Discovery of Orally Bioavailable Ligand Efficient Quinazolindiones as Potent and Selective Tankyrases Inhibitors.

We report herein the discovery of quinazolindiones as potent and selective tankyrase inhibitors. Elucidation of the structure-activity relationship of the lead compound 1g led to truncated analogues that have good potency in cells, pharmacokinetic (PK) properties, and excellent selectivity. Compound 21 exhibited excellent potencies in cells and proliferation studies, good selectivity, in vitro activities, and an excellent PK profile. Compound 21 also inhibited H292 xenograft tumor growth in nude mice. The synthesis, biological, pharmacokinetic, in vivo efficacy studies, and safety profiles of compounds are presented.

Identification and Optimization of Novel Small c-Abl Kinase Activators Using Fragment and HTS Methodologies.

Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.

Enteric RET inhibition attenuates gastrointestinal secretion and motility via cholinergic signaling in rat colonic mucosal preparations.

BACKGROUND: The expression of RET in the developing enteric nervous system (ENS) suggests that RET may contribute to adult intestinal function. ENS cholinergic nerves play a critical role in the control of colonic function through the release of acetylcholine (ACh). In the current study, we hypothesized that a RET-mediated mechanism may regulate colonic ion transport and motility through modulation of cholinergic nerves. METHODS: The effect of RET inhibition on active ion transport was assessed electrophysiologically in rat colonic tissue mounted in Ussing chambers via measurements of short circuit current (Isc) upon electrical field stimulation (EFS) or pharmacologically with cholinergic agonists utilizing a gastrointestinal (GI)-restricted RET inhibitor. We assessed the effect of the RET inhibitor on propulsive motility via quantification of fecal pellet output (FPO) induced by the acetylcholinesterase inhibitor neostigmine. KEY RESULTS: We found that enteric ganglia co-expressed RET and choline acetyltransferase (ChAT) transcripts. In vitro, the RET kinase inhibitor GSK3179106 attenuated the mean increase in Isc induced by either EFS or carbachol but not bethanechol. In vivo, GSK3179106 significantly reduced the prokinetic effect of neostigmine. CONCLUSION AND INFERENCES: Our findings provide evidence that RET-mediated mechanisms regulate colonic function by maintaining cholinergic neuronal function and enabling ACh-evoked chloride secretion and motility. We suggest that modulating the cholinergic control of the colon via a RET inhibitor may represent a novel target for the treatment of intestinal disorders associated with increased secretion and accelerated GI transit such as irritable bowel syndrome with diarrhea (IBS-D).

Exploring the Potential of RET Kinase Inhibition for Irritable Bowel Syndrome: A Preclinical Investigation in Rodent Models of Colonic Hypersensitivity.

Abdominal pain represents a significant complaint in patients with irritable bowel syndrome (IBS). While the etiology of IBS is incompletely understood, prior exposure to gastrointestinal inflammation or psychologic stress is frequently associated with the development of symptoms. Inflammation or stress-induced expression of growth factors or cytokines may contribute to the pathophysiology of IBS. Here, we aimed to investigate the therapeutic potential of inhibiting the receptor of glial cell line-derived neurotrophic factor, rearranged during transfection (RET), in experimental models of inflammation and stress-induced visceral hypersensitivity resembling IBS sequelae. In RET-cyan fluorescent protein [(CFP) RetCFP/+] mice, thoracic and lumbosacral dorsal root ganglia were shown to express RET, which colocalized with calcitonin gene-related peptide. To understand the role of RET in visceral nociception, we employed GSK3179106 as a potent, selective, and gut-restricted RET kinase inhibitor. Colonic hyperalgesia, quantified as exaggerated visceromotor response to graded pressures (0-60 mm Hg) of isobaric colorectal distension (CRD), was produced in multiple rat models induced 1) by colonic irritation, 2) following acute colonic inflammation, 3) by adulthood stress, and 4) by early life stress. In all the rat models, RET inhibition with GSK3179106 attenuated the number of abdominal contractions induced by CRD. Our findings identify a role for RET in visceral nociception. Inhibition of RET kinase with a potent, selective, and gut-restricted small molecule may represent a novel therapeutic strategy for the treatment of IBS through the attenuation of post-inflammatory and stress-induced visceral hypersensitivity.

