Kam Y. J. Zhang

Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF -mutant melanoma

B-RAF is the most frequently mutated protein kinase in human cancers. The finding that oncogenic mutations in BRAF are common in melanoma, followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway, offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients. Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts. Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032 (ref. 5). In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment. This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation, greater than 80% inhibition of ERK phosphorylation in the tumours of patients correlated with clinical response. Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily. These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors.

Germline KRAS mutations cause Noonan syndrome

Noonan syndrome (MIM 163950) is characterized by short stature, facial dysmorphism and cardiac defects. Heterozygous mutations in PTPN11, which encodes SHP-2, cause ∼50% of cases of Noonan syndrome. The SHP-2 phosphatase relays signals from activated receptor complexes to downstream effectors, including Ras. We discovered de novo germline KRAS mutations that introduce V14I, T58I or D153V amino acid substitutions in five individuals with Noonan syndrome and a P34R alteration in a individual with cardio-facio-cutaneous syndrome (MIM 115150), which has overlapping features with Noonan syndrome. Recombinant V14I and T58I K-Ras proteins show defective intrinsic GTP hydrolysis and impaired responsiveness to GTPase activating proteins, render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage–specific manner. These studies establish germline KRAS mutations as a cause of human disease and infer that the constellation of developmental abnormalities seen in Noonan syndrome spectrum is, in large part, due to hyperactive Ras.

Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3.

The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of ΔΨm on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

Density modification for macromolecular phase improvement

Density modification provides a simple and largely automatic tool for improving phase estimates for observed structure factors. The phase information arises from a combination of the known structure factor magnitudes, the current phase estimates, and stereochemical information. The magnitudes, the current phase estimates, and stereochemical information. The addition of these phase information derived from theoretical sources renders new structures amenable to solution, and reduces the effort required to solve other structures. A diverse array of techniques which have been applied to the phase improvement problem are reviewed.

A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design

Cyclic nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes that regulate a variety of cellular processes. We describe a family of potent PDE4 inhibitors discovered using an efficient method for scaffold-based drug design. This method involves an iterative approach starting with low-affinity screening of compounds followed by high-throughput cocrystallography to reveal the molecular basis underlying the activity of the newly identified compounds. Through detailed structural analysis of the interaction of the initially discovered pyrazole carboxylic ester scaffold with PDE4D using X-ray crystallography, we identified three sites of chemical substitution and designed small selective libraries of scaffold derivatives with modifications at these sites. A 4,000-fold increase in the potency of this PDE4 inhibitor was achieved after only two rounds of chemical synthesis and the structural analysis of seven pyrazole derivatives bound to PDE4B or PDE4D, revealing the robustness of this approach for identifying new inhibitors that can be further developed into drug candidates.

Mcl-1 is required for Akata6 B-lymphoma cell survival and is converted to a cell death molecule by efficient caspase-mediated cleavage

Enforced expression of the antiapoptotic Bcl-2 family protein Mcl-1 promotes lymphomagenesis in the mouse; however, the functional role of Mcl-1 in human B-cell lymphoma remains unclear. We demonstrate that Mcl-1 is widely expressed in malignant B-cells, and high-level expression of Mcl-1 is required for B-lymphoma cell survival, since transfection of Mcl-1-specific antisense oligodeoxynucleotides was sufficient to promote apoptosis in Akata6 lymphoma cells. Mcl-1 was efficiently cleaved by caspases at evolutionarily conserved aspartic acid residues in vitro, and during cisplatin-induced apoptosis in B-lymphoma cell lines and spontaneous apoptosis of primary malignant B-cells. Overexpression of the Mcl-1 cleavage product that accumulated during apoptosis was sufficient to kill cells. Therefore, Mcl-1 is an essential survival molecule for B-lymphoma cells and is cleaved by caspases to a death-promoting molecule during apoptosis. In contrast to Mcl-1, Bcl-2 and Bcl-XL were relatively resistant to caspase cleavage in vitro and in intact cells. Interfering with Mcl-1 function appears to be an effective means of inducing apoptosis in Mcl-1-positive B-cell lymphoma, and the unique sensitivity of Mcl-1 to caspase-mediated cleavage suggests an attractive strategy for converting it to a proapoptotic molecule.

COMBINING CONSTRAINTS FOR ELECTRON-DENSITY MODIFICATION

Publisher Summary An integrated procedure, known as SQUASH, that combines the constraints from the correct electron-density distribution, solvent flatness, correct local density shape, and equal molecules has been demonstrated to be a powerful method for macromolecular phase refinement and extension. This chapter describes progress in density modification using SQUASH. The crystallographic phase problem is indeterminate given only the structure–factor amplitudes. It is only through the knowledge of the chemical or physical properties of the electron density that the phases can be retrieved. The characteristic features of electron density can often be expressed as mathematical constraints on the phases. The phasing power of a constraint depends on the number of density points affected and the magnitudes of changes imposed on the electron density. It also depends on the physical nature and accuracy of the constraint and on how rigorously the constraint is applied. The phasing power also increases with the number of independent constraints employed.

Crystal structure of E. coli β–carbonic anhydrase, an enzyme with an unusual pH–dependent activity

Carbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β‐class carbonic anhydrases (β‐CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β‐CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the β‐CA from the chloroplast of the red alga Porphyridium purpureum, the active‐site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β‐CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH‐dependent in the physiological range, and its expression in yeast complements an oxygen‐sensitive phenotype displayed by a β‐CA‐deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β‐CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.

Scaffold-based discovery of indeglitazar, a PPAR pan-active anti-diabetic agent

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARα, PPARγ, and PPARδ. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARγ, to circumvent the clinically observed side effects of full PPARγ agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARγ. Compared with full PPARγ-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).