Novel Class Of Small-molecule Inhibitors Research Articles

Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1.

As a mitotic-specific target widely deregulated in various human cancers, polo-like kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT (Kd ∼ 450 nM). In addition, S-methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.

Design, synthesis, evaluation, and SAR of 4-phenylindoline derivatives, a novel class of small-molecule inhibitors of the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) interaction.

The blockade of the PD-1/PD-L1 immune checkpoint pathway with small molecules is an emerging immunotherapeutic approach. A novel series of 4-phenylindoline derivatives were synthesized, and their inhibitory activity against the PD-1/PD-L1 protein-protein interaction (PPI) was evaluated through a homogenous time-resolved fluorescence (HTRF) assay. Among them, A20 and A22 exhibited potent activity with IC50 values of 17nM and 12nM, respectively. Furthermore, A20 showed the promising inhibitory activity against the PD-1/PD-L1 interaction with the EC50 value of 0.43μM in a co-culture model of PD-L1/TCR Activator-expressing CHO cells and PD-1-expressing Jurkat cells. Besides, the structure-activity relationships (SAR) of the novel synthesized 4-phenylindoline derivatives was concluded, and the binding mode of A22 with the PD-L1 dimer was analyzed by molecular simulation and docking, demonstrating that the N-atom in the side chain of indoline fragment could interact with the amino acid residue of the PD-L1 protein to lead to the potent inhibitory activity. This study provided a new insight for further drug design.

Identification and characterization of the Cucurbitacins, a novel class of small-molecule inhibitors of Tropomyosin receptor kinase a

BackgroundNGF-TrkA is well known to play a key role in propagating and sustaining pruritogenic signals, which form the pathology of chronic pruritus. Inhibition of NGF-TrkA is a known strategy for the treatment of pruritus. In the present paper, we describe the identification, in vitro characterization, structure–activity analysis, and inhibitory evaluation of a novel TrkA inhibitory scaffold exemplified by Cucurbitacins (Cus).MethodsCus were identified as TrkA inhibitors in a large-scale kinase library screen. To obtain structural models of Cus as TrkA inhibitors, AutoDock was used to explore their binding to TrkA. Furthermore, PC12 cell culture systems have been used to study the effects of Cus and traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) extracts on the kinase activity of TrkA.ResultsCus block the phosphorylation of TrkA on several tyrosine sites, including Tyr490, Tyr674/675, and Tyr785, and inhibit downstream Akt and MAPK phosphorylation in response to NGF in PC12 cell model systems. Furthermore, traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) containing Cu extracts were shown to inhibit the phosphorylation of TrkA and Akt. These data reveal mechanisms, at least partly, of the anti-pruritus bioactivity of Cus.ConclusionTaken together, with the recent discovery of the important role of TrkA as a therapeutic target, Cus could be the basis for the design of improved TrkA kinase inhibitors, which could someday help treat pruritus.

Abstract 1296: Arylsulfonamide KCN1 suppresses primary and metastatic growth of uveal melanoma through anti-angiogenic and anti-invasion mechanisms

Abstract Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults with a survival time less than 1 year for patients that develop metastases (about 50%). HIF-1 plays a critical role in UM adaptation to the hypoxic microenvironment, treatment failure, and metastasis. In this study, we evaluated biodistribution and anti-cancer efficacy of arylsulfonamide KCN1, a lead compound in a novel class of small-molecule inhibitors of the hypoxia-inducible factor (HIF-1) pathway in UM models. PET with 11C-labeled KCN1 showed that KCN1 preferentially localizes to the eye and liver, the organ where UM preferentially metastasizes. In survival studies, UM cells were injected into the suprachoroidal space of the right murine eye using a transscleral technique and the eye was enucleated 7-9 days post-inoculation. Starting on Day 1, KCN1 or vehicle only (cremophor EL/ethanol 1:1, diluted with sterile PBS 1:5) were administered daily i.p., 5 x week, until animals reached the endpoint. Main organs were collected and evaluated for metastatic load. We found that KCN1 potently inhibited the growth of primary eye tumors, VEGF expression, angiogenesis in the primary tumor, the number and size of hepatic metastases, and extended mice survival. KCN1 inhibited invasion of UM cells in in vitromodels, such as scratch-wound and Boyden chamber migration assays. To identify possible mediators of hypoxia-HIF-induced metastatic processes inhibited by KCN1 in UM, we tested a series of 20 candidates by qRT-PCR. Among the most consistently HIF-induced/KCN1-inhibited genes were prolyl-4-hydroxylases P4HA1 and 2 that modulate physical properties of the tumor microenvironment by post-translationally modifying collagen. Expression of P4HA1 and 2 is significantly higher in UM patients with metastatic disease and high level of expression correlates with poor prognosis. In summary, our studies suggest KCN1 exerts anti UM activity by inhibiting the primary tumor growth, reducing vascular density in the primary tumor, and the number and size of the hepatic metastases; KCN1 has therefore therapeutic potential for treatment of UM and further clinical development. Citation Format: Stefan Kaluz, Qing Zhang, Hua Yang, Satoru Osuka, Jiyoung Mun, Narra S. Devi, Mark Goodman, Hans E. Grossniklaus, Erwin G. Van Meir. Arylsulfonamide KCN1 suppresses primary and metastatic growth of uveal melanoma through anti-angiogenic and anti-invasion mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1296.

