HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Identification of imidazo[1,2-b]pyridazine TYK2 pseudokinase ligands as potent and selective allosteric inhibitors of TYK2 signalling.

As a member of the Janus (JAK) family of non-receptor tyrosine kinases, TYK2 mediates the signaling of pro-inflammatory cytokines including IL-12, IL-23 and type 1 interferon (IFN), and therefore represents an attractive potential target for treating the various immuno-inflammatory diseases in which these cytokines have been shown to play a role. Following up on our previous report that ligands to the pseudokinase domain (JH2) of TYK2 suppress cytokine-mediated receptor activation of the catalytic (JH1) domain, the imidazo[1,2-b]pyridazine (IZP) 7 was identified as a promising hit compound. Through iterative modification of each of the substituents of the IZP scaffold, the cellular potency was improved while maintaining selectivity over the JH1 domain. These studies led to the discovery of the JH2-selective TYK2 inhibitor 29, which provided encouraging systemic exposures after oral dosing in mice. Phosphodiesterase 4 (PDE4) was identified as an off-target and potential liability of the IZP ligands, and selectivity for TYK2 JH2 over this enzyme was obtained by elaborating along selectivity vectors determined from analyses of X-ray co-crystal structures of representative ligands of the IZP class bound to both proteins.

Regulation of phosphatase activity in bacterial chemotaxis.

Bacterial chemotaxis is the most studied model system for signaling by the widely spread family of two-component regulatory systems. It is controlled by changes in the phosphorylation level of the chemotactic response regulator, CheY, mediated by a histidine kinase (CheA) and a specific phosphatase (CheZ). While it is known that CheA activity is regulated, via the receptors, by chemotactic stimuli, the input that may regulate CheY dephosphorylation by CheZ has not been found. We measured, by using stopped-flow fluorometry, the kinetics of CheZ-mediated dephosphorylation of CheY. The onset of dephosphorylation was delayed by approximately 50 ms after mixing phosphorylated CheY (CheY approximately P) with CheZ, and a distinct overshoot was observed in the approach to the new steady state of CheY approximately P. The delay and overshoot were not observed in a hyperactive mutant CheZ protein (CheZ54RC) that does not support chemotaxis in vivo and appears to be constitutively active. CheZ activity was cooperative with respect to CheY approximately P, with a Hill-coefficient of 2.5. The observed delayed modulation of CheZ activity and its cooperativity suggest that the phosphatase activity is regulated at the level of CheY approximately P-CheZ interaction. This novel kind of interplay between a response regulator and its phosphatase may be involved in signal tuning and in adaptation to chemotactic signals.

Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2.

We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.

Tar-dependent and -independent pattern formation by Salmonella typhimurium.

When Salmonella typhimurium cells were allowed to swarm on either a minimal or complex semisolid medium, patterns of cell aggregates were formed (depending on the thickness of the medium). No patterns were observed with nonchemotactic mutants. The patterns in a minimal medium were not formed by a mutant in the aspartate receptor for chemotaxis (Tar) or by wild-type cells in the presence of alpha-methyl-D,L-aspartate (an aspartate analog), thus resembling the patterns observed earlier in Escherichia coli (E. O. Budrene and H. C. Berg, Nature [London] 349:630-633, 1991) and S. typhimurium (E. O. Budrene and H. C. Berg, Abstracts of Conference II on Bacterial Locomotion and Signal Transduction, 1993). Distinctively, the patterns in a complex medium had a different morphology and, more importantly, were Tar independent. Furthermore, mutations in any one of the genes encoding the methyl-accepting chemotaxis receptors (tsr, tar, trg, or tcp) did not prevent the pattern formation. Addition of saturating concentrations of the ligands of these receptors to wild-type cells did not prevent the pattern formation as well. A tar tsr tcp triple mutant also formed the patterns. Similar results (no negative effect on pattern formation) were obtained with a ptsI mutant (defective in chemotaxis mediated by the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system [PTS]) and with addition of mannitol (a PTS ligand) to wild-type cells. It therefore appears that at least two different pathways are involved in the patterns formed by S. typhimurium: Tar dependent and Tar independent. Like the Tar-dependent patterns observed by Budrene and Berg, the Tar-independent patterns could be triggered by H(2)O(2), suggesting that both pathways of pattern formation may be triggered by oxidative stress.

Oligomerization of the phosphatase CheZ upon interaction with the phosphorylated form of CheY. The signal protein of bacterial chemotaxis.

Earlier studies have suggested that CheZ, the phosphatase of the signaling protein CheY in bacterial chemotaxis, may be in an oligomeric state both when bound to phosphorylated CheY (CheY approximately P) (Blat, Y., and Eisenbach, M. (1994) Biochemistry 33, 902-906) or free (Stock, A., and Stock, J. B. (1987) J. Bacteriol. 169, 3301-3311). The purpose of the current study was to determine the oligomeric state of free CheZ and to investigate whether it changes upon binding to CheY approximately P. By using either one of two different sets of cross-linking agents, free CheZ was found to be a dimer. The formation of the dimer was specific, as it was prevented by SDS which does not interfere with cross-linking mediated by random collisions. The dimeric form of CheZ was confirmed by sedimentation analysis, a cross-linking-free technique. In the presence of CheY approximately P (but not in the presence of non-phosphorylated CheY), a high molecular size cross-linked complex (90-200 kDa) was formed, in which the CheZ:CheY ratio was 2:1. The size of the oligomeric complex was estimated by fluorescence depolarization to be 4-5-fold larger than the dimer, suggesting that its size is in the order of 200 kDa. These results indicate that CheZ oligomerizes upon interaction with CheY approximately P. This phosphorylation-dependent oligomerization may be a mechanism for regulating CheZ activity.

