DJ95 treatment inhibited cancer cell migration, caused morphologic changes to the microtubule network foundation, and severely disrupted mitotic spindle formation of mitotic cells. The high-resolution crystal structure of DJ95 in complex with tubulin protein and the detailed molecular interactions confirmed its direct binding to the colchicine site. In vitro pharmacological screening of DJ95 using SafetyScreen44 (Eurofins Cerep-Panlabs) revealed no significant off-target interactions, and pharmacokinetic analysis showed that DJ95 was maintained at therapeutically relevant plasma concentrations for up to 24 hours in mice. In an A375 xenograft model in nude mice, DJ95 inhibited tumor growth and disrupted tumor vasculature in xenograft tumors. These results demonstrate that DJ95 is potent against a variety of cell lines, demonstrated greater potency to ABC transporter-overexpressing cell lines than existing tubulin inhibitors, directly targets the colchicine binding domain, exhibits significant antitumor efficacy, and demonstrates vascular-disrupting properties. Collectively, these data suggest that DJ95 has great potential as a cancer therapeutic, particularly for multidrug resistance phenotypes, and warrants further development. SIGNIFICANCE STATEMENT: Paclitaxel is a widely used tubulin inhibitor for cancer therapy, but its clinical efficacy is often limited by the development of multidrug resistance. In this study, we reported the preclinical characterization of a new tubulin inhibitor DJ95, and demonstrated its abilities to overcome paclitaxel resistance, disrupt tumor vasculature, and exhibit significant antitumor efficacy.

Purpose: Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand-binding domain (LBD)-binding antagonists, are inactivated by common resistance mechanisms. It is important to develop next-generation mechanistically distinct drugs to treat castration- and drug-resistant prostate cancers. Experimental Design: Second-generation AR pan antagonist UT-34 was selected from a library of compounds and tested in competitive AR binding and transactivation assays. UT-34 was tested using biophysical methods for binding to the AR activation function-1 (AF-1) domain. Western blot, gene expression, and proliferation assays were performed in various AR-positive enzalutamide-sensitive and -resistant prostate cancer cell lines. Pharmacokinetic and xenograft studies were performed in immunocompromised rats and mice. Results: UT-34 inhibits the wild-type and LBD-mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and -resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses, when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist enzalutamide and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-protein–coupled receptor kinase and nuclear receptor family members. Conclusions: Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug.

Microtubule (MT)-targeting agents are highly successful drugs as chemotherapeutic agents, and this is attributed to their ability to target MT dynamics and interfere with critical cellular functions, including, mitosis, cell signaling, intracellular trafficking, and angiogenesis. Because MT dynamics vary in the different stages of the cell cycle, these drugs tend to be the most effective against mitotic cells. While this class of drug has proven to be effective against many cancer types, significant hurdles still exist and include overcoming aspects such as dose limited toxicities and the development of resistance. Newer generations of developed drugs attack these problems and alternative approaches such as the development of dual tubulin and kinase inhibitors are being investigated. This approach offers the potential to show increased efficacy and lower toxicities. This review covers different categories of MT-targeting agents, recent advances in dual inhibitors, and current challenges for this drug target.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive breast cancer with shorter overall survival compared to other breast cancer types. One of the six molecularly-classified TNBC subtypes is the luminal androgen receptor subtype (LAR), which overexpresses androgen receptor (AR) and is dependent on AR for its growth. About 10-20% of TNBCs belong to the LAR subtype. Competitive AR antagonists, enzalutamide and bicalutamide, were effective in preclinical models of LAR TNBC and in clinical trials. This led us to hypothesize that potent selective AR degraders (SARDs), due to their ability to inhibit and degrade the AR, could provide a novel therapeutic approach for the treatment of LAR subtype of TNBC. Description: Western blots, cell line proliferation assays, and gene expression analyses were performed to evaluate novel small molecule SARDs. LAR TNBC cell lines and patient-derived xenografts (PDX) were utilized for in vivo evaluation of the SARDs. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally for four weeks with vehicle, SARD UT-34, SARD UT-105, enzalutamide, or bicalutamide. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. Summary: SARDs bind to the N-terminus of the AR and have been characterized in preclinical advanced prostate cancer models. In this study, the SARDs were evaluated in preclinical models of LAR TNBC. Western blot for AR in LAR MDA-MB-453 cells demonstrated degradation of the AR protein by SARDs at low micromolar concentrations. Gene expression studies showed a complete inhibition of androgen-induced AR target gene transcription by the SARDs. Androgen-induced proliferation of MDA-MB-453 cells was inhibited by SARDs. MDA-MB-453 cells implanted subcutaneously in NOD SCID Gamma female mice grew robustly to 100-300 mm3 in 15-20 days. Treatment of tumor-bearing animals with the SARDs completely inhibited or regressed the tumors. Conclusion: These results support the findings that AR is the driver of MDA-MB-453 cell and tumor growth. SARDs with their unique mechanism of action may provide a new therapeutic option to women affected by the LAR subtype of TNBC. Disclosure: This work was partially supported by Oncternal Therapeutics and by an NCI supplement award to R01 (CA229164S1 to author RN). The SARD program has been licensed to Oncternal Therapeutics, Inc. by the University of Tennessee Research Foundation. Author RN is a consultant to Oncternal Therapeutics. Citation Format: Sarah Asemota, Kirsten Young Young, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Dong-Jin Hwang, Yali He, James B. Breitmeyer, Gunnar F. Kaufmann, Duane D. Miller, Ramesh Narayanan. Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1235.