Abstract 4860: A novel class of small-molecule inhibitors of galectin-1 for prostate cancer therapy

Abstract Galectin-1 (Gal-1) is a multifunctional carbohydrate-binding protein and plays a key role in cancer cell proliferation, apoptosis, cell cycle, tumor angiogenesis and evasion of immune responses. It is also directly involved in the process of angiogenesis and tumor immune escape. An elevated level of Gal-1 has been found in many cancers, including prostate cancer. We show by immunohistochemistry in human prostate tissue that Gal-1 expression level was very low in all non-tumor samples and highly expressed in prostate cancer tissues. In addition, Gal-1 was progressively upregulated from low- , intermediate- to high-grade prostate cancer. Taken together, Gal-1 is an excellent therapeutic target against cancer. Yet, no inhibitors against Gal-1 have been approved by the FDA for cancer treatment due to lack of potent, specific and in vivo effective molecules. We recently discovered a novel small-molecule inhibitor of Gal-1, named LLS2, through a high-throughput one-bead two-compound combinatorial library approach. LLS2 exhibits cytotoxic activity against prostate cancer and ovarian cancer cells. We synthesized a series of LLS2 analogs and performed structure-activity relationship studies, leading to a more potent inhibitor LLS30 that induced apoptosis in castration-resistant prostate cancer cells expressing high levels of Gal-1. Molecular modeling study showed LLS30 bound to the sugar-binding groove of Gal-1. In enzalutamide (ENZ)-resistant 22Rv1 prostate cancer cells expressing high levels of Gal-1 and androgen receptor (AR) splice variants, LLS30 could inhibit expression of AR splice variants and sensitize the cells to ENZ in a dose-dependent manner. Downregulation of Gal-1 by siRNA prevented the expression of alternatively spliced AR variants. More importantly, LLS30 showed potent in vivo antitumor efficacy and synergistic activities with docetaxel and ENZ against PC-3 and 22Rv1 prostate cancer models, respectively. Blood chemistry test from the treated animal showed minimum effect of LLS30 on the liver and kidney functions, implying low toxicity. LLS30 is an excellent drug candidate for preclinical development as a novel prostate cancer therapeutic. Citation Format: Ruiwu Liu, Tsung-Chieh Shih, Sophie Kiss, Xiaocen Li, Ting Wang, Jonathan S. Huynh, Chun-Te Wu, Yong Duan, Paramita M. Ghosh, Kit S. Lam. A novel class of small-molecule inhibitors of galectin-1 for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4860.

Discovery of substituted 1,4-dihydroquinolines as novel class of ABCB1 modulators

Transmembrane efflux pumps are one main cause for multidrug resistance (mdr) of cancer. One hopeful approach to combate the mdr has been the development of inhibitors of the efflux pump activity. A novel class of small-molecule inhibitors of the most important efflux pump ABCB1 (P-glycoprotein) has been discovered. Inhibitory activities are discussed in relation to substituent effects. Most active compounds have been evaluated in first bioanalytical studies to reverse the mdr of an anticancer drug. Cellular toxicity and ABCB1 substrate properties of the compounds were investigated. A cellular induction of relevant efflux pump protein expressions was not observed under inhibitor application, so that our compounds are perspective candidates for further preclinical studies.

Design, synthesis and in vitro and in vivo antitumour activity of 3-benzylideneindolin-2-one derivatives, a novel class of small-molecule inhibitors of the MDM2–p53 interaction

A novel class of small-molecule inhibitors of MDM2–p53 interaction with a (E)-3-benzylideneindolin-2-one scaffold was identified using an integrated virtual screening strategy that combined both pharmacophore- and structure-based approaches. The hit optimisation identified several compounds with more potent activity than the hit compound and the positive drug nutlin-3a, especially compound 1b, which exhibited both the highest binding affinity to MDM2 (Ki = 0.093 μM) and the most potent antiproliferative activity against HCT116 (wild type p53) cells (GI50 = 13.42 μM). Additionally, 1b dose-dependently inhibited tumour growth in BALB/c mice bearing CT26 colon carcinoma, with no visible sign of toxicity. In summary, compound 1b represents a novel and promising lead structure for the development of anticancer drugs as MDM2–p53 interaction disruptors.

High-affinity small-molecule inhibitors of the menin-mixed lineage leukemia (MLL) interaction closely mimic a natural protein-protein interaction.

