Identification of a targetable JAK-STAT enriched androgen receptor and androgen receptor splice variant positive triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.

Metabolism-Guided Selective Androgen Receptor Antagonists: Design, Synthesis, and Biological Evaluation for Activity against Enzalutamide-Resistant Prostate Cancer.

A major challenge for new drug discovery in the area of androgen receptor (AR) antagonists lies in predicting the druggable properties that will enable small molecules to retain their potency and stability during further studies in vitro and in vivo. Indole (compound 8) is a first-in-class AR antagonist with very high potency (IC50 = 0.085 µM) but is metabolically unstable. During the metabolic studies described herein, we synthesized new small molecules that exhibit significantly improved stability while retaining potent antagonistic activity for an AR. This structure-activity relationship (SAR) study of more than 50 compounds classified with three classes (Class I, II, and III) and discovered two compounds (32c and 35i) that are potent AR antagonists (e.g., IC50 = 0.021 µM, T1/2 = 120 min for compound 35i). The new antagonists exhibited improved in vivo pharmacokinetics (PK) with high efficacy antiandrogen activity in Hershberger and antiandrogen Enz-Res tumor xenograft models that overexpress AR (LNCaP-AR).

Inhibiting androgen receptor splice variants with cysteine-selective irreversible covalent inhibitors to treat prostate cancer.

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.

Exploration and Biological Evaluation of Basic Heteromonocyclic Propanamide Derivatives as SARDs for the Treatment of Enzalutamide-Resistant Prostate Cancer.

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.

An Overview of Next-Generation Androgen Receptor-Targeted Therapeutics in Development for the Treatment of Prostate Cancer.

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.

Pyrazol-1-yl-propanamides as SARD and Pan-Antagonists for the Treatment of Enzalutamide-Resistant Prostate Cancer.

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.

Orally Bioavailable Androgen Receptor Degrader, Potential Next-Generation Therapeutic for Enzalutamide-Resistant Prostate Cancer.

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.

Colchicine Binding Site Agent DJ95 Overcomes Drug Resistance and Exhibits Antitumor Efficacy.

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.

Current advances of tubulin inhibitors as dual acting small molecules for cancer therapy.

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.

New Generation of Selective Androgen Receptor Degraders: Our Initial Design, Synthesis, and Biological Evaluation of New Compounds with Enzalutamide-Resistant Prostate Cancer Activity.

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.

Computationally identified novel agonists for GPRC6A.

New insights into G protein coupled receptor regulation of glucose metabolism by ß-cells, skeletal muscle and liver hepatocytes identify GPRC6A as a potential therapeutic target for treating type 2 diabetes mellitus (T2D). Activating GPRC6A with a small molecule drug represents a potential paradigm-shifting opportunity to make significant strides in regulating glucose homeostasis by simultaneously correcting multiple metabolic derangements that underlie T2D, including abnormalities in ß-cell proliferation and insulin secretion and peripheral insulin resistance. Using a computational, structure-based high-throughput screening approach, we identified novel tri-phenyl compounds predicted to bind to the venus fly trap (VFT) and 7-transmembrane (7-TM) domains of GPRC6A. Experimental testing found that these compounds dose-dependently stimulated GPRC6A signaling in a heterologous cell expression system. Additional chemical modifications and functional analysis identified one tri-phenyl lead compound, DJ-V-159 that demonstrated the greatest potency in stimulating insulin secretion in ß-cells and lowering serum glucose in wild-type mice. Collectively, these studies show that GPRC6A is a "druggable" target for developing chemical probes to treat T2DM.

A Potent, Metabolically Stable Tubulin Inhibitor Targets the Colchicine Binding Site and Overcomes Taxane Resistance.

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.

Novel Selective Agents for the Degradation of Androgen Receptor Variants to Treat Castration-Resistant Prostate Cancer.

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.

Current Advances of Tubulin Inhibitors in Nanoparticle Drug Delivery and Vascular Disruption/Angiogenesis.

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.

Structural and Functional Evidence for Testosterone Activation of GPRC6A in Peripheral Tissues.

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.

Structural Optimization of Indole Derivatives Acting at Colchicine Binding Site as Potential Anticancer Agents.

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.

Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding.

The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2 via residues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.

Pharmacokinetics, pharmacodynamics and metabolism of a novel anticancer agent for prostate cancer.

The objective of these studies was to examine the murine pharmacokinetics, pharmacodynamics and metabolism of (3-(1H-indol-2-yl)phenyl)(1H-indol-2-yl)methanone (Indole 15), a novel tubulin inhibitor for the treatment of cancer. We developed HPLC and LC/MS/MS assays to quantitate Indole 15 and characterize its metabolites in vivo. Pharmacokinetic studies were performed after intravenous (IV), oral (PO) and subcutaneous (SC) administration of 10 mg/kg doses to male ICR mice. Urine and fecal samples were also collected over a 72-h period to identify metabolites. Pharmacodynamic studies were conducted by monitoring the tumor size change during a period of two weeks in PC-3 tumor bearing mice after daily IV administration of Indole 15 at doses of 0, 10, 50, 100 and 150 mg/kg. The pooled plasma concentration data after administration via different dose routes was simultaneously fitted by a two-compartmental model. Indole 15 was moderately well absorbed after PO and SC administration, with a bioavailable fraction of 0.27 and 0.72, respectively. The steady state volume distribution (Vss) and clearance (CL) were estimated to be 7.0 l/kg and 4.36 l/h/kg, respectively. The mean data of PC-3 tumor growth in mice was fitted well by a transduction model using fixed plasma pharmacokinetics as a driving function. Analysis of the metabolites in mice indicated that the compound undergoes extensive oxidative metabolism with subsequent sulfation. These studies demonstrate that favorable pharmacokinetic and pharmacodynamic properties of Indole 15 offer hope for achieving pharmacological activity in early clinical trials.

A novel bis-indole destabilizes microtubules and displays potent in vitro and in vivo antitumor activity in prostate cancer.

PURPOSE: Microtubules are one of the most useful subcellular targets in chemotherapy. We identified a novel indole, (3-(1H-indol-2-yl)phenyl)(1H-indol-2-yl)methanone (15), that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models. METHODS: In vitro cancer cell growth inhibition was measured by SRB or MTT assay in human cancer cell lines. Apoptosis induced by 15 was examined in LNCaP and PC-3 cells. Effects of 15 on cell cycle distribution and tubulin were investigated via in vitro models. In vivo toxicity and xenograft efficacy studies were conducted in mice. RESULTS: Indole 15 inhibited the in vitro growth of a number of human cancer cell lines, including drug-resistant cell lines that over-express P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein with IC(50) values in the range of 34-162 nM. Nanomolar concentrations of the compound caused down-regulation of bcl-2, induced PARP cleavage, and induced apoptosis in both LNCaP and PC-3 prostate cancer cells, as confirmed by anti-histone ELISA and DNA laddering. In vitro studies revealed that the compound inhibited polymerization of purified tubulin and induced a strong and concentration-dependent G(2)M arrest in PC-3 cells. In vivo studies in immunodeficient mice bearing PC-3 tumor xenografts showed that the compound effectively inhibited tumor growth. CONCLUSIONS: The potent in vitro and in vivo antitumor activities of this novel indole suggest that drugs with this novel chemical scaffold might be developed for treatment of drug-resistant prostate cancer.

I-387, a novel antimitotic indole, displays a potent in vitro and in vivo antitumor activity with less neurotoxicity.

(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.