In our effort to find small-molecule treatments of advanced prostate cancers (PCs), a novel series of indolyl and indolinyl propanamides (series II and III) were discovered as selective androgen receptor degraders (SARDs). Initial studies of androgen receptor (AR) antagonist (1) and agonist (2) propanamides yielded a tertiary aniline (3) with novel SARD activity but poor metabolic stability. Cyclization to II and III produced submicromolar AR antagonism and protein degradation selective to AR and AR splice variant (AR SV). II and III maintained potency against enzalutamide-resistant (Enz-R) mutant ARs and PC cells and were efficacious in Enz-R xenografts, suggesting their potential to treat advanced PCs. Design, synthesis, and biological activity of novel SARDs that could potentially be used for the treatment of a wide spectrum of PCs including castration-resistant, Enz-R, and/or AR SV-dependent advanced PCs that are often untreatable with known hormone therapies are discussed.

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses of T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca−/− mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.

A new series of indole analogues based on our earlier lead compound, 2-(1H-indol-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine (42), was prepared as tubulin inhibitors in an effort to find a molecule with improved cytotoxic potency and metabolic stability. A series of indolyl-imidazopyridines (IIP) were synthesized and exhibited potent tubulin polymerization inhibitory activity with potent IC50 values ranging from 3 to 175 nM against a panel of human melanoma and prostate cancer cell lines. Among these compounds, the 6-indolyl compound 43 showed improved cytotoxic potency (average IC50 of 9.75 nM vs 55.75 nM) and metabolic stability in human liver microsomes (half-life time was 56.3 min vs. 45.4 min) as compared to previously reported 42. It was also shown to be effective against P-glycoprotein (P-gp) mediated multiple drug resistance (MDR) and taxol resistance.

Androgen receptor (AR) mediates the growth of prostate cancer throughout its course of development, including in abnormal splice variants (AR-SV)-driven advanced stage castration-resistant disease. AR stabilization by androgens makes it distinct from other steroid receptors, which are typically ubiquitinated and degraded by proteasomes after ligand binding. Thus, targeting AR in advanced prostate cancer requires the development of agents that can sustainably degrade variant isoforms for effective therapy. Here we report the discovery and characterization of potent selective AR degraders (SARD) that markedly reduce the activity of wild-type and splice variant isoforms of AR at submicromolar doses. Three SARDs (UT-69, UT-155, and (R)-UT-155) bind the amino-terminal transcriptional activation domain AF-1, which has not been targeted for degradation previously, with two of these SARD (UT-69 and UT-155) also binding the carboxy-terminal ligand binding domain. Despite different mechanisms of action, all three SARDs degraded wild-type AR and inhibited AR function, exhibiting greater inhibitory potency than the approved AR antagonists. Collectively, our results introduce a new candidate class of next-generation therapeutics to manage advanced prostate cancer. Cancer Res; 77(22); 6282–98. ©2017 AACR.

