Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

Scaffold hopping towards potent and selective JAK3 inhibitors: discovery of novel C-5 substituted pyrrolopyrazines.

The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.

Steady-state kinetic characterization of kinase activity and requirements for Mg2+ of interleukin-1 receptor-associated kinase-4.

Interleukin-1 receptor-associated kinase-4 (IRAK-4) is a Ser/Thr-specific protein kinase that plays a critical role in intracellular signaling cascades mediated by Toll-like and interleukin-1 (IL-1) receptors. Despite a growing body of information on the physiological functions of IRAK-4, its kinase activity remains poorly studied. The present study entails characterization of the steady-state kinetic properties and Mg(2+) requirements of full-length, recombinant human IRAK-4 preactivated by incubation with MgATP. In the presence of 20 mM Mg(2+), activated IRAK-4 herein is demonstrated to phosphorylate a peptide substrate (IRAK-1 peptide), derived from the activation loop of IRAK-1, with a k(cat) of 30 +/- 2.9 s(-1) and K(m) values of 668 +/- 120 and 852 +/- 273 microM for ATP and the peptide, respectively. Two-substrate, dead-end and product inhibition data, using analogues of ATP, are consistent with both a sequential ordered kinetic mechanism with ATP binding to the enzyme prior to the peptide and a sequential random mechanism. Investigation of the Mg(2+) requirements for phosphoryl transfer activity of IRAK-4 revealed that more than one Mg(2+) ion interacts with the enzyme and that the enzyme is maximally active in the presence of 5-10 mM free Mg(2+). While one divalent metal, as part of a chelate complex with ATP, is essential for catalysis, kinetic evidence is provided to show that uncomplexed Mg(2+) further enhances the catalytic activity of IRAK-4 by bringing about an approximately 3-fold increase in k(cat) and an approximately 6-fold reduction in the K(m) for ATP and by rendering the interaction between the nucleotide and peptide substrate binding sites less antagonistic.

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex.

RNA-dependent RNA polymerases (RdRPs) from nonsegmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication, and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP. We determined that the intact L protein is essential for RdRP activity, and in truncated L protein constructs, RdRP activity is abrogated due to their deficiency in RNA template binding. These results are in agreement with the observation of an RNA template-binding tunnel at the interface of RdRP and capping domains in RSV and vesicular stomatitis virus (VSV) L protein cryo-EM structures. We also describe nonradiometric assays for measuring RNA binding and RNA polymerization activity of RSV RdRP, which are amenable to compound screening and profiling.

Design and Discovery of N-(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic.

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.

Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice.

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice-stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.

Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.

Design and Discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers.

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation.

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis.

3-Amido pyrrolopyrazine JAK kinase inhibitors: development of a JAK3 vs JAK1 selective inhibitor and evaluation in cellular and in vivo models.

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.

A modeling-derived hypothesis on chronicity in respiratory diseases: desensitized pathogen recognition secondary to hyperactive IRAK/TRAF6 signaling.

Several chronic respiratory diseases exhibit hyperactive immune responses in the lung: abundant inflammatory mediators; infiltrating neutrophils, macrophages, lymphocytes and other immune cells; and increased level of proteases. Such diseases include cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and severe/neutrophilic asthma. Paradoxically, patients with these diseases are also susceptible to detrimental bacterial infection and colonization. In this paper, we seek to explain how a positive feedback mechanism via IL-8 could lead to desensitization of epithelial cells to pathogen recognition thus perpetuating bacterial colonization and chronic disease states in the lung. Such insight was obtained from mathematical modeling of the IRAK/TRAF6 signaling module, and is consistent with existing clinical evidence. The potential implications for targeted treatment regimes for these persistent respiratory diseases are explored.

The kinase activities of interleukin-1 receptor associated kinase (IRAK)-1 and 4 are redundant in the control of inflammatory cytokine expression in human cells.

IRAK-1 and IRAK-4 are protein kinases that mediate signaling by Toll/IL1/Plant R (TIR) domain-containing receptors including the IL-1, IL-18, and Toll-like receptors (TLRs). Although well studied in mouse systems, the mechanism by which they function in human systems is less clear. To extend our knowledge of how these proteins regulate inflammatory signaling in human cells, we genetically and pharmacologically manipulated IRAK-1 and IRAK-4 kinase activities in vitro. Ablation of IRAK-4 expression in human umbilical vein endothelial cells (HUVEC) with siRNA suppressed IL-1beta induced IL-6 and IL-8 production whereas IRAK-1 siRNA suppressed TNFalpha induced but not IL-1beta induced cytokine production. Complementation of IRAK-4-depleted cells with a kinase-inactive allele restored IL-1beta induced cytokine gene expression suggesting that the IRAK-4 kinase activity is dispensable relative to its scaffolding function. Consistent with this finding, an IRAK-4 selective kinase inhibitor (RO6245) that inhibited IRAK-1 degradation failed to block IL-1beta induced cytokine production. In contrast, an inhibitor of both IRAK-1 and IRAK-4 (RO0884) reduced IL-1beta induced p38 MAP kinase, c-Jun N-terminal kinase activation, and IL-6 production in HUVEC. RO0884 also antagonized IL-1beta, TNFalpha, and TLR-mediated cytokine production in human fibroblast-like synoviocytes and peripheral blood mononuclear cells. Therefore in human cells the non-kinase functions of IRAK-4 are essential, whereas the kinase activity of IRAK-4 appears redundant with that of IRAK-1. Pharmacologic inhibition of both kinases appears necessary to block pro-inflammatory cytokine production.

A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase.

Structure of a kinesin microtubule depolymerization machine.

With their ability to depolymerize microtubules (MTs), KinI kinesins are the rogue members of the kinesin family. Here we present the 1.6 A crystal structure of a KinI motor core from Plasmodium falciparum, which is sufficient for depolymerization in vitro. Unlike all published kinesin structures to date, nucleotide is not present, and there are noticeable differences in loop regions L6 and L10 (the plus-end tip), L2 and L8 and in switch II (L11 and helix4); otherwise, the pKinI structure is very similar to previous kinesin structures. KinI-conserved amino acids were mutated to alanine, and studied for their effects on depolymerization and ATP hydrolysis. Notably, mutation of three residues in L2 appears to primarily affect depolymerization, rather than general MT binding or ATP hydrolysis. The results of this study confirm the suspected importance of loop 2 for KinI function, and provide evidence that KinI is specialized to hydrolyze ATP after initiating depolymerization.