A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

The crystal structure of PF-8, the DNA polymerase accessory subunit from Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 A. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

Synthesis and characterization of an N-acylsulfonamide inhibitor of human asparagine synthetase.

[structure: see text] The synthesis of N-acylsulfonamide 6, which is an analogue of beta-aspartyl-AMP, is described. This compound appears to be the first and only potent inhibitor of human asparagine synthetase that has been described to date. The N-acylsulfonamide 6 exhibits slow-onset inhibition kinetics, with a K(i) of 728 nM. Preparation and characterization of two additional N-acylsulfonamide analogues has also demonstrated the importance of hydrogen-bonding interactions in the recognition of the AS inhibitor with the enzyme. These observations provide the basis for the discovery of new compounds with application in the treatment of drug-resistant leukemia.

Thiadiazolidinones: a new class of alanine racemase inhibitors with antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen and a major cause of hospital-acquired infections. New antibacterial agents that have not been compromised by bacterial resistance are needed to treat MRSA-related infections. We chose the S. aureus cell wall synthesis enzyme, alanine racemase (Alr) as the target for a high-throughput screening effort to obtain novel enzyme inhibitors, which inhibit bacterial growth. Among the 'hits' identified was a thiadiazolidinone with chemical properties attractive for lead development. This study evaluated the mode of action, antimicrobial activities, and mammalian cell cytotoxicity of the thiadiazolidinone family in order to assess its potential for development as a therapeutic agent against MRSA. The thiadiazolidones inhibited Alr activity with 50% inhibitory concentrations (IC50) ranging from 0.36 to 6.4 µM, and they appear to inhibit the enzyme irreversibly. The series inhibited the growth of S. aureus, including MRSA strains, with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 µg/ml. The antimicrobial activity showed selectivity against Gram-positive bacteria and fungi, but not Gram-negative bacteria. The series inhibited human HeLa cell proliferation. Lead development centering on the thiadiazolidinone series would require additional medicinal chemistry efforts to enhance the antibacterial activity and minimize mammalian cell toxicity.

Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach.

Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that (i) screen for compounds that block vaccinia DNA synthesis, (ii) eliminate trivial inhibitors, e.g. DNA intercalators, and (iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola.

Identification of non-nucleoside DNA synthesis inhibitors of vaccinia virus by high-throughput screening.

Variola virus, the causative agent of smallpox, is a potential bioweapon. The development of new antiviral compounds for smallpox prophylaxis and treatment is critical, especially because the virus can acquire resistance to the drugs that are currently available. We have identified novel small chemical inhibitors that target DNA synthesis of vaccinia, the prototypical poxvirus. Robotic high-throughput screening of 49663 compounds and follow-up studies identified very potent inhibitors of vaccinia DNA synthesis, with IC 50 values as low as 0.5 microM. Cell-based assays showed that 16 inhibitors effectively blocked vaccinia infection with minimal cytotoxicity. Three inhibitors had selectivity indexes that approximate that of cidofovir. These new non-nucleoside inhibitors are expected to interfere with components of the vaccinia DNA synthesis apparatus that are distinct from cidofovir. On the basis of the high sequence similarity between the proteins of vaccinia and variola viruses, these new inhibitors are anticipated to be equally effective against smallpox.

Processivity factor of KSHV contains a nuclear localization signal and binding domains for transporting viral DNA polymerase into the nucleus.

Kaposi's sarcoma-associated human herpesvirus (KSHV) encodes a processivity factor (PF-8, ORF59) that forms homodimers and binds to viral DNA polymerase (Pol-8, ORF9). PF-8 is essential for stabilizing Pol-8 on template DNA so that Pol-8 can incorporate nucleotides continuously. Here, the intracellular interaction of these two viral proteins was examined by confocal immunofluorescence microscopy. When individually expressed, PF-8 was observed exclusively in the nucleus, whereas Pol-8 was found only in the cytoplasm. However, when co-expressed, Pol-8 was co-translocated with PF-8 into the nucleus. Mutational analysis revealed that PF-8 contains a nuclear localization signal (NLS) as well as domains located at the N-terminus and the C-proximal regions that are required for Pol-8 binding. This study suggests that the mechanism that enables PF-8 to transport Pol-8 into the nucleus is the first critical step required for Pol-8 and PF-8 to function processively in KSHV DNA synthesis.

Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase.

Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.

Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.