Development Of Selective Small Molecule Inhibitors Research Articles

Kir5.1 channels: potential role in epilepsy and seizure disorders.

Inwardly rectifying potassium (Kir) channels are broadly expressed in many mammalian organ systems, where they contribute to critical physiological functions. However, the importance and function of the Kir5.1 channel (encoded by the KCNJ16 gene) have not been fully recognized. This review focuses on the recent advances in understanding the expression patterns and functional roles of Kir5.1 channels in fundamental physiological systems vital to potassium homeostasis and neurological disorders. Recent studies have described the role of Kir5.1-forming Kir channels in mouse and rat lines with mutations in the Kcnj16 gene. The animal research reveals distinct renal and neurological phenotypes, including pH and electrolyte imbalances, blunted ventilatory responses to hypercapnia/hypoxia, and seizure disorders. Furthermore, it was confirmed that these phenotypes are reminiscent of those in patient cohorts in which mutations in the KCNJ16 gene have also been identified, further suggesting a critical role for Kir5.1 channels in homeostatic/neural systems health and disease. Future studies that focus on the many functional roles of these channels, expanded genetic screening in human patients, and the development of selective small-molecule inhibitors for Kir5.1 channels, will continue to increase our understanding of this unique Kir channel family member.

The cGAS-STING pathway as a therapeutic target in inflammatory diseases.

The cGAS–STING signalling pathway has emerged as a key mediator of inflammation in the settings of infection, cellular stress and tissue damage. Underlying this broad involvement of the cGAS–STING pathway is its capacity to sense and regulate the cellular response towards microbial and host-derived DNAs, which serve as ubiquitous danger-associated molecules. Insights into the structural and molecular biology of the cGAS–STING pathway have enabled the development of selective small-molecule inhibitors with the potential to target the cGAS–STING axis in a number of inflammatory diseases in humans. Here, we outline the principal elements of the cGAS–STING signalling cascade and discuss the general mechanisms underlying the association of cGAS–STING activity with various autoinflammatory, autoimmune and degenerative diseases. Finally, we outline the chemical nature of recently developed cGAS and STING antagonists and summarize their potential clinical applications.

Kinetic parameters of human aspartate/asparagine–β-hydroxylase suggest that it has a possible function in oxygen sensing

Human aspartate/asparagine–β-hydroxylase (AspH) is a 2-oxoglutarate (2OG)–dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor–like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1–2, 3–4, 5–6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases.

Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer.

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.

Abstract 3011: Development of selective small-molecule inhibitors of cellular MALT1 protease activity