Antimitotics that target tubulin are among the most useful chemotherapeutic drugs, but their clinical activity is often limited by the development of multidrug resistance. We recently discovered the novel small-molecule DJ101 as a potent and metabolically stable tubulin inhibitor that can circumvent the drug efflux pumps responsible for multidrug resistance of existing tubulin inhibitors. In this study, we determined the mechanism of action of this drug. The basis for its activity was illuminated by solving the crystal structure of DJ101 in complex with tubulin at a resolution of 2.8Å. Investigations of the potency of DJ101 in a panel of human metastatic melanoma cell lines harboring major clinically relevant mutations defined IC50 values of 7–10 nmol/L. In cells, DJ101 disrupted microtubule networks, suppressed anchorage-dependent melanoma colony formation, and impaired cancer cell migration. In melanoma-bearing mice, DJ101 administration inhibited tumor growth and reduced lung metastasis in the absence of observable toxicity. DJ101 also completely inhibited tumor growth in a paclitaxel-resistant xenograft mouse model of human prostate cancer (PC-3/TxR), where paclitaxel was minimally effective. Our findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation tubulin inhibitor for cancer therapy. Significance: These findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation antitubulin drug for cancer therapy. Cancer Res; 78(1); 265–77. ©2017 AACR.

Current treatment options for advanced castration-resistant prostate cancers (CRPC) are effective for a brief period before becoming refractory. It is important to use relevant in vivo models for candidate selection in the development of next-generation mechanistically-distinct drugs to treat castration- and drug- resistant prostate cancers. Here, we describe a series of AR pan-antagonists (selective AR degraders (SARDs)) that degrade the AR and AR splice variants. SARDs inhibit the wild-type and LBD mutant ARs comparably and inhibit the in vitro proliferation and in vivo growth of enzalutamide-sensitive and resistant prostate cancer xenografts. Xenograft studies conducted in immune-compromised SRG rats (Sprague Dawley Rag2-/- Il2rg -/-; Hera Biolabs) with three lead SARDs demonstrated regression of enzalutamide-sensitive and -resistant VCaP tumors both in castrated and in intact rats. SRG rats provide the benefit of abundant blood samples for weekly evaluation of rising PSA, which also indicated that the SARDs inhibit the rising PSA, while enzalutamide was inactive in enzalutamide-resistant model. Drug metabolism and pharmacokinetic (DMPK) studies, also conducted with SRG and wild-type rats and in combination with efficacy, indicate that the molecules possess all the necessary drug-like properties. The molecules exhibit a broad safety margin with no cross-reactivity with G-Protein Coupled Receptor, kinase, and nuclear receptor family members. Collectively, the SARDs exhibit the properties necessary for a next-generation prostate cancer drug and that use of SRG rats facilitated thorough characterization of their in vivo properties. Citation Format: Suriyan Udhayasuriyan Ponnusamy, Fallon K Noto, Bisoye Towobola Adedeji, Yali He, Dong-Jin Hwang, Sam Moody, Thirumagal Thiyagarajan, Tseten Yeshi Jamling, Duane D Miller, Ramesh Narayanan. Potential next-generation androgen receptor-targeted therapeutic for enzalutamide-resistant prostate cancer; In vivo characterization in immune-compromised SRG rats [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B037. doi:10.1158/1535-7163.TARG-19-B037

Extensive research over the last decade has resulted in a number of highly potent tubulin polymerization inhibitors acting either as microtubule stabilizing agents (MSAs) or microtubule destabilizing agents (MDAs). These inhibitors have potent cytotoxicity against a broad spectrum of human tumor cell lines. In addition to cytotoxicity, a number of these tubulin inhibitors have exhibited abilities to inhibit formation of new blood vessels as well as disrupt existing blood vessels. Tubulin inhibitors as a vascular disrupting agents (VDAs), mainly from the MDA family, induce rapid tumor vessel occlusion and massive tumor necrosis. Thus, tubulin inhibitors have become increasingly popular in the field of tumor vasculature. However, their pharmaceutical application is halted by a number of limitations including poor solubility and toxicity. Thus, recently, there has been considerable interests in the nanoparticle drug delivery of tubulin inhibitors to circumvent those limitations. This article reviews recent advances in nanoparticle based drug delivery for tubulin inhibitors as well as their tumor vasculature disruption properties.

