Jayvardhan Pandit

Identification, characterization, and crystal structure of the Omega class glutathione transferases.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species andCaenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-Å resolution and has a characteristic GST fold (Protein Data Bank entry code 1eem). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215–900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long α-helices. The subunits cross at the GAF-B/catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215–900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215–900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.

Design of selective, ATP-competitive inhibitors of Akt.

This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from an elaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that was modestly selective and medicinal chemistry optimization of that lead to provide more selective analogues. Analysis of the data suggested that highly lipophilic analogues would likely suffer from poor overall properties. Central to the discussion is the concept of optimization of lipophilic efficiency and the ability to balance overall druglike propeties with the careful control of lipophilicity in the lead series. Discovery of the nonracemic amide series and subsequent modification produced an advanced analogue that performed well in advanced preclinical assays, including xenograft tumor growth inhibition studies, and this analogue was nominated for clinical development.

Use of structure-based design to discover a potent, selective, in vivo active phosphodiesterase 10A inhibitor lead series for the treatment of schizophrenia.

Utilizing structure-based virtual library design and scoring, a novel chimeric series of phosphodiesterase 10A (PDE10A) inhibitors was discovered by synergizing binding site interactions and ADME properties of two chemotypes. Virtual libraries were docked and scored for potential binding ability, followed by visual inspection to prioritize analogs for parallel and directed synthesis. The process yielded highly potent and selective compounds such as 16. New X-ray cocrystal structures enabled rational design of substituents that resulted in the successful optimization of physical properties to produce in vivo activity and to modulate microsomal clearance and permeability.

Discovery and Optimization of a Novel Spiropyrrolidine Inhibitor of β-Secretase (BACE1) through Fragment-Based Drug Design

The aspartyl protease β-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aβ-peptide, a major component of plaques in the brains of Alzheimers disease (AD) patients, and inhibition of this enzyme has emerged as a major strategy for pharmacologic intervention in AD. An X-ray-based fragment screen of Pfizers proprietary fragment collection has resulted in the identification of a novel BACE binder featuring spiropyrrolidine framework. Although exhibiting only weak inhibitory activity against the BACE enzyme, the small compound was verified by biophysical and NMR-based methods as a bona fide BACE inhibitor. Subsequent optimization of the lead compound, relying heavily on structure-based drug design and computational prediction of physiochemical properties, resulted in a nearly 1000-fold improvement in potency while maintaining ligand efficiency and properties predictive of good permeability and low P-gp liability.

Structure-Based Design of Irreversible Human KAT II Inhibitors: Discovery of New Potency-Enhancing Interactions

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structure-activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The use of k inact/K i to assess potency was critical for understanding the SAR in this series and for identifying compounds with improved pharmacodynamic profiles.

High-resolution crystals and preliminary X-ray diffraction studies of a catalytic RNA.

High-resolution single crystals of a catalytic RNA molecule derived from the sequence of the satellite RNA of tobacco ringspot virus have been obtained. The unit-cell volumes of the RNA crystals vary depending on the crystallization conditions and temperature. The best crystal form, when flash frozen, has space group P1 with unit-cell dimensions a = 53.08, b = 71.81, c = 28.03 A, alpha = 98.43, beta = 104.32 and gamma = 74.54 degrees. This form diffracts to a resolution of 2.4 A. A heavy-atom derivative search is in progress.

Discovery of hydroxamate bioisosteres as KAT II inhibitors with improved oral bioavailability and pharmacokinetics

A series of kynurenine aminotransferase II (KAT II) inhibitors has been developed replacing the hydroxamate motif with a bioisostere. Triazolinones or triazoles have proven to be effective replacements with significantly improved pharmacokinetics including reduced clearance and increased bioavailability. An X-ray crystal structure of an inhibitor bound in KAT II confirms that the irreversible binding to the co-factor is maintained and that the heterocycles make productive hydrogen bonds to the arginine-399.

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

The Discovery and Characterization of the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Potentiator N-{(3S,4S)-4-[4-(5-Cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242)

A unique tetrahydrofuran ether class of highly potent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiators has been identified using rational and structure-based drug design. An acyclic lead compound, containing an ether-linked isopropylsulfonamide and biphenyl group, was pharmacologically augmented by converting it to a conformationally constrained tetrahydrofuran to improve key interactions with the human GluA2 ligand-binding domain. Subsequent replacement of the distal phenyl motif with 2-cyanothiophene to enhance its potency, selectivity, and metabolic stability afforded N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242, 3), whose preclinical characterization suggests an adequate therapeutic index, aided by low projected human oral pharmacokinetic variability, for clinical studies exploring its ability to attenuate cognitive deficits in patients with schizophrenia.