Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.

The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Annexin V is an abundant eukaryotic protein that binds phospholipid membranes in a Ca2؉ -dependent manner. In the present studies, site-directed mutagenesis was combined with x-ray crystallography and solution liposome binding assays to probe the functional role of a cluster of interfacial basic residues in annexin V. Four mutants were investigated: R23E, K27E, R61E, and R149E. All four mutants exhibited a significant reduction in adsorption to phospholipid membranes relative to the wild-type protein, and the R23E mutation was the most deleterious. Crystal structures of wild-type and mutant proteins were similar except for local changes in salt bridges involving basic cluster residues. The combined data indicate that Arg 23 is a major determinant for interfacial phospholipid binding and participates in an intermolecular salt bridge that is key for trimer formation on the membrane surface. Together, crystallographic and solution data provide evidence that the interfacial basic cluster is a locus where trimerization is synergistically coupled to membrane phospholipid binding.Annexins comprise a large family of homologous eukaryotic proteins with a common core region that promotes Ca 2ϩ -dependent binding to phospholipid membrane surfaces (for a review, see Refs. 1-5). This property underlies many proposed in vivo annexin functions, including membrane trafficking, cell signaling, and roles in inflammatory and coagulation processes. Annexin V is an abundant protein that binds preferentially to acidic phospholipid membranes in the presence of Ca 2ϩ . The annexin-phospholipid association is of high affinity but is reversible with the removal of Ca 2ϩ . When bound to the membrane surface, annexin V molecules assemble laterally into well organized trimers and higher order arrays of trimers (6). This lateral assembly promotes spontaneous two-dimensional crystallization of annexin V on membrane surfaces, a process that is inhibited by extreme membrane surface curvature (7,8). Annexin-coated membrane surfaces undergo changes in their properties, becoming markedly rigid as the crystallization domains increase in size (9 -11). This membrane-bound layer of annexin V, which has anticoagulant properties, is believed to play a functional role in processes associated with the protein. In placenta, reduced levels of annexin V occur with anti-phospholipid syndrome, which causes hypercoagulation and recurrent miscarriage. Rand and co-workers (12, 13) have proposed a mechanism for anti-phospholipid syndrome in which the disease process strips off the protective layer of annexin V at the maternal-fetal interface, permitting excessive thrombosis to occur.The mechanism of membrane adsorption of annexin V has generated considerable interest from many disciplines, from clinical to structural biology. The protein provides an excellent model for elucidating complex interfacial behavior by peripheral membrane proteins. The molecular structure of the 35-kDa protein has been well characterized in both soluble and membrane-bound forms by x-ray ...