Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.
Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d ) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.
Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of
ketopantoate to pantoate on the pantothenate (vitamin B5) biosynthetic pathway. The Escherichia coli
panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three
chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K
M(NADPH)
of 20 μM, a K
M(ketopantoate) of 60 μM, and a k
cat of 40 s-1. Regular prismatic KPR crystals were prepared
using the hanging drop technique. They belonged to the tetragonal space group P42212, with cell
parameters: a = b = 103.7 Å and c = 55.7 Å, accommodating one enzyme molecule per asymmetric
unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using
the SeMet protein, for which data were collected to 2.3 Å resolution. The native data were collected to
1.7 Å resolution and used to refine the final structure. The secondary structure comprises 12 α-helices,
three 310-helices, and 11 β-strands. The enzyme is monomeric and has two domains separated by a cleft.
The N-terminal domain has an αβ-fold of the Rossmann type. The C-terminal domain (residues 170−291) is composed of eight α-helices. KPR is shown to be a member of the 6-phosphogluconate
dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme−NADPH−ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.
Single crystal X-ray diffraction is the technique of choice for studying the interactions of small organic molecules with proteins by determining their three-dimensional structures; however the requirement for highly purified protein and lack of process automation have traditionally limited its use in this field. Despite these shortcomings, the use of crystal structures of therapeutically relevant drug targets in pharmaceutical research has increased significantly over the last decade. The application of structure-based drug design has resulted in several marketed drugs and is now an established discipline in most pharmaceutical companies. Furthermore, the recently published full genome sequences of Homo sapiens and a number of micro-organisms have provided a plethora of new potential drug targets that could be utilised in structure-based drug design programs. In order to take maximum advantage of this explosion of information, techniques have been developed to automate and speed up the various procedures required to obtain protein crystals of suitable quality, to collect and process the raw X-ray diffraction data into usable structural information, and to use three-dimensional protein structure as a basis for drug discovery and lead optimisation. This tutorial review covers the various technologies involved in the process pipeline for high-throughput protein crystallography as it is currently being applied to drug discovery. It is aimed at synthetic and computational chemists, as well as structural biologists, in both academia and industry, who are interested in structure-based drug design.
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