Nicotinamide phosphoribosyltransferase (NMPRTase) has a crucial role in the salvage pathway of NAD+ biosynthesis, and a potent inhibitor of NMPRTase, FK866, can reduce cellular NAD+ levels and induce apoptosis in tumors. We have determined the crystal structures at up to 2.1-A resolution of human and murine NMPRTase, alone and in complex with the reaction product nicotinamide mononucleotide or the inhibitor FK866. The structures suggest that Asp219 is a determinant of substrate specificity of NMPRTase, which is confirmed by our mutagenesis studies. FK866 is bound in a tunnel at the interface of the NMPRTase dimer, and mutations in this binding site can abolish the inhibition by FK866. Contrary to current knowledge, the structures show that FK866 should compete directly with the nicotinamide substrate. Our structural and biochemical studies provide a starting point for the development of new anticancer agents.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

Nicotinamide adenine dinucleotide (NAD(+)) has crucial roles in many cellular processes, both as a coenzyme for redox reactions and as a substrate to donate ADP-ribose units. Enzymes involved in NAD(+) metabolism are attractive targets for drug discovery against a variety of human diseases, including cancer, multiple sclerosis, neurodegeneration and Huntington's disease. A small-molecule inhibitor of nicotinamide phosphoribosyltransferase, an enzyme in the salvage pathway of NAD(+) biosynthesis, is presently in clinical trials against cancer. An analog of a kynurenine pathway intermediate is efficacious against multiple sclerosis in an animal model. Indoleamine 2,3-dioxygenase plays an important role in immune evasion by cancer cells and other disease processes. Inhibitors against kynurenine 3-hydroxylase can reduce the production of neurotoxic metabolites while increasing the production of neuroprotective compounds. This review summarizes the existing knowledge on NAD(+) metabolic enzymes, with emphasis on their relevance for drug discovery.

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-Å resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix ␣D in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor. Toll-like receptors (TLRs)1 and interleukin-1 receptors (IL1Rs) have crucial roles in host immune and inflammatory responses (1). A total of 10 TLRs have been identified from the human genome. They recognize conserved molecular patterns in pathogenic microbial organisms, and this recognition is indispensable for the activation of the innate immune systems as well as the adaptive immune systems in vertebrates (2, 3). Ten different IL-1Rs are present in the human genome. IL-1R type I (IL-1RI) and IL-1R accessory protein (IL-1RAcP) mediate the pleiotropic effects of the proinflammatory cytokine IL-1, whereas IL-18R and AcPL function as receptors for the IL-18 cytokine (4). As their names imply, IL-1RI and IL-18R have high affinities for their ligands, whereas the accessory proteins (IL-1RAcP and AcPL) cannot bind the ligand directly.The TLRs and IL-1Rs have entirely different extracellular domains. The TLRs contain many copies of the leucine-rich repeat (LRR), whereas the IL-1Rs contain three copies of the Ig-like domains. However, these two families of receptors share a conserved intracellular domain, known as the Toll/interleukin-1 receptor (TIR) domain. This domain is also present in several cytoplasmic molecules, such as MyD88, TIRAP/MAL, TRIF/TICAM, and TIRP/TRAM, which function as signal adapters for these receptors (5).TIR domains contain 150 -200 amino acid residues, and their sequences are weakly conserved among the TLRs, IL-1Rs, and the adapter molecules (Fig. 1). The amino acid sequence identity between any pair of TIR domains is generally about 25%. The authentic...