Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.
Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.
Heat shock protein (Hsp)90 is emerging as an important therapeutic target for the treatment of cancer. Two analogues of the Hsp90 inhibitor geldanamycin are currently in clinical trials. Geldanamycin (GA) and its analogues have been reported to bind purified Hsp90 with low micromolar potency, in stark contrast to their low nanomolar antiproliferative activity in cell culture and their potent antitumor activity in animal models. Several models have been proposed to account for the Ϸ100-fold-greater potency in cell culture, including that GA analogues bind with greater affinity to a five-protein Hsp90 complex than to Hsp90 alone. We have determined that GA and the fluorescent analogue BODIPY-GA (BDGA) both demonstrate slow, tight binding to purified Hsp90. BDGA, used to characterize the kinetics of ligand-Hsp90 interactions, was found to bind Hsp90␣ with k off ؍ 2.5 ؋ 10 ؊3 min ؊1 , t 1/2 ؍ 4.6 h, and Ki* ؍ 10 nM. It was found that BDGA binds to a functional multiprotein Hsp90 complex with kinetics and affinity identical to that of Hsp90 alone. Also, BDGA binds to Hsp90 from multiple cell lysates in a time-dependent manner with similar kinetics. Therefore, our results indicate that the high potency of GA in cell culture and in vivo can be accounted for by its timedependent, tight binding to Hsp90 alone. In the broader context, these studies highlight the essentiality of detailed biochemical characterization of drug-target interactions for the effective translation of in vitro pharmacology to cellular and in vivo efficacy.benzoquinone ansamycin ͉ time-dependent inhibition ͉ BODIPY-geldananmycin H eat shock protein (Hsp)90 is a ubiquitous, highly expressed molecular chaperone protein capable of sensing cellular stress (1). In cells, Hsp90 functions as a multiprotein chaperone complex, with cochaperones that include Hsp70, Hsp40, and Hop (2). Upon ATP binding and hydrolysis, this intermediate complex forms a mature chaperone complex containing p23, which catalyzes the conformational maturation of ''client protein'' substrates (3, 4). Multiple client proteins of the Hsp90 chaperone complex are involved in signal-transduction pathways, cell-cycle regulation, and apoptosis pathways commonly deregulated in cancer (5, 6). Although essential for cellular viability, the pharmacological inhibition of this chaperone has emerged as an attractive approach for inhibition of tumorigenesis (7-10).The natural product geldanamycin (GA), a benzoquinone ansamycin, binds to Hsp90, inhibits its ATPase activity (11), and decreases cellular levels of client proteins involved in cancer cell survival, such as mutated p53, mutated B-Raf, Akt, Bcr-Abl, and ErbB2 (10). GA has potent antiproliferative activity in many cell lines in culture (12) and inhibits tumor growth in mouse xenograft models (10). Two GA analogues, 17-allylamino,17-demethoxygeldanamycin (17-AAG) and , are currently in multiple clinical trials for the treatment of cancer (7, 13, 14).GA binds to the N-terminal ATP-binding domain of Hsp90 and inhibits ATP binding and ATP...
Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.
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