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One of the challenges for targeting B-Raf(V600E) with small molecule inhibitors had been achieving adequate selectivity over the wild-type protein B-Raf(WT), as inhibition of the latter has been associated with hyperplasia in normal tissues. Recent studies suggest that B-Raf inhibitors inducing the 'DFG-in/αC-helix-out' conformation (Type IIB) likely will exhibit improved selectivity for B-Raf(V600E). To explore this hypothesis, we transformed Type IIA inhibitor (1) into a series of Type IIB inhibitors (sulfonamides and sulfamides 4-6) and examined the SAR. Three selectivity indices were introduced to facilitate the analyses: the B-Raf(V600E)/B-Raf(WT) biochemical ((b)S), cellular ((c)S) selectivity, and the phospho-ERK activation ((p)A). Our data indicates that α-branched sulfonamides and sulfamides show higher selectivities than the linear derivatives. We rationalized this finding based on analysis of structural information from the literature and provided evidence for a monomeric B-Raf-inhibitor complex previously hypothesized to be responsible for the desired B-Raf(V600E) selectivity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors., (©2011 AACR.)

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., (V600E)B-Raf A375, IC(50) on cells = 2 nmol/L; ED(50) on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor-induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients., ((c) 2010 AACR.)

The discovery and optimization of a novel series of aminoisoquinolines as potent, selective, and efficacious inhibitors of the mutant B-Raf pathway is presented. The N-linked pyridylpyrimidine benzamide 2 was identified as a potent, modestly selective inhibitor of the B-Raf enzyme. Replacement of the benzamide with an aminoisoquinoline core significantly improved kinase selectivity and imparted favorable pharmacokinetic properties, leading to the identification of 1 as a potent antitumor agent in xenograft models.

The cluster [Rh(6)(CO)(16)] reacts with (PPN)NO(2) [PPN = N(PPh(3))(2)] in dichloromethane to form the known cluster [Rh(6)N(CO)(15)](-) in which the nitrogen atom is encapsulated within a trigonal prismatic Rh(6) cluster. A kinetic study shows that the reactions proceed in six kinetically distinguishable steps followed by another four steps that are kinetically unidentifiable but that can nevertheless be identified speculatively on the basis of analogies to other reactions of carbonyl clusters. Stopped-flow studies show that the initial step involves bimolecular nucleophilic attack on a carbon atom of a CO ligand by an oxygen atom in the NO(2)(-) ion. This is followed by CO loss (via a [CO]-dependent and a [CO]-independent path) with formation of [Rh(6)(CO)(14)(OC[combining low line]ON[combining low line]O)](-). Activation parameters have been obtained for all three steps. [Rh(6)(CO)(14)(OC[combining low line]ON[combining low line]O)](-) has been characterized spectroscopically and has the C and N atoms coordinated to one Rh atom. Reaction of this cluster with CO leads to the fairly rapid establishment of an equilibrium with the adduct [Rh(6)(CO)(n)(OC[combining low line]ON[combining low line]O)](-), where n is most probably 15. Both participants in this equilibrium react more slowly with loss of CO(2), and these reactions show negligible dependence on temperature and are therefore largely entropy controlled. The overall reaction is very solvent dependent and numerous side reactions can be identified if the conditions of reaction are not closely controlled.