(3-(1H-indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel synthetic compound that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models. I-387 inhibited the in vitro growth of several human cancer cell lines with IC50 values in the range of 15 to 39 nmol/L. Nanomolar concentrations of the compound induced apoptosis and caused phosphorylation of the antiapoptotic protein Bcl-2. I-387 induced a strong and concentration-dependent G2-M arrest in PC-3 cells by constitutive activation of Cdc2/cyclin B1 complex and destabilized polymerization of purified tubulin in vitro by binding to the colchicine-binding site. In vivo, I-387 treatment effectively inhibited tumor growth in mice bearing PC-3 tumor xenografts. In vitro studies of nerve growth factor–dependent neurite outgrowth in PC12 pheochromocytoma cells and in vivo studies of mouse behavior showed that I-387 was less neurotoxic than vinblastine and vincristine, tubulin destabilizers with known neurotoxicity. Interestingly, multidrug-resistant cell lines that overexpressed P-glycoprotein (P-gp), multidrug resistance–associated proteins, and breast cancer resistance protein were rendered resistant to docetaxel, vinblastine, SN-38, and doxorubicin, but not to I-387. I-387 dosed at 10 mg/kg was equally effective with 76% tumor growth inhibition in xenograft models using MES-SA uterine sarcoma cells and MES-SA/DX5 cells overexpressing P-gp. In contrast, docetaxel and vinblastine were not effective in MES-SA/DX5 xenograft models. The potent in vitro and in vivo antitumor activity of I-387 suggests that it may represent a new antimitotic agent for management of various malignancies, particularly for patients with drug-resistant cancer. Mol Cancer Ther; 9(11); 2859–68. ©2010 AACR.

Glucocorticoids are the most widely used antiinflammatory drugs in the world. However, prolonged use of glucocorticoids results in undesirable side effects such as muscle wasting, osteoporosis, and diabetes. Skeletal muscle wasting, which currently has no approved therapy, is a debilitating condition resulting from either reduced muscle protein synthesis or increased degradation. The imbalance in protein synthesis could occur from increased expression and function of muscle-specific ubiquitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle ring finger 1 (MuRF1), or decreased function of the IGF-I and phosphatidylinositol-3 kinase/Akt kinase pathways. We examined the effects of a nonsteroidal tissue selective androgen receptor modulator (SARM) and testosterone on glucocorticoid-induced muscle atrophy and castration-induced muscle atrophy. The SARM and testosterone propionate blocked the dexamethasone-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including Forkhead box O (FoxO). Dexamethasone caused a significant up-regulation in the expression of ubiquitin ligases, but testosterone propionate and SARM administration blocked this effect by phosphorylating FoxO. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of MAFbx and MuRF1 and down-regulation of IGF-I, all of which was attenuated by a SARM. The results suggest that levator ani atrophy caused by hypogonadism may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on up-regulation of MAFbx and MuRF1. Our studies provide the first evidence that glucocorticoid- and hypogonadism-induced muscle atrophy are mediated by distinct but overlapping mechanisms and that SARMs may provide a more effective and selective pharmacological approach to prevent glucocorticoid-induced muscle loss than steroidal androgen therapy.