Discovery of a First-in-Class Gut-Restricted RET Kinase Inhibitor as a Clinical Candidate for the Treatment of IBS.

Abdominal pain and abnormal bowel habits represent major symptoms for irritable bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our screening effort and subsequent structure-activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small molecule RET kinase inhibitor with a RET IC50 of 0.3 nM and is efficacious in vivo.

TLR2 agonism reverses chemotherapy-induced neutropenia in Macaca fascicularis.

Neutropenia is a common consequence of radiation and chemotherapy in cancer patients. The resulting immunocompromised patients become highly susceptible to potentially life-threatening infections. Granulocyte colony-stimulating factor (G-CSF) is known to stimulate neutrophil production and is widely used as a treatment of chemotherapy-induced neutropenia. A small-molecule G-CSF secretagogue without a requirement for refrigerated supply chain would offer a more convenient and cost-effective treatment of chemotherapy-induced neutropenia. Bacterial lipopeptides activate innate immune responses through Toll-like receptor 2 (TLR2) and induce the release of cytokines, including G-CSF, from macrophages, monocytes, and endothelial. Pam2CSK4 is a synthetic lipopeptide that effectively mimics bacterial lipoproteins known to activate TLR2 receptor signaling through the TLR2/6 heterodimer. Substrate-based drug design led to the discovery of GSK3277329, which stimulated the release of G-CSF in activated THP-1 cells, peripheral blood mononuclear cells, and human umbilical vein endothelial cells. When administered subcutaneously to cynomolgus monkeys (Macaca fascicularis), GSK3277329 caused systemic elevation of G-CSF and interleukin-6 (IL-6), but not IL-1ß or tumor necrosis factor α, indicating a selective cytokine-stimulation profile. Repeat daily injections of GSK3277329 in healthy monkeys also raised circulating neutrophils above the normal range over a 1-week treatment period. More importantly, repeated daily injections of GSK3277329 over a 2-week period restored neutrophil loss in monkeys given chemotherapy treatment (cyclophosphamide, Cytoxan). These data demonstrate preclinical in vivo proof of concept that TLR2 agonism can drive both G-CSF induction and subsequent neutrophil elevation in the cynomolgus monkey and could be a therapeutic strategy for the treatment of chemotherapy-induced neutropenia.

Development of a high-throughput screen to detect inhibitors of TRPS1 sumoylation.

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK1201TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC50 values of 12.5 µM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.

Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site.

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

Matrix metalloproteinase-activated doxorubicin prodrugs inhibit HT1080 xenograft growth better than doxorubicin with less toxicity.

Matrix metalloproteinase (MMP)-activated prodrugs were formed by coupling MMP-cleavable peptides to doxorubicin. The resulting conjugates were excellent in vitro substrates for MMP-2, -9, and -14. HT1080, a fibrosarcoma cell line, was used as a model system to test these prodrugs because these cells, like tumor stromal fibroblasts, expressed several MMPs. In cultured HT1080 cells, simple MMP-cleavable peptides were primarily metabolized by neprilysin, a membrane-bound metalloproteinase. MMP-selective metabolism in cultured HT1080 cells was obtained by designing conjugates that were good MMP substrates but poor neprilysin substrates. To determine how conjugates were metabolized in animals, MMP-selective conjugates were given to mice with HT1080 xenografts and the distribution of doxorubicin was determined. These studies showed that MMP-selective conjugates were preferentially metabolized in HT1080 xenografts, relative to heart and plasma, leading to 10-fold increases in the tumor/heart ratio of doxorubicin. The doxorubicin deposited by a MMP-selective prodrug, compound 6, was more effective than doxorubicin at reducing HT1080 xenograft growth. In particular, compound 6 cured 8 of 10 mice with HT1080 xenografts at doses below the maximum tolerated dose, whereas doxorubicin cured 2 of 20 mice at its maximum tolerated dose. Compound 6 was less toxic than doxorubicin at this efficacious dose because mice treated with compound 6 had no detectable changes in body weight or reticulocytes, a marker for marrow toxicity. Hence, MMP-activated doxorubicin prodrugs have a much higher therapeutic index than doxorubicin using HT1080 xenografts as a preclinical model.

