Identification of genes that confer tumor cell resistance to the aurora B kinase inhibitor, AZD1152.

AZD1152 is a highly selective Aurora B kinase inhibitor currently undergoing Phase I and II clinical evaluation in patients with acute myelogenous leukemia and advanced solid malignancies. We have established two AZD1152-resistant cell lines from SW620 colon and MiaPaCa pancreatic carcinoma lines, which are >100-fold resistant to the active metabolite of AZD1152, AZD1152 HQPA and interestingly, cross-resistant to the pan-Aurora kinase inhibitor, VX-680/MK0457. Using whole-genome microarray analysis and comparative genomic hybridization, we were able to identify MDR1 and BCRP as the causative genes that underlie AZD1152 HQPA-resistance in these models. Furthermore, the upregulation of either of these genes is sufficient to render in vivo tumor growth insensitive to AZD1152. Finally, the upregulation of MDR1 or BCRP is predictive of tumor cell sensitivity to this agent, both in vitro and in vivo. The data provide a genetic basis for resistance to Aurora kinase inhibitors, which could be utilized to predict clinical response to therapy.

Discovering novel ligands for macromolecules using X-ray crystallographic screening.

The need to decrease the time scale for clinical compound discovery has led to innovations at several stages in the process, including genomics/proteomics for target identification, ultrahigh-throughput screening for lead identification, and structure-based drug design and combinatorial chemistry for lead optimization. A critical juncture in the process is the identification of a proper lead compound, because a poor choice may generate costly difficulties at later stages. Lead compounds are commonly identified from high-throughput screens of large compound libraries, derived from known substrates/inhibitors, or identified in computational prescreeusing X-ray crystal structures. Structural information is often consulted to efficiently optimize leads, but under the current paradigm, such data require preidentification and confirmation of compound binding. Here, we describe a new X-ray crystallography-driven screening technique that combines the steps of lead identification, structural assessment, and optimization. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information. The utility of the method is demonstrated in the discovery and optimization of a new orally available class of urokinase inhibitors for the treatment of cancer.

Improving the in vivo duration of 5-lipoxygenase inhibitors: application of an in vitro glucuronosyltransferase assay.

An in vitro glucuronidation assay was used to optimize a series of N-hydroxyurea-containing 5-lipoxygenase inhibitors for metabolic stability. The glucuronidation of these compounds in cynomolgus monkey microsomes followed Michaelis-Menten kinetics allowing calculation of V(max) and K(M). The V(max) values ranged from 0.02 to 7.9 nmol/min/mg microsomal protein, a 400-fold difference, whereas K(M) ranged from 204 to 2500 microM, only a 12-fold difference. In vitro intrinsic clearance values (CL(int) were calculated for 18 compounds tested in the kinetic assay and compared with the in vivo plasma clearance (CL(p)) calculated from intravenous studies done in cynomolgus monkeys. These initial results suggested a relationship between the in vitro CL(int) and in vivo duration as defined by CL(p). A more rapid in vitro assay was developed in a 96-well format using a single concentration of substrate (100 microM) from which a glucuronidation rate was calculated. The results from this assay for 40 compounds correlated with in vivo plasma clearance (r = 0.57). This more efficient assay was used to test more than 100 compounds and develop structure-metabolism relationships based on metabolic stability and improved duration. The culmination of this effort contributed to the discovery of ABT-761, a 5-lipoxygenase inhibitor with in vivo duration in monkey improved 40-fold over thefirst generation inhibitor. Further studies performed in human liver microsomes demonstrated a similar trend that was corroborated by the 8-fold increase in duration after oral dosing in humans observed with ABT-761.

Aryl ketones as novel replacements for the C-terminal amide bond of succinyl hydroxamate MMP inhibitors.

A series of succinyl hydroxamate MMP inhibitors were prepared incorporating an aryl amino ketone moiety in place of the more typical C-terminal amino acid amides. Compounds of the C-terminal ketone series displayed potent inhibition of MMPs. Several compounds of the series were shown to be orally bioavailable.

Biaryl ether retrohydroxamates as potent, long-lived, orally bioavailable MMP inhibitors.

A novel series of biaryl ether reverse hydroxamate MMP inhibitors has been developed. These compounds are potent MMP-2 inhibitors with limited activity against MMP-1. Select members of this series exhibit excellent pharmacokinetic properties with long elimination half-lives (7 h) and high oral bioavailability (100%).

Discovery and characterization of the potent, selective and orally bioavailable MMP inhibitor ABT-770.

Modification of the biphenyl portion of MMP inhibitor 2a gave analogue 2i which is greater than 1000-fold selective against MMP-2 versus MMP-1. The stereospecific synthesis of both enantiomers of 2i was achieved beginning with (S)- or (R)-benzyl glycidyl ether. The (S)-enantiomer, 11 (ABT-770), is orally bioavailable and efficacious in an in vivo model of tumor growth.