Women experience a decline in estrogen and androgen levels after natural or surgically induced menopause, effects that are associated with a loss of sexual desire and bone mineral density. Studies in our laboratories have shown the beneficial effects of selective androgen receptor modulators (SARMs) in the treatment of osteoporosis and muscle wasting in animal models. A series of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-cyano-3-trifluoromethyl-phenyl)-propionamide analogs was synthesized to evaluate the effects of B-ring substitutions on in vitro and in vivo pharmacologic activity, especially female sexual motivation. The androgen receptor (AR) relative binding affinities ranged from 0.1 to 26.5% (relative to dihydrotestosterone) and demonstrated a range of agonist activity at 100 nM. In vivo pharmacologic activity was first assessed by using male rats. Structural modifications to the B-ring significantly affected the selectivity of the SARMs, demonstrating that single-atom substitutions can dramatically and unexpectedly influence activity in androgenic (i.e., prostate) and anabolic (i.e., muscle) tissues. (S)-N-(4-cyano-3-trifluoromethyl-phenyl)-3-(3-fluoro,4-chlorophenoxy)-2-hydroxy-2-methyl-propanamide (S-23) displayed full agonist activity in androgenic and anabolic tissues; however, the remaining SARMs were more prostate-sparing, selectively maintaining the size of the levator ani muscle in castrated rats. The partner-preference paradigm was used to evaluate the effects of SARMs on female sexual motivation. With the exception of two four-halo substituted analogs, the SARMs increased sexual motivation in ovariectomized rats, with potency and efficacy comparable with testosterone propionate. These results indicate that the AR is important in regulating female libido given the nonaromatizable nature of SARMs and it could be a superior alternative to steroidal testosterone preparations in the treatment of hypoactive sexual desire disorder.

Nonsteroidal estrogens have been known since the 1930s. However, the relatively recent (1996) discovery of estrogen receptor subtype beta (ERbeta) suggested a possible paradigm shift away from SERM-like selectivity. Selective ERbeta agonism would potentially allow expansion of estrogenic targeting into new indications (discussed herein) currently precluded by the thrombogenic and hyperproliferative effects of nonselective estrogens.ERbeta agonist design has been very successful. Pharmacophores for ERbeta selective nonsteroidal estrogens are generally diphenolic compounds that achieve an inter-phenolic distance and geometry similar to 17beta-estradiol with few restraints on the nature of the element linking the phenols (or phenol mimetics). The tremendously chemodiverse ERbeta agonist patent literature is reviewed, segregating the agonists into structurally similar compounds based on their interphenolic linking elements.A comprehensive understanding of the chemotype landscape of this field and an assessment of its maturation.Subtype selective ERbeta agonist therapy seems very promising. However, more clinical testing is needed to firmly establish its therapeutic potential. At this point, ERbeta is a promising target in search of an indication.

Interest in the development and therapeutic potential of nonsteroidal tissue-selective androgen receptor modulators (SARMs) has increased dramatically within the past decade. Rapidly expanding knowledge of nuclear hormone receptor structure and function and successful proof-of-principle clinical trials with SARMs have revived an almost dormant search for improved androgens. This Award Address attempts to chronicle the landmark discoveries (with emphasis on our work), organize the SARM landscape into clinically relevant bins, and provide insight into the clinical prospects for SARMs. 1.1. Origins of Androgen Use. An early (1889) and unusual experiment in androgen therapy was performed by Charles Edouard Brown-Sequard, age 72. He administered a testicular extract to himself and reported that he felt “increased vigor and capacity for work”. Despite retrospective suggestions that any effect was purely placebo, this report resulted in widespread use of testicular extracts throughout Europe and North America for several decades. Attempts to isolate the active components of testicular extract failed until 1935 when testosterone (17 hydroxy-4-andosten-3-one) was isolated from bull testes. Shortly thereafter, its synthesis was reported. In the same year, extracts of urine from males were shown to cause nitrogen retention, an indicator of anabolic metabolism. Testosterone was the first anabolic androgen to be used clinically, but its use is limited by its androgenicity and pharmacokinetic (PK) issues. 1 In the latter half of the 20th century, the chemical scaffold of testosterone was modified extensively, producing many

Diabetes is a disease that affects over 150 million people worldwide for which there are multiple oral and injectable medications. Because of trends in obesity and sedentary lifestyles, diabetes rates in both developed and developing countries are increasing at an alarming rate. Current medications are not adequately effective in maintaining long-term glycemic control in most patients, even when used in combination, leaving diabetics susceptible to developing life threatening and debilitating complications such as cardiovascular disease, blindness, kidney complications, and amputations. Consequently, there is a critical need for more potent pharmacotherapies with novel mechanisms of action. A panel of 20 emerging diabetes targets is presented, and small molecule modulators for each target will be discussed.

