A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) calponin homology (CH)-domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of β-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t1/2) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the calponin homology (CH)-domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess “substrate trapping” properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC20 in vitro, truncated SMTNL1 lacking the CH-domain was ineffective. In summary, our findings suggest an important role for the CH-domain in mediating the effects of SMTNL1 on smooth muscle contraction.

p90 ribosomal S6 kinases (RSKs), containing two distinct kinase catalytic domains, are phosphorylated and activated by extracellular signal-regulated kinase (ERK). The amino-terminal kinase domain (NTD) of RSK phosphorylates exogenous substrates, whereas the carboxyl-terminal kinase domain (CTD) autophosphorylates Ser-386. A conserved putative autoinhibitory alpha helix is present in the carboxyl-terminal tail of the RSK isozymes (697 HLVKGAMAATYSALNR 712 of RSK2). Here, we demonstrate that truncation (⌬␣) or mutation (Y707A) of this helix in RSK2 resulted in constitutive activation of the CTD. In vivo, both mutants enhanced basal Ser-386 autophosphorylation by the CTD above that of wild type (WT). The enhanced Ser-386 autophosphorylation was attributed to disinhibition of the CTD because a CTD dead mutation (K451A) eliminated Ser-386 autophosphorylation even in conjunction with ⌬␣ and Y707A. Constitutive activity of the CTD appears to enhance NTD activity even in the absence of ERK phosphorylation because basal phosphorylation of S6 peptide by ⌬␣ and Y707A was ϳ4-fold above that of WT. A RSK phosphorylation motif antibody detected a 140-kDa protein (pp140) that was phosphorylated upon epidermal growth factor or insulin treatment. Ectopic expression of ⌬␣ or Y707A resulted in increased basal phosphorylation of pp140 compared with that of WT, presenting the possibility that pp140 is a novel RSK substrate. Thus, it is clear that the CTD regulates NTD activity in vivo as well as in vitro.p90 ribosomal S6 kinase (RSK) 1 is a member of a growing subfamily of mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) that contain two distinct kinase catalytic domains in a single polypeptide chain (see Fig. 1A). The three mammalian isozymes of RSK (RSK1, RSK2, RSK3), encoded by separate genes (1), are phosphorylated and activated in vivo by extracelluar signal-regulated kinase (ERK).The amino-terminal kinase domain (NTD) of RSK, residues 68 -327 of human RSK2 (see Fig. 1A), is most closely related to p70 S6 kinase with regard to primary structure. To date, only the NTD has been shown to phosphorylate exogenous substrates for RSK, including the cAMP response element binding protein (2, 3), c-Fos (4, 5) and the estrogen receptor (6). The list of substrates suggests that RSK plays a role in transcriptional regulation. The carboxyl-terminal kinase domain (CTD) of RSK, residues 422-679 in RSK2 (see Fig. 1A), is related to calmodulin-dependent protein kinases (CaMKs) and autophosphorylates Ser-386 in the linker region between the two kinase domains (7).Activation of RSK in vivo requires interaction between ERK and the ERK-docking site located in the extreme carboxyl terminus of RSK (8,9). RSK activation also requires ERK phosphorylation of Thr-577 in the CTD activation loop and Ser-369 in the linker, as well as autophosphorylation of Ser-386 by the CTD (see Fig. 1A) (7). Attenuation of CTD activity by mutation of Thr-577 or the ATP binding pocket generates an enzyme that cannot be fully activated (7,10,11). Thus...