Reactions of the halides X- (X- = chloride, bromide or iodide) with the substituted cluster Rh6(CO)15(PPh3) in oxygen-free chloroform lead to [Rh5(CO)14(PPh3)]-, Rh(CO)2(PPh3)2X and [Rh(CO)2X2]- in the molar ratios 2:1: approximately 13. Oxidation by the solvent is assumed to lead to most of the Rh(I) product, and the stoichiometry for reactions with I- can be defined as 4Rh6(CO)15(PPh3) + 27I- + 12CHCl3 --> 2[Rh5(CO)14(PPh3)]- + Rh(CO)2(PPh3)2I + 13[Rh(CO)2I2]- + 6C2H2Cl4 + 4CO + 12Cl-. This can be rationalized quite simply with the aid of a few generally justifiable assumptions. Rate constants for reactions with bromide increase to a limiting value with increasing [Br-] in a way that shows that breaking of one Rh-Rh bond, with an unusual closo to nido structural change, is rate determining. This opening of the cluster might be spontaneous or solvent induced. To complete the reaction, the bromide has to compete with the reverse nido to closo change. The same closo to nido change is also a major rate determining step for reactions with P(OPh)3 in oxygen-free solutions, and for reactions with bromide in oxygenated solutions in the presence of trifluoroacetic and some other acids. The limiting rates increase slightly with increasing basicity of the ligands P(p-XC6H4)3 along the series X = F3C, Cl, F, H and MeO. Activation parameters for these reactions are reported.

Studies of CO-dissociative substitution reactions of the complexes Ru(CO)4L (L = a wide variety of P-donor ligands) have been extended and analysis of the results by the QALE methodology has been refined (QALE = quantitative analysis of ligand effects). Rates increase substantially with increasing size of L, mainly as a consequence of increasingly favourable activation entropies. These can be associated with increasing Ru-CO bond breaking that is compensated enthalpically by increasing Ru-P bond making allowed by release of steric strain. Explicit allowance for pi-acidity shows that these effects are just significant while sigma-donor and aryl effects are negligible. However, pendent hydrogen atoms, attached directly to the phosphorus atoms, have a pronounced and unique positive effect on the rates, with significant kinetic isotope effects (KIE). This is associated with the novel occurrence of direct Ru-H or incipient Ru-(eta2-P-H) agostic bond making as the CO ligand departs.

Tripyrrolylphosphine reacts with the cluster Rh6CO15(NCMe) to afford the disubstituted Rh6CO14(mu2)-P(NC4H4)3) derivative (2) via the Rh6CO15P(NC4H4)3 intermediate (1) with eta(1)-P coordination. In the solid state, 2 has the phosphine occupying a bridging position where it is bonded to two neighboring Rh atoms in the Rh(6) octahedron through the P-atom and an approximately tetrahedral alpha-carbon atom of one of the pyrrolyl rings. This can be described by the interaction of an electron pair, associated with a negative charge on one of the canonical forms of the NC(4)H(4) ring, with the adjacent Rh center. (1)H NMR spectra show that the solid-state structure is retained in solution, but the phosphine is not rigid, and three distinctive dynamic processes are observed. Each of these represents independent hindered rotation of inequivalent pyrrolyl rings about P-N bonds, the ring involved in the interaction with the Rh(6) skeleton displaying the highest activation barrier with deltaH = 15.8 +/- 0.1 kcal mol(-1) and deltaS = 1.4 +/- 0.3 cal K(-1) mol(-1). The assignment has been confirmed by 1H TOCSY and EXSY experiments, and a mechanism is proposed. The formation of 2 from 1 is reversible in the presence of CO, which is highly unusual for bridged clusters. The kinetics of the forward and reverse reactions have been studied, and the values of DeltaH degrees and DeltaS degrees for formation of 2 (+1.3 +/- 0.5 kcal mol(-1) and -9 +/- 2 cal K(-1) mol (-1), respectively) show that the Rh-C bond in the bridge is comparable in strength with the Rh-CO bond it replaces. The intrinsic entropy of 2 is exceptionally unfavorable, overcoming the favorable entropy caused by CO release, and this allows the reversibility of bridge formation. The reactions proceed via a reactive intermediate that may involve agostic bonding of the ring. The reverse reaction has an exceedingly unfavorable activation entropy that emphasizes the unique nature of 2.