The pharmacologic effects of (S)-N-(4-cyano-3-trifluoromethyl-phenyl)-3-(3-fluoro, 4-chlorophenoxy)-2-hydroxy-2-methyl-propanamide (S-23) were characterized in male rats as an animal model of hormonal male contraception. S-23 showed high binding affinity (inhibitory constant = 1.7 ± 0.2 nm) and was identified as a full agonist in vitro. In castrated male rats, the ED50 of S-23 in the prostate and levator ani muscle was 0.43 and 0.079 mg/d, respectively. In intact male rats treated for 14 d, S-23 alone suppressed LH levels by greater than 50% at doses greater than 0.1 mg/d, with corresponding decreases in the size of the prostate but increases in the size of levator ani muscle. In intact male rats treated for up to 10 wk with S-23 and estradiol benzoate (EB; necessary to maintain sexual behavior in rats), S-23 showed biphasic effects on androgenic tissues and spermatogenesis by suppressing serum concentrations of LH and FSH. EB alone showed no effect on spermatogenesis. In the EB + S-23 (0.1 mg/d) group, four of six animals showed no sperm in the testis and zero pregnancies (none of six) in mating trials. After termination of treatment, infertility was fully reversible, with a 100% pregnancy rate observed after 100 d of recovery. S-23 increased bone mineral density and lean mass but reduced fat mass in a dose-dependent manner. This is the first study to show that a selective androgen receptor modulator combined with EB is an effective and reversible regimen for hormonal male contraception in rats. The beneficial effects of S-23 on the muscle, tissue selectivity, and favorable pharmacokinetic properties make it a strong candidate for use in oral male contraception. An aryl-propionamide selective androgen receptor modulator (S-23) is an effective and reversible agent for hormonal male contraception in rats.

Hydrogen-blending effects in flame structure and NO emission behaviour are numerically studied with detailed chemistry in methane–air counterflow diffusion flames. The composition of fuel is systematically changed from pure methane to the blending fuel of methane–hydrogen through H2 molar addition up to 30%. Flame structure, which can be described representatively as a fuel consumption layer and a H2–CO consumption layer, is shown to be changed considerably in hydrogen-blending methane flames, compared to pure methane flames. The differences are displayed through maximum flame temperature, the overlap of fuel and oxygen, and the behaviours of the production rates of major species. Hydrogen-blending into hydrocarbon fuel can be a promising technology to reduce both the CO and CO2 emissions supposing that NOx emission should be reduced through some technologies in industrial burners. These drastic changes of flame structure affect NO emission behaviour considerably. The changes of thermal NO and prompt NO are also provided according to hydrogen-blending. Importantly contributing reaction steps to prompt NO are addressed in pure methane and hydrogen-blending methane flames. Copyright © 2006 John Wiley & Sons, Ltd.

Experiments in low-strain-rate methane–air counterflow diffusion flames diluted with nitrogen have been conducted to study flame extinction behavior and edge flame oscillation in which flame length is less than the burner diameter and thus lateral conductive heat loss, in addition to radiative loss, could be high at low global strain rates. The critical mole fraction at flame extinction is examined in terms of velocity ratio and global strain rate. Onset conditions of the edge flame oscillation and the relevant modes are also provided with global strain rate and nitrogen mole fraction in the fuel stream or in terms of fuel Lewis number. It is observed that flame length is intimately relevant to lateral heat loss, and this affects flame extinction and edge flame oscillation considerably. Lateral heat loss causes flame oscillation even at fuel Lewis number less than unity. Edge flame oscillations, which result from the advancing and retreating edge flame motion of the outer flame edge of low-strain-rate flames, are categorized into three modes: a growing, a decaying, and a harmonic-oscillation mode. A flame stability map based on the flame oscillation modes is also provided for low-strain-rate flames. The important contribution of lateral heat loss even to edge flame oscillation is clarified finally.

