Isaac M. Westwood

Targeting HSP70 The second potentially druggable heat shock protein and molecular chaperone

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.

Selective small molecule inhibitors of the potential breast cancer marker, human arylamine N-acetyltransferase 1, and its murine homologue, mouse arylamine N-acetyltransferase 2.

The identification, synthesis and evaluation of a series of rhodanine and thiazolidin-2,4-dione derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 is described. The most potent inhibitors identified have submicromolar activity and inhibit both the recombinant proteins and human NAT1 in ZR-75 cell lysates in a competitive manner. (1)H NMR studies on purified mouse Nat2 demonstrate that the inhibitors bind within the putative active site of the enzyme.

Prospective virtual screening with Ultrafast Shape Recognition: the identification of novel inhibitors of arylamine N-acetyltransferases

There is currently a shortage of chemical molecules that can be used as bioactive probes to study molecular targets and potentially as starting points for drug discovery. One inexpensive way to address this problem is to use computational methods to screen a comprehensive database of small molecules to discover novel structures that could lead to alternative and better bioactive probes. Despite that pleasing logic the results have been somewhat mixed. Here we describe a virtual screening technique based on ligand–receptor shape complementarity, Ultrafast Shape Recognition (USR). USR is specifically applied to identify novel inhibitors of arylamine N-acetyltransferases by computationally screening almost 700 million molecular conformers in a time- and resource-efficient manner. A small number of the predicted active compounds were purchased and tested obtaining a confirmed hit rate of 40 per cent which is an outstanding result for a prospective virtual screening.

Arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs), known as drug- and carcinogen-metabolising enzymes, have had historic roles in cellular metabolism, carcinogenesis and pharmacogenetics, including epidemiological studies of disease susceptibility. NAT research in the past 5 years builds on that history and additionally paves the way for establishing the following new concepts in biology and opportunities in drug discovery: i) NAT polymorphisms can be used as tools in molecular anthropology to study human evolution; ii) tracing NAT protein synthesis and degradation within cells is providing insight into protein folding in cell biology; iii) studies on control of NAT gene expression may help to understand the increase in the human NAT isoenzyme, NAT1, in breast cancer; iv) a NAT homologue in mycobacteria plays an essential role in cell-wall synthesis and mycobacterial survival inside host macrophage, thus identifying a novel biochemical pathway; v) transgenic mice, with genetic modifications of all Nat genes, provide in vivo tools for drug metabolism; and vi) structures of NAT isoenzymes provide essential in silico tools for drug discovery.

Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs).

The synthesis and inhibitory activity of a series of 5-substituted-(1,1-dioxo-2,3-dihydro-1H-1 lambda(6)-benzo[e][1,2]thiazin-4-ylidene)-thiazolidine-2,4-dione derivatives as competitive inhibitors of recombinant bacterial arylamine-N-acetyltransferases (NATs) are described. The most potent NAT inhibitors are those that contain planar hydrophobic substituents on the sultam nitrogen.

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Insights into the Conformational Variability and Regulation of Human Nek2 Kinase

Summary The Nek family of serine/threonine kinases regulates centrosome and cilia function; in addition, several of its members are potential targets for drug discovery. Nek2 is dimeric, is cell cycle regulated and functions in the separation of centrosomes at G2/M. Here, we report the crystal structures of wild-type human Nek2 kinase domain bound to ADP at 1.55-Å resolution and T175A mutant in apo form as well as that bound to a non-hydrolyzable ATP analog. These show that regions of the Nek2 structure around the nucleotide-binding site can adopt several different but well-defined conformations. None of the conformations was the same as that observed for the previously reported inhibitor-bound structure, and the two nucleotides stabilized two conformations. The structures suggest mechanisms for the auto-inhibition of Nek2 that we have tested by mutagenesis. Comparison of the structures with Aurora-A and Cdk2 gives insight into the structural mechanism of Nek2 activation. The production of specific inhibitors that target individual kinases of the human genome is an urgent challenge in drug discovery, and Nek2 is especially promising as a cancer target. We not only identify potential challenges to the task of producing Nek2 inhibitors but also propose that the conformational variability provides an opportunity for the design of Nek2 selective inhibitors because one of the conformations may provide a unique target.

Structure and Mechanism of Arylamine N-Acetyltransferases

Arylamine N-acetyltransferases (NATs) are a family of phase II drug-metabolising enzymes which are important in the biotransformation of various aromatic and heterocyclic amines and hydroxylamines, arylhydrazines and arylhydrazides. NATs are present in a wide range of eukaryotes and prokaryotes. Humans have two functional NAT isoforms, both of which are highly polymorphic. The pharmacogenetics of NATs is an area which has been extensively studied. The determination of the X-ray crystal structure of NAT from Salmonella typhimurium led to the identification of the catalytically essential triad of residues: Cys-His-Asp, which is present in all functional NAT enzymes. Recent co-crystallisation data and in silico docking studies of NAT from Mycobacterium smegmatis with substrates and inhibitors have aided the identification of important contact residues within the active site. The X-ray crystal structures of four prokaryotic NAT proteins have now been determined, and these have been used to generate structural models of eukaryotic NATs, providing valuable insight into their active-site architecture. In addition to aiding crystallographic experiments, recent progress in the production of recombinant prokaryotic and eukaryotic NATs has allowed comparative studies of the kinetics and activity profiles of these enzymes. In this review we present an overview of recent structural and activity studies on NAT enzymes, and we outline how in silico methods may be used to predict NAT protein-ligand interactions based on the current knowledge.

Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules—triazoles—in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.

Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1

There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.