Mutants with defective phosphatase activity show no phosphorylation-dependent oligomerization of CheZ. The phosphatase of bacterial chemotaxis.

CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis. The mechanism by which the activity of CheZ is regulated is not known. We used cheZ mutants of Salmonella typhimurium, which had been isolated by Sockett et al. (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V. M., and Macnab, R. M. (1992) J. Bacteriol. 174, 793-806), for cloning the mutant cheZ genes, overexpressing and purifying their products. We then measured the phosphatase activity, binding to CheY and to phosphorylated CheY (CheY approximately P), and CheY approximately P dependent oligomerization of the mutant CheZ proteins. While all the mutant proteins were defective in their phosphatase activity, they bound to CheY and CheY approximately P as well as wild-type CheZ. However, unlike wild-type CheZ, all the four mutant proteins failed to oligomerize upon interaction with CheY approximately P. On the basis of these and earlier results it is suggested that (i) oligomerization is required for the phosphatase activity of CheZ, (ii) the region defined by residues 141-145 plays an important role in mediating CheZ oligomerization and CheY approximately P dephosphorylation but is not necessary for the binding to CheY approximately P, (iii) the oligomerization and hence the phosphatase activity are regulated by the level of CheY approximately P, and (iv) this regulation plays a role in the adaptation to chemotactic stimuli.

Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region.

Sister chromatid cohesion is mediated by evolutionary conserved chromosomal proteins, termed "cohesins." Using an extension of chromatin immunoprecipitation, we have analyzed the distribution of cohesins Mcd1/ Sccl and Smc1 along yeast chromosome III. Both proteins occur preferentially at the same approximately 23 positions. Sites in a approximately 50 kb region around the centromere give especially intense signals. Prominent centric region binding appears to emerge from a more even distribution, probably by differential loss of cohesins along the chromosome arms. Cohesin binding peaks correspond closely to peaks of high local AT composition, a base composition periodicity of approximately 15 kb that is distinct from the approximately 50 kb periodicity of base composition isochores, consistent with axis association of cohesins. The methodology described can be used to analyze the distribution of any DNA-binding protein and, via microchips, along entire genomes.

Conserved C-terminus of the phosphatase CheZ is a binding domain for the chemotactic response regulator CheY.

CheZ is the phosphatase of the chemotactic response regulator, CheY. There are three conserved domains on CheZ. Here we determined the function of the C-terminal domain (residues 202-214). A truncated form of CheZ, missing residues 202-214, hardly bound to the phosphorylated form of CheY. Conversely, a peptide composed of the last 19 amino acid residues of the CheZ (residues 196-214), generated by tryptic digestion, bound specifically to the phosphorylated form of CheY. This was demonstrated by both fluorescence depolarization of the peptide (labeled with fluorescein) and cross-linking. It is concluded that the conserved C-terminus of CheZ functions as a CheY-binding domain.

Phosphorylation-dependent binding of the chemotaxis signal molecule CheY to its phosphatase, CheZ.

Bacterial chemotaxis is accomplished by regulating the direction of flagellar rotation. The primary target of the control appears to be CheY, a diffusible clockwise-signal molecule which interacts with the switch at the base of the flagellar motor and causes clockwise rotation. The regulatory mechanism appears to be phosphorylation/dephosphorylation of CheY. Here we demonstrate that CheZ, which accelerates the dephosphorylation of CheY, binds to CheY (immobilized on CNBr-activated Sepharose beads), that the binding to phosphorylated CheY is higher by over 2 orders of magnitude than the binding to nonphosphorylated CheY, and that the binding to both the phosphorylated and nonphosphorylated forms of CheY is significantly higher in the presence of Mg2+. We also show that the mutant proteins CheY13DK, CheY57DE, and CheY109KR bind CheZ to the same extent as wild-type CheY. The extent of the binding of these mutant proteins was not, however, increased in the presence of acetyl phosphate, the phosphorylating agent. The results indicate that neither a conformation which has a clockwise-causing activity in vivo nor phosphorylation is sufficient, alone, for maximal binding of CheZ to CheY and that Mg2+ is required for the binding of these proteins as well as for the phosphorylation and dephosphorylation of CheY.

Signal termination in bacterial chemotaxis: CheZ mediates dephosphorylation of free rather than switch-bound CheY.

Chemotaxis in bacteria is controlled by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein CheY. When phosphorylated, CheY binds to FliM, which is one of the proteins that constitute the "gear box" (or "switch") of the flagellar motor. Consequently, the motor shifts from the default direction of rotation, counterclockwise, to clockwise rotation. This biased rotation is terminated when CheY is dephosphorylated either spontaneously or, faster, by a specific phosphatase, CheZ. Logically, one might expect CheZ to act directly on FliM-bound CheY. However, here we provide direct biochemical evidence that, in contrast to this expectation, phosphorylated CheY (CheY approximately P), bound to FliM, is protected from dephosphorylation by CheZ. The complex between CheY approximately P and FliM was trapped by cross-linking with dimethylsuberimidate, and its susceptibility to CheZ was measured. CheY approximately P complexed with FliM, unlike free CheY approximately P, was not dephosphorylated by CheZ. However, it did undergo spontaneous dephosphorylation. Nonspecific cross-linked CheY dimers, measured as a control, were dephosphorylated by CheZ. No significant binding between CheZ and any of the switch proteins was detected. It is concluded that, in the termination mechanism of signal transduction in bacterial chemotaxis, CheZ acts only on free CheY approximately P. We suggest that CheZ affects switch-bound CheY approximately P by shifting the equilibrium between bound and free CheY approximately P.