We recently reported two nonsteroidal androgen receptor (AR) ligands that demonstrate tissue-selective pharmacological activity, identifying these S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide analogs as the first members of a new class of drugs known as selective androgen receptor modulators. The purpose of these studies was to explore additional structure-activity relationships of selective androgen receptor modulators to enhance their AR binding affinity, AR-mediated transcriptional activation, and in vivo pharmacological activity. The AR binding affinity (K(i)) of 29 novel synthetic AR ligands was determined by a radioligand competitive binding assay and ranged from 1.0-51 nM. Compounds with electron-withdrawing substituents at the para- and meta-positions of the B-ring demonstrated the highest AR binding affinity. The AR-mediated transcriptional activation was determined using a cotransfection assay in CV-1 cells. Most compounds with two substituents in the B-ring maintained or improved their functional activity in vitro. However, compounds with three halogen substituents exhibited significant regioselectivity. Fifteen compounds were selected to examine their pharmacological activity in castrated rats. In vivo pharmacological activity and selectivity were significantly changed by structural modification in the B-ring. Compounds with halogen groups at the para- and meta-positions of the B-ring displayed the highest pharmacological activity. Incorporating substituents at the ortho-position of the B-ring resulted in poor pharmacological activity. In vitro and in vivo agonist activities were partially correlated. In conclusion, novel selective androgen receptor modulators with improved in vivo pharmacological activity can be designed and synthesized based on the structure-activity relationship identified in these studies.

The androgen receptor (AR) is a nuclear hormone receptor that, upon binding to testosterone, dihydrotestosterone (DHT) and other endogenous androgens, supports the development, growth and maintenance of masculine features through activation of anabolic and androgenic metabolism. The AR has been demonstrated to be a productive therapeutic target. AR ligands in clinical practice include androgenic steroids, antiandrogenic steroids and antiandrogenic nonsteroidals. Of primary importance for this review are nonsteroidal selective AR modulators (SARMs) that have tissue-specific agonist and/or antagonist activities. The AR has a myriad of peripheral and central functions that can be modulated in a pleiotropic and tissue-specific fashion using the increasingly diverse collection of SARMs discussed herein. This suggests that SARMs will have a high level of clinical utility for a wide variety of health conditions. The patent literature focusing on SARMs is reviewed and reveals multiple chemical classes in various ...

Selective androgen receptor modulators (SARMs) have many potential therapeutic applications, including male hypogonadism, osteoporosis, muscle-wasting diseases, sexual libido, and contraception. A series of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamides bearing a four-halogen substituent in the B-ring that displayed in vivo activity were identified in our previous study. Interestingly, in vivo pharmacological activity was not correlated with in vitro androgen receptor (AR) binding affinity. In this study, analysis of the area under the concentration-time curve-response relationship demonstrated that the discrepancy between in vitro and in vivo pharmacological activity of these halogen-substituted SARMs was due to differences in systemic exposure rather than intrinsic pharmacological activity. Studies also suggested that two simple criteria (i.e., Ki10 nM and lower in vivo clearance) could be used to identify efficacious and potent SARMs. We tested this hypothesis using a series of four compounds incorporating either a nitro or cyano substituent at the para-position of the A- and B-aromatic rings. The S-3-(4-nitrophenoxy) and S-3-(4-cyanophenoxy) 2-hydroxy-2-methyl-N-(4-nitro-3-trifluromethylphenyl) propionamides (S-19 and S-20, respectively) and S-3-(4-nitrophenoxy) and S-3-(4-cyanophenoxy) 2-hydroxy-2-methyl-N-(4-cyano-3-trifluromethylphenyl) propionamides (S-21 and S-22, respectively) demonstrated high AR binding affinity, with Ki values ranging from 2.0 to 3.8 nM. Pharmacokinetic studies of selected compounds showed that the in vivo clearance of S-22 was the slowest followed sequentially by S-20, S-21, and S-19. The dose-response relationships for S-22 showed that S-22 exerted efficacious and selective activity in anabolic tissues at dose rates as low as 0.03 mg/day, indicative of the high potency of this compound in anabolic tissue (relative potency 4.41) and its potential for clinical use in androgen deficiency-related disorders.

