Rigidification Dramatically Improves Inhibitor Selectivity for RAF Kinases.

One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAFV600E-harboring cell lines consistent with its kinome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.

Effects of rigidity on the selectivity of protein kinase inhibitors.

Established strategies for discovering selective kinase inhibitors are target-centric as they often target certain structural or reactive features in the target kinase. In the absence of such prominent features, there is a lack of general methods for discovering selective inhibitors. Here we describe a new strategy that exploits conformational flexibility of kinases for achieving selective kinase inhibition. Through ring closure, we designed and synthesized a panel of isoquinoline-containing compounds as rigidified analogs of an amidophenyl-containing parent compound. These analogs potently inhibit kinases including Abl and BRAF but have diminished inhibition against some other kinases compared to the parent compound. Sequence analysis reveals that many of the kinases that are potently inhibited by the isoquonoline-containing compounds contain a long insertion within their catalytic domains. A crystal structure of one rigid compound bound to BRAF confirmed its binding mode. Our findings highlight the potential of a novel strategy of rigidification for improving the selectivity of kinase inhibitors.

A Chemical-Genetic Approach Reveals the Distinct Roles of GSK3α and GSK3ß in Regulating Embryonic Stem Cell Fate.

Glycogen synthase kinase 3 (GSK3) plays a central role in diverse cellular processes. GSK3 has two mammalian isozymes, GSK3α and GSK3ß, whose functions remain ill-defined because of a lack of inhibitors that can distinguish between the two highly homologous isozymes. Here, we show that GSK3α and GSK3ß can be selectively inhibited in mouse embryonic stem cells (ESCs) using a chemical-genetic approach. Selective inhibition of GSK3ß is sufficient to maintain mouse ESC self-renewal, whereas GSK3α inhibition promotes mouse ESC differentiation toward neural lineages. Genome-wide transcriptional analysis reveals that GSK3α and GSK3ß have distinct sets of downstream targets. Furthermore, selective inhibition of individual GSK3 isozymes yields distinct phenotypes from gene deletion, highlighting the power of the chemical-genetic approach in dissecting kinase catalytic functions from the protein's scaffolding functions. Our study opens new avenues for defining GSK3 isozyme-specific functions in various cellular processes.

Synthesis, docking simulation, biological evaluations and 3D-QSAR study of 5-Aryl-6-(4-methylsulfonyl)-3-(metylthio)-1,2,4-triazine as selective cyclooxygenase-2 inhibitors.

A series of 5-Aryl-6-(4-methylsulfonyl)-3-(metylthio)-1,2,4-triazine derivatives were synthesized and their COX-1/COX-2 inhibitory activity as well as in vivo anti-inflammatory and analgesic effects were evaluated. All of compounds showed strong inhibition of COX-2 with IC50 values in the range of 0.1-0.2µM and in most cases had stronger anti-inflammatory and analgesic effects than indomethacin at doses 3 and 6mg/kg. Among them, 5-(4-chlorophenyl)-6-(4-(methylsulfonyl) phenyl)-3-(methylthio)-1,2,4-triazine (9c) was the most potent and selective COX-2 compound; its selectivity index of 395 was comparable to celecoxib (SI=405). Evaluation of anti-inflammatory and analgesic effects of 9c showed its higher potency than indomethacin and hence could be considered as a promising lead candidate for further drug development. Furthermore, the affinity data of these compounds were rationalized through enzyme docking simulation and 3D-QSAR study by k-Nearest Neighbour Molecular Field Analysis.

Synthesis, cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors.

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G2/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.

Design, synthesis, cytotoxic evaluation and tubulin inhibitory activity of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazole derivatives.

A new series of 4-aryl-5-(3,4,5-trimethoxyphenyl)-2-alkylthio-1H-imidazoles were synthesized and their cytotoxic activities in vitro against four different cell lines (HT-29, MCF-7, NIH-3T3, AGS) were evaluated. Compound 6g bearing 3,4,5-trimethoxyphenyl moiety on ring A and 4-methoxy substituent on ring B displayed potent cytotoxic activity against all cell lines. Flow cytometry analysis and microtubule polymerization assay confirmed that cytotoxic activities of this compound were related to inhibitory effect against microtubules polymerization. Molecular modeling studies revealed that compound 6g could strongly bind to the colchicine binding site of α,ß-tubulin through hydrogen bond interactions with Thrα179 and Cysß241.

