Target-based Assays and Screening Strategies

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

Abstract

- Small molecule antagonists of the Natriuretic peptide receptor 1 (Npr1). Pruritic agents (endogenously produced or exogenously encountered) stimulate pruritic neurons via connections to the CNS and produce the perception of itch. Our collaborator, Dr. M. Hoon’s (NIDCR, NIH) laboratory identified the spinal cord receptor for natriuretic polypeptide b (Nppb), Npr1 as a target for the treatment of itch. Recently Npr1 assays were employed in large-scale quantitative high throughput screening (qHTS) to identify novel Npr1 antagonists to investigate the potential of pharmacological treatments of chronic itch, a condition that results in long-term unremitting urge to scratch that significantly degrades the quality of life for sufferers. While demonstrating efficacy in preclinical murine models of itch, the original lead candidates, which were postulated to inhibit the receptor’s guanylyl cyclase activity, also were active on the hNpr2 subtype, which has a highly conserved guanylate binding site with hNpr1. To identify Npr1 antagonists with greater subtype selectivity, the project team conducted additional qHTS on >120,000 small molecules from the NCATS compound libraries and standard HTS on 150,000 natural product fractions from the first release of the NCI Natural Products library. Furthermore, the team developed several new orthogonal assays to confirm compound potency and selectivity of compounds identified in the primary qHTS. These assays included a cGMP Homogeneous Time-Resolved Fluorescence (HTRF) assay and high-throughput Npr1 Cellular Thermal Shift Assay (CETSA) assay. Of the compounds selected, the most potent exhibited both Npr1 and 2 activities, while Npr1-specific active compounds were among the lower potency candidates. The next phase of the program will incorporate a medicinal chemistry effort to optimize the potency and selectivity of the lead candidate compounds. - Mass spectrometry-based HTS for isomerization-mediating enzyme drug targets. Two of the infectious disease drug targets under investigation in our program, cofactor-independent phosphoglycerate mutase (iPGM) and chorismate mutase (CM) catalyze isomerization reactions. iPGM interconverts the glycolytic metabolites 3- and 2-phosphoglycerates, whereas CM catalyzes a Claisen rearrangement of chorismate to prephenate, the first committed step in aromatic amino acid biosynthesis. The reactants and products of both these drug targets are not directly measurable in HTS assay formats using current technology. The enzyme targets either require a series of additional enzymes, for example, to drive 2-phosphoglycerate to ATP detectable using ATP-dependent luciferase bioluminescence, or as for the product of CM, caustic post-assay derivatization steps to yield phenylpyruvate having an absorbance measurable in standard microtiter plate readers. Mass spectrometry (MS) is highly effective and widely used in detecting and measuring metabolites and is increasingly being applied to HTS strategies. However, the discrimination of isomers in HTS settings using MS has not been described. In collaboration with Dr. T. Covey’s group (SCIEX), a MS-based platform combining differential ion mobility spectrometry (DMS) MS, having the capability to resolve positional isomers and diastereomers, with high-speed sample transfer from 1536-well assay plates using acoustic ejection technology MS systems, referred to as DAEMS was developed and tested. We have demonstrated the DAEMS platform technology to detect substrate/product (S/P) ratios for iPGM and CM, as well for the S/P ratios for other enzymes and potential drug target isomerases, including triosephosphate isomerase and aconitase. As nearly a third of the metabolome is composed of isomeric combinations the DAEMS technology should be enabling for future drug discovery programs. - Novel metallo-beta-lactamase (MBL) inhibitors from machine leaning models. Antibiotic-resistant infections lead to >35,000 U.S. deaths yearly and remain a significant and increasing public health challenge. This is due to the widespread use of antibiotics that has led to the emergence of bacterial resistance, for example, from beta-lactamases which hydrolyze and thereby inactivate a broad range of clinically used beta-lactam drugs. Bacteria have evolved two beta-lactamase enzyme classes, serine-beta-lactamases (SBL) and metallo-beta-lactamases (MBL). SBLs are the more clinically relevant enzyme for which FDA-approved drugs exist (e.g., clavulanic acid) and are used in combination with antibiotics like carbapenems, which are often a last resort against resistant bacteria. No clinically approved broad-spectrum MBL inhibitors currently exist. To help address this gap we established a collaboration with Prof. M. Crowder’s (Miami U.) laboratory and NCATS to develop machine leaning-enabled screening paradigms to discover novel MBL inhibitor chemotypes. We have focused on the New Delhi MBL, the most prevalent MBL worldwide, which can hydrolyze the last resort carbapenems. The NIH Genesis library of 76,369 compounds was analyzed using the optimized virtual screening prediction tool and the highest 2816 predicted probability compounds were assayed against the beta-lactamase activity of the New Delhi MBL, NDM-1 using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. - Pharmacologic recuse of Hedgehog autoprocessing mutants. The Sonic Hedgehog (SHh) signaling pathway is essential to embryonic cell differentiation as Hh protein concentration gradients are critical to proper embryonic morphologic development. Pathway malfunction can result in compromised fetal growth and in cancer. Drug development efforts targeting human SHh autoprocessing, however, have been limited by access to pure SHh target protein, its resistance to crystallography, and assay complications from its unique autocatalytic post-translational mechanism. SHh contains an N-terminal signaling domain (SHhN) and a C-terminal autoprocessing domain (SHhC), that undergoes an intramolecular cleavage at a specific site between domains. Cleavage occurs in two steps: first, a conserved cysteine and glycine sequence reorganize the protein backbone into a thioester intermediate. In a second step, the hydroxyl group of a non-covalently bound cholesterol ligand within the SHhC domain directs a thioester transesterification. This releases the SHhN domain, now covalently modified with cholesterol via an ester linkage, from the SHhC domain. Fundamentally, the SHhC domain is a single turnover enzyme facilitating intramolecular peptide backbone cleavage and subsequent cholesterol transfer to the SHhN domain. This cholesterol modification is crucial for the proper localization, transport, and signaling activity of SHh. Holoprosencephaly, a congenital disorder affecting fetal brain development, results from mutation in SHhC including the D46A (or D303A) mutation. This mutation prevents SHh autoprocessing and interaction with cholesterol, ultimately affecting the SHh signaling pathway. In collaboration with Prof. B. Callahan’s (Binghamton U.) laboratory, we have developed an innovative bioluminescent cell-based qHTS assay for identifying ligands capable of rescuing SHh D46A autoprocessing that is scalable to 1536-well microtiter plate format. A subsequent qHTS campaign has identified a novel synthetic non-sterol cell-permeable ligand class capable of facilitating SHh D46A autoprocessing. The study provides evidence for an SHh allosteric site with access to the hedgehog protein’s internal thioester that can act as pharmacological correctors of function disrupting point mutants of a critically important family of developmental morphogens and oncogenic factors.