Todd L. Graybill

Discovery of thieno[3,2-d]pyrimidine-6-carboxamides as potent inhibitors of SIRT1, SIRT2, and SIRT3.

The sirtuins SIRT1, SIRT2, and SIRT3 are NAD(+) dependent deacetylases that are considered potential targets for metabolic, inflammatory, oncologic, and neurodegenerative disorders. Encoded library technology (ELT) was used to affinity screen a 1.2 million heterocycle enriched library of DNA encoded small molecules, which identified pan-inhibitors of SIRT1/2/3 with nanomolar potency (e.g., 11c: IC50 = 3.6, 2.7, and 4.0 nM for SIRT1, SIRT2, and SIRT3, respectively). Subsequent SAR studies to improve physiochemical properties identified the potent drug like analogues 28 and 31. Crystallographic studies of 11c, 28, and 31 bound in the SIRT3 active site revealed that the common carboxamide binds in the nicotinamide C-pocket and the aliphatic portions of the inhibitors extend through the substrate channel, explaining the observable SAR. These pan SIRT1/2/3 inhibitors, representing a novel chemotype, are significantly more potent than currently available inhibitors, which makes them valuable tools for sirtuin research.

Camphor sulfonamide derivatives as novel, potent and selective CXCR3 antagonists.

A series of N-arylpiperazine camphor sulfonamides was discovered as novel CXCR3 antagonists. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in the identification of potent and selective CXCR3 antagonists are described.

Tetrahydro-4-quinolinamines identified as novel P2Y1 receptor antagonists

High-throughput screening of the GSK compound collection against the P2Y(1) receptor identified a novel series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet aggregation.

Synthesis and evaluation of azapeptide-derived inhibitors of serine and cysteine proteases

Abstract Azapeptide analogues ii of the α-halomethyl ketones i were synthesized and evaluated as potential inhibitors of serine and cysteine proteases. Inhibitors ii discriminate between the two classes of protease, demonstrating selectivity for cysteine proteases. Azaglycines 1–3 and 5 displayed time-dependent inactivation (kobs/[I] = 200–1500 M−1s−1) of cathepsin B and calpain.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Structural and stereochemical requirements of time-dependent inactivators of the interleukin-1β converting enzyme

Abstract Structural and stereochemical requirements of substrate based time-dependent inactivators of interleukin-1β converting enzyme were investigated. Hydrophobic amino acids with L-stereochemistry are preferred at the P 2 and P 3 positions. It appears that both D-and L-Asp are accepted by the enzyme at the P I position.

Characterization of a continuous fluorogenic assay for calpain I. Kinetic evaluation of peptide aldehydes, halomethyl ketones and (acyloxy)methyl ketones as inhibitors of the enzyme

Z-Leu-Arg-(7-methoxynaphthyl)amide (1) is a substrate for calpain I. The specificity constant for 1 (kcatKm = 1405 ± 40 M−1s−1) is 10x greater than for any previously reported fluorogenic substrate. Using this substrate, a sensitive, continuous fluorogenic assay was developed permitting the identification of Z-(D)Ala-Leu-Phe-(OCO-2,6-Fl2-Ph) (69) as the first selective (>100-fold versus cathepsins B and L) time-dependent inhibitor of the enzyme.

Encoded Library Technology Screening of Hepatitis C Virus NS4B Yields a Small-Molecule Compound Series with In Vitro Replicon Activity

ABSTRACT To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets.

A convenient ‘catch, cyclize, and release’ preparation of 3-thio-1,2,4-triazoles mediated by polymer-bound BEMP

Abstract A robust ‘catch, cyclize, and release’ preparation of 3-thioalkyl-1,2,4-triazoles mediated by the polymer-bound base P-BEMP is described. This reengineered synthesis combines the chemical efficiency of the classical synthesis (three steps; three diversity points) with the practical benefits of resin-bound reagents (use of excess reagents to drive reactions to completion, no purification of intermediates, automation-friendly). Key advantages/limitations of this scheme, reagent compatibility, and the results of a representative 64-member combinatorial library are described and presented herein.

Inhibition of human erythrocyte calpain I by novel quinolinecarboxamides

Abstract 1,4-Dihydro-4-oxo-3-quinolinecarboxamides are a class of non-peptide reversible inhibitor of human erythrocyte Calpain I. The preparation and in vitro evaluation of these compounds are discussed.