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

GR231118, BW1911U90, Bis(31/31')[[Cys31, Trp32, Nva34] neuropeptide Y(31-36)] (T-190) and [Trp-Arg-Nva-Arg-Tyr]2-NH2 (T-241) are peptide analogs of the C-terminus of neuropeptide Y that have recently been shown to be antagonists of the neuropeptide Y Y1 receptor. In this study, the activity of these peptides at each of the cloned neuropeptide Y receptor subtypes is determined in radioligand binding assays and in functional assays (inhibition of forskolin-stimulated cAMP formation). GR231118 is a potent antagonist at the human and rat neuropeptide Y Y1 receptors (pA2 = 10.5 and 10.0, respectively; pKi = 10.2 and 10.4, respectively), a potent agonist at the human neuropeptide Y Y4 receptor (pEC50 = 8.6; pKi = 9.6) and a weak agonist at the human and rat neuropeptide Y Y2 and Y5 receptors. GR231118 also has high affinity for the mouse neuropeptide Y Y6 receptor (pKi = 8.8). Therefore, GR231118 is a relatively selective neuropeptide Y Y1 receptor antagonist, but has appreciable activity at the neuropeptide Y Y4 and Y6 receptors as well. BW1911U90, T-190 and T-241 are moderately potent neuropeptide Y Y1 receptor antagonists (pA2 = 7.1, 5.8 and 6.5, respectively; pKi = 8.3, 6.5 and 6.8, respectively) and neuropeptide Y Y4 receptor agonists (pEC50 = 6.8, 6.3 and 6.6, respectively; pKi; 8.3, 7.7 and 8.3, respectively). These data suggest that the C-terminus of neuropeptide Y and related peptides is sufficient for activation of the neuropeptide Y Y4 receptor, but is not sufficient for activation of the neuropeptide Y Y1 receptor. Because BW1911U90, T-190 and T-241 are significantly less potent at the cloned human neuropeptide Y Y1 receptor than at the neuropeptide Y receptor in human erythroleukemia cells, these cells may express a novel neuropeptide Y receptor with high affinity for these peptides.

When deprived of nerve growth factor (NGF), developing sympathetic neurons die by apoptosis. This death is associated with an increase in the level of c-Jun protein and is blocked by expression of a c-Jun dominant negative mutant. Here we have investigated whether NGF withdrawal activates Jun kinases, a family of stress-activated protein kinases that can stimulate the transcriptional activity of c-Jun by phosphorylating serines 63 and 73 in the transactivation domain and which can activate c-jun gene expression. We found that sympathetic neurons contained high basal levels of Jun kinase activity that increased further after NGF deprivation. In contrast, p38 kinase, another stress-activated protein kinase that can also stimulate c-jun gene expression, was not activated after NGF withdrawal. Consistent with Jun kinase activation, we found using a phospho-c-Jun-specific antibody that c-Jun was phosphorylated on serine 63 after NGF withdrawal. Furthermore, expression of a constitutively active form of MEK kinase 1 (MEKK1), which strongly activates the Jun kinase pathway, increased c-Jun protein levels and c-Jun phosphorylation and induced apoptosis in the presence of NGF. This death could be prevented by co-expression of SEKAL, a dominant negative mutant of SAPK/ERK kinase 1 (SEK1), an activator of Jun kinase that is a target of MEKK1. In contrast, expression of SEKAL alone did not prevent c-Jun expression, increases in c-Jun phosphorylation, or cell death after NGF withdrawal. Thus, activation of Jun kinase and increases in c-Jun phosphorylation and c-Jun protein levels occur at the same time after NGF withdrawal, but c-Jun levels and phosphorylation are regulated by an SEK1-independent pathway.

Sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis in its absence. We have investigated the pattern of expression of the Jun and Fos family of transcription factors in dying sympathetic neurons using antibodies specific for each family member. When sympathetic neurons are deprived of NGF, the level of c-Jun protein significantly increases, whereas the levels of the other members of the Jun and Fos family remain relatively constant. c-Jun also becomes more phosphorylated, probably on its amino terminal transactivation domain. When microinjected into sympathetic neurons, an expression vector for a c-Jun dominant negative mutant protects them against NGF withdrawal-induced death, indicating that AP-1 activity is essential for neuronal cell death. Furthermore, overexpression of the full-length c-Jun protein is, in itself, sufficient to induce apoptosis in sympathetic neurons.