Synthesis, cyclooxygenase inhibitory effects, and molecular modeling study of 4-aryl-5-(4-(methylsulfonyl)phenyl)-2-alkylthio and -2-alkylsulfonyl-1H-imidazole derivatives.

A series of 4-aryl-5-(4-(methylsulfonyl)phenyl)-2-alkylthio and 2-alkylsulfonyl-1H-imidazole derivatives were synthesized. All compounds were tested in human blood assay to determine COX-1 and COX-2 inhibitory potency and selectivity. Among the synthesized compounds, 2-alkylthio series were more potent and selective than 2-sulfonylalkyl derivatives. In molecular modeling, interaction of 2-sulfonylalkyl moiety with Arg120 in COX-1 and an extra hydrogen bond with Tyr341 in COX-2 increased the residence time of ligands in the active site in 2-sulfonylalkyl and 2-alkylthio analogs, respectively.

Synthesis and SAR study of 4,5-diaryl-1H-imidazole-2(3H)-thione derivatives, as potent 15-lipoxygenase inhibitors.

A series of 4,5-diaryl-1H-imidazole-2(3H)-thione was synthesized and their inhibitory potency against soybean 15-lipoxygenase and free radical scavenging activities were determined. Compound 11 showed the best IC(50) for 15-LOX inhibition (IC(50) = 4.7 µM) and free radical scavenging activity (IC(50) = 14 µM). Methylation of SH at C(2) position of imidazole has dramatically decreased the 15-LOX inhibition and radical scavenging activity as it can be observed in the inactive compound 14 (IC(50) >250 µM). Structure activity similarity (SAS) showed that the most important chemical modification in this series was methylation of SH group and Docking studies revealed a proper orientation for SH group towards Fe core of the 15-LOX active site. Therefore it was concluded that iron chelating could be a possible mechanism for enzyme inhibition in this series of compounds.

Synthesis and SAR studies of 3-allyl-4-prenyloxyaniline amides as potent 15-lipoxygenase inhibitors.

15-Lipoxygenases are one of the nonheme iron-containing proteins with ability of unsaturated lipid peroxidation in animals and plants. The critical role of the enzymes in formation of inflammations, sensitivities and some of cancers has been demonstrated in mammalians. Importance of the 15-lipoxygenases leads to development of mechanistic studies, products analysis and synthesis of their inhibitors. In this work new series of the 3-allyl-4-allyoxyaniline amides and 3-allyl-4-prenyloxyaniline amides were designed, synthesized and their inhibitory potency against soybean 15-lipoxygenase were determined. Among the synthetic amides, 3-allyl-4-(farnesyloxy)-adamantanilide showed the most potent inhibitory activity by IC(50) value of 0.69 µM. SAR studies showed that in spite of prenyl length increases, the effects of the amide size and its electronic properties on the inhibitory potency became predominant. The SAR studies was also showed that the orientation of allyl and prenyloxy moieties toward Fe core of the SLO active site pocket is the most suitable location for enzyme inhibition.

Synthesis of new series of α-cyclodextrin esters as dopamine carrier molecule.

A new series of amphiphilic α-cyclodextrins were synthesized by grafting N-acylated amino acids [valine, leucine, phenylalanine, methionine, and tryptophan (3a-e)] to the primary hydroxyl groups via ester bond formation. The synthetic pathway involves selective hexa-bromination of the primary hydroxyls followed by per-substitution with the carboxylate moiety of the N-acetyl residues in the presence of DBU (1,8-diazabicyclo[5,4,0]undec-7-ene). The ability of the synthetic compounds for the extraction of dopamine was studied. The results showed a considerable ability of some of the amphiphilic compounds for the extraction of dopamine into octanol phase from water. To complete the study, the binding affinity of dopamine toward the synthetic host molecules was calculated by using of the molecular docking technique.