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.

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Inhibitors of the chaperone Hsp90 are potentially useful as chemotherapeutic agents in cancer. This paper describes an application of fragment screening to Hsp90 using a combination of NMR and high throughput X-ray crystallography. The screening identified an aminopyrimidine with affinity in the high micromolar range and subsequent structure-based design allowed its optimization into a low nanomolar series with good ligand efficiency. A phenolic chemotype was also identified in fragment screening and was found to bind with affinity close to 1 mM. This fragment was optimized using structure based design into a resorcinol lead which has subnanomolar affinity for Hsp90, excellent cell potency, and good ligand efficiency. This fragment to lead campaign improved affinity for Hsp90 by over 1,000,000-fold with the addition of only six heavy atoms. The companion paper (DOI: 10.1021/jm100060b) describes how the resorcinol lead was optimized into a compound that is now in clinical trials for the treatment of cancer.

Data from both our own and literature studies of the biochemistry and inhibition of influenza virus endonuclease was combined with data on the mechanism of action and the likely active site mechanism to propose a pharmacophore. The pharmacophore was used to design a novel structural class of inhibitors, some of which were found to have activities similar to that of known influenza endonuclease inhibitors and were also antiviral in cell culture.

Mapping interactions at protein-ligand binding sites is an important aspect of understanding many biological reactions and a key part of drug design. In this paper, we have used a fragment-based approach to probe "hot spots" at the cofactor-binding site of a model dehydrogenase, Escherichia coli ketopantoate reductase. Our strategy involved the breaking down of NADPH (Kd = 300 nM) into smaller fragments and the biophysical characterization of their binding using WaterLOGSY NMR spectroscopy, isothermal titration calorimetry (ITC), and inhibition studies. The weak binding affinities of fragments were measured by direct ITC titrations under low c value conditions. The 2'-phosphate and the reduced nicotinamide groups were found to contribute a large part of the binding energy. A combination of ITC and site-directed mutagenesis enabled us to locate the fragments at separate hot spots on opposite ends of the cofactor-binding site. This study has identified structural determinants for cofactor recognition that represent a blueprint for future inhibitor design.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.

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