SUMMARY Numerical study with momentum-balanced boundary conditions has been conducted to grasp chemical effects of added CO2, to either fuel- or oxidizer-side on flame structure and NO emission behaviour in CH4–O2–N2 diffusion flames. Cautious investigation is made for the comparison among the behaviours of principal chain branching and important H-removal key reactions. This describes successfully the reason why flame temperatures for fuel-side dilution are higher than those for oxidizer-side dilution. The role of the principal chain branching reaction is also recognized to be important even in the change of major flame structure caused by chemical effects. The importantly contributing reaction steps to NO production are examined. The reduced production rates of thermal NO and prompt NO due to chemical effects are much more remarkable for fuel-side dilution. It is also found that the reaction step, H+NO+M=HNO þ M plays a decisive role of the formation and destruction of prompt NO. Copyright # 2005 John Wiley & Sons, Ltd.

SUMMARY Numerical study on flame structure and NO emission behaviour has been conducted to grasp chemical effects of added H2O on either fuel- or oxidizer-side in CH4–O2–N2 counterflow diffusion flames. An artificial species, which has the same thermodynamic, transport, and radiation properties of added H2O, is introduced to feasibly isolate the chemical effects. Special concern is focused on the important role of remarkably produced OH radicals due to chemical effects of added H2O on flame structure and NO emission. The reason why the difference of behaviours between the principal chain branching reaction rate and flame temperature appear is attributed to the drastic change of reaction step (R120) from the production to the consumption of OH. It is also, however, seen that the most important contribution of produced OH due to chemical effects of added H2O is through reaction step (R127). The importantly contributing reaction steps to NO production are also examined. The production rates of thermal NO and prompt NO are suppressed by chemical effects of added H2O. The contribution of the reaction steps related to HNO intermediate species to the production of prompt NO is also stressed. Copyright # 2004 John Wiley & Sons, Ltd.

SUMMARY A numerical study with momentum-balanced boundary conditions has been conducted to grasp the chemical effects of added CO2 to fuel- and oxidizer-sides on flame structure and NO emission behaviour in H2–O2 diffusion flames with varying flame location. A reaction mechanism is proposed to show better agreements with experimental results in CO2-added hydrogen flames. Oxidizer-side dilution results in significantly higher flame temperatures and NO emission. Flame location is dramatically changed due to high diffusivity of hydrogen according to variation of the composition of fuel- and oxidizer-sides. This affects flame structure and NO emission considerably especially the chemical effects of added CO2. The present work also displays separately thermal contribution and prompt NO emission due to the chemical effects caused by thermal dissociation of added CO2 in NO emission behaviour. It is found that flame temperature and the flame location affect the contribution of thermal and prompt NO due to chemical effects considerably in NO emission behaviour. Copyright # 2004 John Wiley & Sons, Ltd.

A numerical study has been conducted to clearly grasp the impact of chemical effects caused by added CO 2 and of flame location on flame structure and NO emission behaviour. Flame location affects the major source reaction of CO formation. CO 2 + H → CO + OH and the H-removal reaction, CH 4 + H → CH 3 + H 2 . It is, as a result, seen that the reduction of maximum flame temperature due to chemical effects for fuel-side dilution is mainly caused by the competition of the principal chain branching reaction with the reaction, CH 4 + H → CH 3 + H 2 . while that for fuel-side dilution is attributed to the competition of the principal chain branching reaction with the reaction, CO 2 + H → CO + OH. The importance of the NNH mechanism for NO production, where the reaction pathway is NNH -> NH → HNO, is recognized. In C-related reactions most of NO is the direct outcome of (R171) and the contribution of (R171) becomes more and more important with increasing amount of added CO 2 as much as the reaction step (R171) competes with the key reaction of thermal mechanism, (R237), for N atom. This indicates a possibility that NO emission in hydrogen flames diluted with CO 2 shows less dependent behaviour upon flame temperature.