Abstract. A set of screening conditions for initial experiments in protein crystallization has been developed, tested, and is herein presented. These solution and precipitant conditions are empirically derived based on known or published crystallization conditions of various proteins in the past, so as to sample as large a range of buffer, pH, additive and precipitant variables as possible, using small amounts of proteins. The 50 crystallization conditions have been tested on 15 previously crystallized proteins, all of which were also crystallized in at least one form by this screen. This method is also shown to be highly successful in the crystallization of proteins which had not previously been crystallized.

BRAF V600E 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-Raf V600E 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-Raf V600E 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-Raf V600E -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-Raf V600E -positive cells. In B-Raf V600E -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-Raf V600E -driven tumors.cancer ͉ cell signaling ͉ melanoma ͉ phosphorylation ͉ protein kinases O ncogenic mutations in the BRAF gene (1) correlate with increased severity and decreased response to chemotherapy in a wide variety of human tumors (2-4). Hence, direct therapeutic inhibition of oncogenic B-Raf kinase activity affords an avenue to treat these tumors. The therapeutic approach of targeting oncogenic kinase activity has proved very valuable in oncology (5, 6). Recently, we have described the technique termed scaffold-based drug discovery, a strategy for identifying small molecule inhibitors of cyclic nucleotide phosphodiesterases (7). Here, we describe an expansion of this strategy to discover a scaffold targeting protein kinases, and we report the elaboration of this scaffold into the potent and selective B-Raf V600E inhibitor PLX4720. Because a majority of all melanomas harbor an activating missense mutation (V600E) in the B-Raf oncogene (1), targeted inhibition of the V600E gene product is a particularly rational therapeutic goal in this otherwise therapy-resistant tumor type. Previous generations of B-Raf inhibitors possess Raf inhibitory activity at low nanomolar concentrations (8-13); however, the relative therapeutic efficacy of such inhibitors has been hampered by the lack of bioavailability or by the number of nonspecific targets that are also affected (14, 15). The development of highly specific and effectual inhibitors of the BRAF V600E gene product would provide insight into the true therapeutic rele...

The principal heat-shock proteins that have chaperone activity (that is, they protect newly made proteins from misfolding) belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs can form large multimeric structures and have a wide range of cellular functions, including endowing cells with thermotolerance in vivo and being able to act as molecular chaperones in vitro; sHSPs do this by forming stable complexes with folding intermediates of their protein substrates. However, there is little information available about these structures or the mechanism by which substrates are protected from thermal denaturation by sHSPs. Here we report the crystal structure of a small heat-shock protein from Methanococcus jannaschii, a hyperthermophilic archaeon. The monomeric folding unit is a composite beta-sandwich in which one of the beta-strands comes from a neighbouring molecule. Twenty-four monomers form a hollow spherical complex of octahedral symmetry, with eight trigonal and six square 'windows'. The sphere has an outer diameter of 120 A and an inner diameter of 65 A.

Ras proteins participate as a molecular switch in the early steps of the signal transduction pathway that is associated with cell growth and differentiation. When the protein is in its GTP complexed form it is active in signal transduction, whereas it is inactive in its GDP complexed form. A comparison of eight three-dimensional structures of ras proteins in four different crystal lattices, five with a nonhydrolyzable GTP analog and three with GDP, reveals that the "on" and "off" states of the switch are distinguished by conformational differences that span a length of more than 40 A, and are induced by the gamma-phosphate. The most significant differences are localized in two regions: residues 30 to 38 (the switch I region) in the second loop and residues 60 to 76 (the switch II region) consisting of the fourth loop and the short alpha-helix that follows the loop. Both regions are highly exposed and form a continuous strip on the molecular surface most likely to be the recognition sites for the effector and receptor molecule(or molecules). The conformational differences also provide a structural basis for understanding the biological and biochemical changes of the proteins due to oncogenic mutations, autophosphorylation, and GTP hydrolysis, and for understanding the interactions with other proteins.