Paul L. Richardson

Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-RS.

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6′–38′) complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-RS) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-RS with a bend at residue A18′ and makes extensive hydrophobic and electrostatic interactions along the exposed β-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-RS. These results present a structural basis for hPAC1-RS selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-RS receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.

α-Keto amides as inhibitors of histone deacetylase

α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The α-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.

Indole amide hydroxamic acids as potent inhibitors of histone deacetylases.

A series of hydroxamic acid-based HDAC inhibitors with an indole amide residue at the terminus have been synthesized and evaluated. Compounds with a 2-indole amide moiety have been found as the most active inhibitors among the different regioisomers. Introduction of substituents on the indole ring further improved the potency and generated a series of very potent inhibitors with significant antiproliferative activity. A representative compound in the series, 7b, has been found to be orally active in tumor growth inhibition model.

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide(1) is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3-41microM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

Discovery, identification, and characterization of candidate pharmacodynamic markers of methionine aminopeptidase-2 inhibition.

The catalytic activity of methionine aminopeptidase-2 (MetAP2) has been pharmacologically linked to cell growth, angiogenesis, and tumor progression, making this an attractive target for cancer therapy. An assay for monitoring specific protein changes in response to MetAP2 inhibition, allowing pharmacokinetic (PK)/pharmacodynamic (PD) models to be established, could dramatically improve clinical decision-making. Candidate MetAP2-specific protein substrates were discovered from undigested cell culture-derived proteomes by MALDI-/SELDI-MS profiling and a biochemical method using (35)S-Met labeled protein lysates. Substrates were identified either as intact proteins by FT-ICR-MS or applying in-gel protease digestions followed by LC-MS/MS. The combination of these approaches led to the discovery of novel MetAP2-specific substrates including thioredoxin-1 (Trx-1), SH3 binding glutamic acid rich-like protein (SH3BGRL), and eukaryotic elongation factor-2 (eEF2). These studies also confirmed glyceraldehye 3-phosphate dehydrogenase (GAPDH) and cyclophillin A (CypA) as MetAP2 substrates. Additional data in support of these proteins as MetAP2-specific substrates were provided by in vitro MetAP1/MetAP2 enzyme assays with the corresponding N-terminal derived peptides and 1D/2D Western analyses of cellular and tissue lysates. FT-ICR-MS characterization of all intact species of the 18 kDa substrate, CypA, enabled a SELDI-MS cell-based assay to be developed for correlating N-terminal processing and inhibition of proliferation. The MetAP2-specific protein substrates discovered in this study have diverse properties that should facilitate the development of reagents for testing in preclinical and clinical environments.

SAR of amino pyrrolidines as potent and novel protein-protein interaction inhibitors of the PRC2 complex through EED binding.

Herein we disclose SAR studies of a series of dimethylamino pyrrolidines which we recently reported as novel inhibitors of the PRC2 complex through disruption of EED/H3K27me3 binding. Modification of the indole and benzyl moieties of screening hit 1 provided analogs with substantially improved binding and cellular activities. This work culminated in the identification of compound 2, our nanomolar proof-of-concept (PoC) inhibitor which provided on-target tumor growth inhibition in a mouse xenograft model. X-ray crystal structures of several inhibitors bound in the EED active-site are also discussed.

Target Identification of Compounds from a Cell Viability Phenotypic Screen Using a Bead/Lysate-Based Affinity Capture Platform

The pharmaceutical industry has been continually challenged by dwindling target diversity. To obviate this trend, phenotypic screens have been adopted, complementing target-centric screening approaches. Phenotypic screens identify drug leads using clinically relevant and translatable mechanisms, remaining agnostic to targets. While target anonymity is advantageous early in the drug discovery process, it poses challenges to hit progression, including the development of backup series, retaining desired pharmacology during optimization, discovery of markers, and understanding mechanism-driven toxicity. Consequently, significant effort has been expended to elaborate the targets and mechanisms at work for promising screening hits. Affinity capture is commonly leveraged, where the compounds are linked to beads and targets are abstracted from cell homogenates. This technique has proven effective for identifying targets of kinase, PARP, and HDAC inhibitors, and examples of new targets have been reported. Herein, a three-pronged approach to target deconvolution by affinity capture is described, including the implementation of a uniqueness index that helps discriminate between bona fide targets and background. The effectiveness of this approach is demonstrated using characterized compounds that act on known and noncanonical target classes. The platform is subsequently applied to phenotypic screening hits, identifying candidate targets. The success rate of bead-based affinity capture is discussed.

Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5-13 (C10A)

B‐type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high‐affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5‐13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three‐dimensional structure by X‐ray diffraction to 2.1 Å resolution. The structure reveals the detailed interactions that five of the complementarity‐determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, ΔG = −54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion‐limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine‐scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides. Proteins 2009.

A uniquely modified RNA: Introduction of a single RNA cleavage agent into the M1 ribozyme

Abstract We describe an efficient, four-step synthesis of an N 4 -modified cytidine phosphoramidite reagent ( 1 ) that permits nonnative functionality to be introduced into a synthetic oligoribonucleotide. This reagent was used to prepare a 377-nt E. coli M1 ribozyme equipped with a single RNA cleavage agent at nucleotide 11. We describe an efficient, four-step synthesis of an N 4 -modified cytidine phosphoramidite reagent ( 1 ) that permits nonnative functionality to be introduced into a synthetic oligoribonucleotide. This reagent was used to prepare a 377-nt E. coli M1 ribozyme equipped with a single RNA cleavage agent at nucleotide 11.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors.

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236–45. ©2017 AACR.