Abstract Background: The aim of this study was to develop potent, selective and cell-permeable inhibitors of MALT1 protease activity. MALT1 is an Arg-specific protease that cleaves multiple substrates, acting as a key component in T- and B-cell receptor signaling in lymphocytes. MALT1 protease activity is reported to mediate normal and disease-associated lymphocyte proliferation and survival as part of the CARMA-1/BCL-10/MALT1 (CBM) signaling complex, making MALT1 an attractive therapeutic target. Constitutive MALT1 activity is a hallmark of Diffuse Large B Cell Lymphoma of the activated B cell type (ABC-DLBCL). This aberrant activation is observed as a consequence of mutations in CARMA-1/CARD11, and in multiple regulatory proteins upstream of the CBM complex. Methods: MALT1 enzyme assay with Ac-Leu-Arg-Ser-Arg-AMC as substrate was used to measure Ki. Selectivity towards other Arg-specific proteases was measured by inhibition of Trypsin and Thrombin. For determination of cellular potency the ABC-DLBCL cell line OCI-LY3, carrying the L251P mutation of CARMA-1/CARD11, was used. A MALT1 cell assay to measure A20 substrate cleavage was adapted to capillary electrophoresis and quantification. MALT1 down-stream signaling was measured by inhibition of IL-6 expression. Results: Published inhibitors of MALT1 were profiled for potency, selectivity, DMPK properties and activity in cell-based assays, and showed several drawbacks such as low MALT1 activity, low cell permeability and/or selectivity. We developed several novel reversible small molecule inhibitors of MALT1 with Ki <20 nM and high selectivity against Trypsin and Thrombin. Compounds show inhibition of intracellular MALT1 activity with potencies <300nM in the A20 cleavage assay, as well as inhibition of IL-6 expression. Intracellular inhibition of MALT1 activity requires both potent inhibition of MALT1 and cell permeability (estimated by Caco-2 permeability), e.g. compound A with MALT1 Ki = 16nM, Trypsin Ki>100μM, Thrombin Ki>100μM, and Caco-2 Papp = 1×10−6cm/s shows intracellular A20 cleavage IC50 = 1100nM, whereas compound B with MALT1 Ki = 47nM and Caco-2 Papp = 5.6×10−6cm/s, displays intracellular A20 cleavage IC50 = 600nM. We observe a clear dissociation between intracellular inhibition of MALT1 and inhibition of cell proliferation of DLBCL cell lines of ABC-type with constitutive MALT1 activity, e.g. compound C (MALT1 Ki = 35nM, Trypsin Ki>100μM, Thrombin Ki>100μM and intracellular A20 cleavage IC50 = 260nM) inhibits the proliferation of OCI-LY3 cells and TMD8 cells with CC50 values of 73μM and 96μM respectively, after 5 days exposure. Conclusion: Inhibition of MALT1 protease activity did not translate into significant anti-proliferative effects in DLBCL cell lines. These new MALT1 inhibitors provide the opportunity to test other therapeutic hypotheses involving the targeting of MALT1 protease activity. Citation Format: Fredrik G. Oberg, Sofia Karlstrom, Ian Henderson, Ellen Hewitt, Anders Kallin, Susanne Sedig, Jimmy Lindberg, Anna-Karin Sohlenius-Sternbeck, Richard Bethell, Mark Albertella. Development of selective small-molecule inhibitors of cellular MALT1 protease activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3011.

Mechanisms of histone lysine-modifying enzymes: A computational perspective on the role of the protein environment

Epigenetic pathways are involved in a wide range of diseases, including cancer and neurological disorders. Specifically, histone modifying and reading processes are the most broadly studied and are targeted by several licensed drugs. Although there have been significant advances in understanding the mechanistic aspects underlying epigenetic regulation, the development of selective small-molecule inhibitors remains a challenge.Experimentally, it is generally difficult to elucidate the atomistic basis for substrate recognition, as well as the sequence of events involved in binding and the subsequent chemical processes. In this regard, computational modelling is particularly valuable, since it can provide structural features (including transition state structures along with kinetic and thermodynamic parameters) that enable both qualitative and quantitative evaluation of the mechanistic details involved. Here, we summarize knowledge gained from computational modelling studies elucidating the role of the protein environment in histone-lysine modifying and reading mechanisms. We give a perspective on the importance of calculations to aid and advance the understanding of these processes and for the future development of selective inhibitors for epigenetic regulators.

Directed Discovery of Agents Targeting the Met Tyrosine Kinase Domain by Virtual Screening

Hepatocyte growth factor (HGF) is an important regulator of normal development and homeostasis, and dysregulated signaling through the HGF receptor, Met, contributes to tumorigenesis, tumor progression, and metastasis in numerous human malignancies. The development of selective small-molecule inhibitors of oncogenic tyrosine kinases (TK) has led to well-tolerated, targeted therapies for a growing number of cancer types. To identify selective Met TK inhibitors, we used a high-throughput virtual screen of the 13.5 million compound ChemNavigator database to find compounds most likely to bind to the Met ATP binding site and to form several critical interactions with binding site residues predicted to stabilize the kinase domain in its inactive conformation. Subsequent biological screening of 70 in silico hit structures using cell-free and intact cell assays identified three active compounds with micromolar IC(50) values. The predicted binding modes and target selectivity of these compounds are discussed and compared to other known Met TK inhibitors.