Purpose: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase.Experimental Design: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments.Results: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET-dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET-dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time-and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers.Conclusions: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment.

Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5 -phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and of L-␤-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5 -phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, Ϸ25 Å from the active site. In this mode of pyridoxal-5 -phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5 -phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.A denosylcobalamin (AdoCbl; coenzyme B 12 ) is nature's biochemical radical reservoir, capable of catalyzing challenging chemical reactions by way of H atom abstraction and the generation of free-radical intermediates (1-3). AdoCbldependent isomerases catalyze 1,2 shifts between an H atom and a functional group such as -OH, -NH 3 ϩ , -(CO)S-coenzyme A, or other carbon-based groups. The catalytic power of AdoCbl lies in the homolytic cleavage of its weak (Ϸ30 kcal͞mol) organometallic C-Co bond, formed between an octahedral Co(III) center with five N coordinations and a 5Ј-deoxyadenosyl (Ado) axial ligand. C-Co bond homolysis results in the transient formation of cob(II)alamin and 5Ј-deoxyadenosyl radical (Ado • ). Ado • abstracts an H atom from the substrate, forming a substrate radical and 5Ј-deoxyadenosine (AdoH). To close the catalytic cycle, substrate reabstracts the H atom from AdoH, and recombination of cob(II)alamin and Ado • accompanies product formation. Amazingly, the enzymatic rate of C-Co bond homolysis is enhanced by a factor of Ϸ10 12 over nonenzymatic homolysis (4, 5). AdoCbl-dependent isomerases are often present in catabolic pathways and can serve to rearrange the substrate's carbon skeleton and͞or functional groups for further degradation. One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6, 7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8-10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8, 11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig. 1A). In 5,6-LAM, AdoCbl is the source of the transient Ado • , whereas, in 2,3-L...

The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

Cysteine desulfurases (CDs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that cleave sulfur from cysteine via an enzyme cysteinyl persulfide intermediate. In vitro studies of these enzymes have generally employed dithiothreitol as a cosubstrate to reductively cleave the persulfide intermediate, and it has been suggested that persulfide cleavage is the rate-limiting step for catalysis. In this study, the kinetics and mechanisms of cleavage of the persulfide intermediate in Slr0387 (CD-0387), a sequence group I (NifS/IscS-like) cysteine desulfurase from Synechocystis sp. PCC 6803, by physiological and nonphysiological reductants have been examined, and the extent to which this step is rate-limiting for catalysis has been determined. The observations that dithiols such as dithiothreitol (DTT) cleave the persulfide with approximately 100-fold greater efficiency than structurally similar monothiols such as 2-mercaptoethanol (2-ME), that cleavage by DTT exhibits saturation kinetics, and that the dependence of the observed first-order rate constant for persulfide cleavage by DTT on the concentration of the dithiol corresponds precisely with that for formation of a complex between DTT and the PLP cofactor of the resting enzyme suggest that persulfide cleavage by dithiols occurs by prior formation of a complex, in which addition of one thiol to the cofactor positions the second thiol for attack. This conclusion and the observation that a second molecule of L-cysteine can bind to the cofactor in the persulfide form of CD-0387 explain why several CDs are subject to potent inhibition by L-cysteine during turnover with DTT: binding of L-cysteine prevents formation of the PLP-DTT adduct and renders the dithiol no better than a monothiol, which must react with the persulfide in bimolecular fashion. Consistent with this rationale, catalysis by CD-0387 with 2-ME as cosubstrate, while less efficient, is not subject to potent inhibition by L-cysteine. The similarity of the maximum rate constant for persulfide cleavage by DTT to k(cat) suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that k(cat) is approximately 10-fold greater when tris-(2-carboxyethyl)phosphine (TCEP), the most efficient persulfide cleaver identified, is used as the reducing cosubstrate. The faster turnover with TCEP provides a chemical model for activation of CD-0387 and other CDs by the presence of accessory factors that serve as efficient acceptors of the persulfide sulfur.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase 1 study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies.