Novel Molecular Modalities for Pharmacological Probe and Therapeutic Lead Discovery

National Center For Advancing Translational Sciences

Investigators

Abstract

- Synthetic macrocyclic peptides (MCPs) affinity-selected using a protein target of interest from nucleic acid-encoded libraries containing vast numbers of possible peptide sequences represent a future therapeutic modality. MCPs often display molecular target selectivity and potency in line with monoclonal antibodies but are closer to small molecule drugs in molecular weight, offering the potential for similar pharmacokinetic bioactivity. The first orally available MCPs are only now entering clinical trials with the aim of advancing approaches to treatments currently served by antibody-based therapies. - Ipglycermides are a class of synthetic MCPs discovered with our collaborator Prof. H. Suga (U. Tokyo) that potently inhibit the glycolytic enzyme activity of cofactor-independent phosphoglycerate mutases (iPGMs). Because iPGM, found in many infectious organisms, is a structurally unrelated isozyme of the important human glycolytic dPGM enzyme, iPGM has been considered a potential drug target for selectively inhibiting glycolysis in pathogenic microorganisms. Ipglycermides are composed of between 11 to 14 amino acids and contain either an obligate 8- or 13-membered macrocycle formed through a thioether bridge connecting the Tyr1 alpha-acetamide and either the Cys8 or Cys13 sulfhydryl side chain. - While cyclic peptides found in nature have evolved to be bioavailable, laboratory generated MCPs are primarily enriched in vitro for affinity using a resin-bound target protein. In collaboration with Prof. S. Lokey (U. California Santa Cruz) we initiated a collaboration to enrich new ipglycermide MCP chemotypes from his DNA-encoded cyclic peptide libraries (DEL) designed to bias libraries toward improving membrane permeability. Preliminary DEL selection experiments using iPGM and next-generation sequencing (NGS) data from the enriched MCPs is ongoing. - Additional strategies for exploring the chemical architecture of MCPs are being studied through our collaboration with Dr. G. Copeland (BRT Biotechnologies, Inc.). Here, non-encoded MCP libraries designed to sample the large chemical space represented by nucleic acid-encoded MCP libraries are created to enable testing with functional assays in 1536-well microtiter plate qHTS format. The orthogonal pooling strategy allows both a larger number of individual MCPs for testing (i.e., tested as pooled mixtures) and ‘decoding’ of active wells (i.e., based on positioning the same MCP in different wells in different mixtures) to identify specific MCPs showing bioactivity. Using a pooled 10,000 macrocyclic peptide library, we screened the iPGM coupled-enzyme functional assay and readily identified the ipglycermide Ce-2d samples that were included in the library as positive controls. - Chorismides are a class of synthetic MCPs discovered and investigated with our collaborators Prof. H. Suga (U. Tokyo) and Prof. Scott Lovell (U. Kansas) that potently inhibit secreted Mycobacterium tuberculosis chorismate mutase (*MtbCM). Chorismate mutase (CM) represents a branching point in the metabolic shikimate pathway that is utilized by bacteria, fungi, and plants to biosynthesize aromatic amino acids. Notably absent in mammals, CM is unique to pathogenic microorganisms underpinning its drug target potential. We have the first MCP inhibitors of *MtbCM, two distinct chemotypes, one binding at the CM active site and the other to a newly defined interdimer allosteric site, as elucidated by X-ray crystallography. -Currently, *MtbCM activity is measured using an acid/base-mediated product derivatization absorbance assay incompatible with high throughput screening (HTS). To remove the HTS assay bottleneck we coupled a chorismide to a fluorescent probe and developed a suite of four new alternative fluorescence-based ligand-displacement assays to facilitate the discovery of new CM small molecule inhibitors. - Natural products (NP), which have evolved over eons to encode biological activity, are one of medicine’s oldest and most enduring sources of drugs. The chemotypes represented by NPs are among the most versatile, targeting a range of pathogenic microbes that continue to create a significant worldwide health and economic burden. - In our collaboration with Prof. D. Sherman (U. Michigan) and Dr. X-z Su (NIAID, NIH) we have contributed to the investigation of the NP Premarineosin A and its analogs that have been obtained by coupling metabolic engineering with late-stage derivatization. Premarineosin A is a potent antimalarial NP and has also been observed in our laboratory to have antinematode activity. Premarineosin A has been underexplored due to limited availability and synthetic complexity; a limitation being addressed in this project by the Sherman laboratory. The evaluation of Premarineosin A and its analogs on mammalian cell line viability, intraerythrocytic P. falciparum replication and C. elegans life-stages have been initiated to map the structure-activity relationship associated with the bioactivity of this NP series.