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

Abstract 5678: Design of GRP78 inhibitors as novel therapeutics for breast cancer.

Abstract Cancer cells exploit cellular stress response mechanisms, such as the unfolded protein response, to survive through cytotoxic treatments. A key mediator of the unfolded protein response, the 78kDa glucose regulated protein (GRP78) chaperone, has frequently been found to be overexpressed in breast cancer. In addition to providing a growth advantage to cancer cells, GRP78 induction leads to drug resistance. Suppressing GRP78 levels using siRNA has been shown to slow cancer cell progression and overcome drug resistance. Another approach to inactivating GRP78 is by inhibiting its ATPase activity. Currently, there are only few selective GRP78 inhibitors in development despite ample evidence of its critical role in cancer cell survival and drug resistance. Here, we report a novel class of small-molecule inhibitors of GRP78 ATPase activity. We designed a focused library of compounds based on a structure-based docking approach and screened for inhibition of GRP78 ATPase activity. Based of the initial hit, we further optimized several analogues with more potent activity and selectivity for GRP78 inhibition (IC50 <5μM). The lead compound, KR-240, induced CHOP significantly under conditions of ER stress and activated the pro-apoptotic arm of the unfolded protein response. KR-240 showed moderate cytotoxicity against a panel of breast cancer cell lines and increased sensitivity to chemotherapeutic agents, further supporting the role for GRP78 in cancer survival and drug resistance. Overall, our results indicate that our novel GRP78 inhibitor, KR-240 has the potential for further development as an anticancer agent. Citation Format: Kavya Ramkumar, Bikash Debnath, Amy S. Lee, Nouri Neamati. Design of GRP78 inhibitors as novel therapeutics for breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5678. doi:10.1158/1538-7445.AM2013-5678

Pharmacologic Targeting of Bacterial β-Glucuronidase Alleviates Nonsteroidal Anti-Inflammatory Drug-Induced Enteropathy in Mice

Small intestinal mucosal injury is a frequent adverse effect caused by nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms are not completely understood, but topical (luminal) effects have been implicated. Many carboxylic acid-containing NSAIDs, including diclofenac (DCF), are metabolized to acyl glucuronides (AGs), and/or ether glucuronides after ring hydroxylation, and exported into the biliary tree. In the gut, these conjugates are cleaved by bacterial β-glucuronidase, releasing the potentially harmful aglycone. We first confirmed that DCF-AG was an excellent substrate for purified Escherichia coli β-D-glucuronidase. Using a previously characterized novel bacteria-specific β-glucuronidase inhibitor (Inhibitor-1), we then found that the enzymatic hydrolysis of DCF-AG in vitro was inhibited concentration dependently (IC₅₀ ∼164 nM). We next hypothesized that pharmacologic inhibition of bacterial β-glucuronidase would reduce exposure of enterocytes to the aglycone and, as a result, alleviate enteropathy. C57BL/6J mice were administered an ulcerogenic dose of DCF (60 mg/kg i.p.) with or without oral pretreatment with Inhibitor-1 (10 μg per mouse, b.i.d.). Whereas DCF alone caused the formation of numerous large ulcers in the distal parts of the small intestine and increased (2-fold) the intestinal permeability to fluorescein isothiocyanate-dextran, Inhibitor-1 cotreatment significantly alleviated mucosal injury and reduced all parameters of enteropathy. Pharmacokinetic profiling of DCF plasma levels in mice revealed that Inhibitor-1 coadministration did not significantly alter the C(max), half-life, or area under the plasma concentration versus time curve of DCF. Thus, highly selective pharmacologic targeting of luminal bacterial β-D-glucuronidase by a novel class of small-molecule inhibitors protects against DCF-induced enteropathy without altering systemic drug exposure.

Structural Modification of the P2‘ Position of 2,7-Dialkyl-Substituted 5(S)-Amino-4(S)-hydroxy-8-phenyl-octanecarboxamides: The Discovery of Aliskiren, a Potent Nonpeptide Human Renin Inhibitor Active after Once Daily Dosing in Marmosets

Due to its function in the rate limiting initial step of the renin-angiotensin system, renin is a particularly promising target for drugs designed to control hypertension, a growing risk to health worldwide. Despite vast efforts over more than two decades, no orally efficacious renin inhibitor had reached the market. As a result of a structure-based topological design approach, we have identified a novel class of small-molecule inhibitors with good oral blood-pressure lowering effects in primates. Further lead optimization aimed for improvement of in vivo potency and duration of action, mainly by P2' modifications at the hydroxyethylene transition-state isostere. These efforts resulted in the discovery of aliskiren (46, CGP060536B, SPP100), a highly potent, selective inhibitor of renin, demonstrating excellent efficacy in sodium-depleted marmosets after oral administration, with sustained duration of action in reducing dose-dependently mean arterial blood pressure. Aliskiren has recently received regulatory approval by the U.S. Food and Drug Administration for the treatment of hypertension.