Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.
bDengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via highthroughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC 50 ) of 1.03 ؎ 0.09 M. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future. D engue virus (DENV) (serotypes 1 to 4) belongs to the familyFlaviviridae, a group of enveloped RNA viruses that includes the genera Hepacivirus, Flavivirus, and Pestivirus. The genus Flavivirus consists of arthropod-borne disease agents such as yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), and DENV (1). More than 70 members of the Flavivirus genus are important human pathogens that cause significant morbidity and mortality (2). DENV is a public health threat to an estimated 2.5 billion people living in areas where dengue is epidemic, leading to 50 to 100 million human infections each year (3, 4). DENV infection frequently leads to dengue fever, life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) (5-7). Approximately 500,000 cases of DHF and DSS have been reported among more than 100 countries, causing approximately 12,500 deaths per year (3). Despite the tremendous efforts invested in anti-DENV research, no clinically approved vaccine or antiviral therapeutic agents are available for humans, and disease treatment is limited to supportive care (4,8,9). Considering the spread of this epidemic and the severity of DENV, an effective anti-DENV drug is urgently needed.DENV is an enveloped RNA virus consisting of a 10.7-kb single-stranded, positive-polarity RNA genome associated with multiple copies of capsid proteins. DENV RNA is translated as a single polyprotein upon entering the host cell and is cleaved by host proteases and the virus-encoded two-component protease (NS...
Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the Abbreviations: Ala-Pro-AFC, H-Ala-Pro-7-amino-4-trifluoromethylcoumarin; BRET, bioluminescence resonance energy transfer; DPP, dipeptidyl peptidase; DPP-IV, dipeptidyl peptidase IV; EOM, enzyme overlay membrane; FBS, fetal bovine serum; GFP, green fluorescent protein; Gly-Pro-AMC, H-Gly-Pro-7-amino-4-methylcoumarin; GPCR, G-Protein coupled receptor; PBS, phosphate buffered saline; PDB, protein data bank; PDBTM, protein data bank of transmembrane proteins; PFO, perfluorooctanoate; Rluc, renilla luciferase; SI, sucrase isomaltase; TM, transmembrane; TMD, transmembrane domain; WT, wild-type.Additional Supporting Information may be found in the online version of this article.Kuei-Min Chung and Jai-Hong Cheng contributed equally to this article. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM.
Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15 , which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15 . Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15 , which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.
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