Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a "tough target" to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a noncatalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases, which is absent in other histone demethylase families, that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely, 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2- b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the cocrystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the noncovalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.

PURPOSE: Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers. METHODS: PK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC-MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice. RESULTS: Metarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC0-∞ of 14400 ng h/mL, Cmax of 810 ng/mL and half-life (t1/2) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC50 of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC0-24h ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC0-24h and C24h. AUC0-24h MD to AUC0-24h SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors. CONCLUSIONS: Metarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.

A new series of quinazoline-based analogs as potent bromodomain-containing protein 4 (BRD4) inhibitors is described. The structure-activity relationships on 2- and 4-position of quinazoline ring, and the substitution at 6-position that mimic the acetylated lysine are discussed. A co-crystallized structure of 48 (CN750) with BRD4 (BD1) including key inhibitor-protein interactions is also highlighted. Together with preliminary rodent pharmacokinetic results, a new lead (65, CN427) is identified which is suitable for further lead optimization.

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.

VT-1129 is a novel fungal-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and the desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose (LD-MD) strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model LD-MD doses to generate different steady-state concentrations. Mice were inoculated intracranially with C. neoformans, and oral treatment began 1 day later. Treatment consisted of placebo, three VT-1129 LD-MD groups: LD of 1, 3, or 30 mg/kg on day 1, followed by MD of 0.15, 0.5, or 5 mg/kg once daily, respectively. In the fungal burden arm, therapy continued for 14 days, and brains were collected on day 15 for fungal burden assessment. In the survival arm, treatment continued for 10 days after which mice were monitored off-therapy until day 30. VT-1129 plasma and brain concentrations were also measured. Each VT-1129 dose significantly improved survival and reduced fungal burden compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions (R2 = 0.72 and 0.67, respectively), with plasma concentration of 1 μg/mL yielding a reduction of ∼5 log10 CFU/g. With the highest LD-MD dose, fungal burden was undetectable in half of the mice in the fungal burden arm, and in a quarter of the mice in the survival arm, 20 days after final dose. These data support an LD-MD strategy for quickly reaching highly efficacious VT-1129 concentrations in treating cryptococcal meningitis.

A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of RXFP1 and is a potent activator of anti-fibrotic genes.

The Assay Guidance Manual (AGM) is an eBook of best-practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through pre-clinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists. This article is protected by copyright. All rights reserved.

Aldehyde dehydrogenases (ALDHs) are responsible for the metabolism of aldehydes (exogenous and endogenous) and possess vital physiological and toxicological functions in areas such as CNS, inflammation, metabolic disorders, and cancers. Overexpression of certain ALDHs (e.g., ALDH1A1) is an important biomarker in cancers and cancer stem cells (CSCs) indicating the potential need for the identification and development of small molecule ALDH inhibitors. Herein, a newly designed series of quinoline-based analogs of ALDH1A1 inhibitors is described. Extensive medicinal chemistry optimization and biological characterization led to the identification of analogs with significantly improved enzymatic and cellular ALDH inhibition. Selected analogs, e.g., 86 (NCT-505) and 91 (NCT-506), demonstrated target engagement in a cellular thermal shift assay (CETSA), inhibited the formation of 3D spheroid cultures of OV-90 cancer cells, and potentiated the cytotoxicity of paclitaxel in SKOV-3-TR, a paclitaxel resistant ovarian cancer cell line. Lead compounds also exhibit high specificity over other ALDH isozymes and unrelated dehydrogenases. The in vitro ADME profiles and pharmacokinetic evaluation of selected analogs are also highlighted.

Hepatitis C virus (HCV) is a small, single-stranded, positive sense RNA virus that infects more than an estimated 70 million people worldwide. Left untreated, persistent infection of HCV often results in chronic hepatitis, cirrhosis or liver failure with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can now provide curative treatment, however the high cost of these therapies limit their access and impact in some of the most at-risk populations, there remains an unmet therapeutic need for effective, shorter-duration and affordable treatment options. Recently, we disclosed the anti-HCV activity of the cheap, over-the-counter antihistamine chlorcyclizine targeting viral entry. Following our hit-to-lead optimization campaign, here we report an evaluation of preclinical in vitro ADME and in vivo pharmacokinetic profiles of several lead compounds, which led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this lead compound into existing DAA regimens has the potential to improve efficacy, affordability and accessibility of treatment.