A prostate-specific antigen (PSA)-activated vinblastine prodrug selectively kills PSA-secreting cells in vivo.

Currently, there is no therapy for men with androgen-refractory prostate cancer that substantially extends survival. This report characterizes by in vitro and in vivo techniques a new chemotherapeutic that is composed of desacetyl-vinblastine covalently linked to a peptide that contains a peptide bond that can be hydrolyzed by prostate-specific antigen (PSA). This compound (referred to as vinblastine-conjugate) is minimally toxic to cells in culture which do not express PSA. In the presence of PSA, the peptide moiety is hydrolyzed, generating several highly toxic metabolites that contain vinblastine. Animals bearing PSA-positive human prostate tumors that were treated with the vinblastine-conjugate experienced a >99% reduction in PSA serum level. In contrast, animals bearing PSA-positive human prostate tumors treated with the cytotoxic metabolites derived from the PSA hydrolysis of the vinblastine-conjugate showed a nonsignificant change in both PSA and tumor weight values. The cell killing activity of the vinblastine-conjugate is PSA dependent because animals bearing non-PSA-producing human tumor xenografts had a nonsignificant increase in tumor weight after vinblastine-conjugate treatment. Exploratory efficacy/toxicity studies in LNCaP tumor-bearing nude mice were conducted with animals treated for 5 consecutive days with various doses of either the vinblastine-conjugate or a PSA-generated toxic metabolite (desacetyl-vinblastine). The desacetyl-vinblastine treatment resulted in 10-70% mortality with a very slight effect on tumor growth. In contrast, vinblastine-conjugate treatments resulted in no mortality, good to excellent antitumor efficacy, very slight to slight peripheral neuropathy and myelopathy, and slight to severe testicular degeneration. Similar treatment of beagle dogs with the vinblastine-conjugate showed even less toxicity. These data support the use of the PSA-hydrolyzable vinblastine-conjugate as an experimental therapy for prostate cancer in man.

Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity.

Chemotherapy of prostate cancer with antimitotic agents such as vinblastine and doxorubicin is only marginally effective, due to dose-limiting systemic toxicity. Herein we report the development of peptidyl conjugate 5 of the cytotoxic agent vinblastine (1), along with the results of its in vitro and in vivo evaluation as a pro-drug targeted at prostate cancer cells. Prostate-derived tumors are known to produce significant amounts of prostate specific antigen (PSA), a serine protease with chymotrypsin-like properties. Earlier work in these laboratories established that an appropriately engineered peptidyl pro-drug will release active cytotoxic agent strictly within the microenvironment of the tumor tissue (Garsky, V. M., et al. J. Med.Chem. 2001, 44, 4216-4224). Conjugate 5, which features an octapeptide segment attached by an ester linkage at the 4-position of vinblastine (1), undergoes rapid cleavage by PSA (T(1/2) = 12 min) between the Gln and Ser residues. In nude mouse xenograft studies, 5 reduced circulating PSA levels by 99% and tumor weight by 85% at a dose just below its MTD. By contrast, the putative end-point metabolite, the cytotoxic agent des-acetyl vinblastine (1b), was ineffective in reducing PSA levels and tumor burden at its maximum tolerated doses. Additional data from metabolism studies on 5 support the supervention of a novel in vivo processing mechanism, the spontaneous release of 1b from a dipeptidyl intermediate driven by favorable diketopiperazine formation.