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Singh S.N.,University of Connecticut | Bakshi K.,University of Kansas | Mercier R.W.,Center for Drug Discovery | Makriyannis A.,Center for Drug Discovery | Pavlopoulos S.,Center for Drug Discovery
Biochemistry | Year: 2011

Internalization of G-protein-coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB1 5P 454-473). Complexes formed between the peptide and human arrestin-2 (wt-arr2 1-418) were compared to those formed with a truncated arrestin-2 mutant (tr-arr2 1-382) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The pentaphosphopeptide CB1 5P 454-473 adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a heptaphosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh 7P 330-348), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB1 5P 454-473 peptide binds to tr-arr2 1-382 with higher affinity than to the full-length wt-arr2 1-418. As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred NOE data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB1 5P 454-473 peptide. © 2011 American Chemical Society.

News Article | September 23, 2016

Researchers with the Virginia Tech Center for Drug Discovery have identified a compound that blocks the growth of a fungus that causes deadly lung infections and allergic reactions in people with compromised immune systems. The research team targeted the switch that allows the fungus Aspergillus fumigatus to survive in iron-deficient conditions like the human body. Specifically, they targeted an enzyme known as SidA, which is essential for the synthesis of molecules called siderophores that are made during infection to steal iron from human proteins. Furthermore, by performing high-throughput screening in the center’s Drug Discovery Screening Laboratory, they found a compound called Celastrol that blocks the growth of iron-producing organelles in the fungus. The results were published in the journal ACS Chemical Biology. “This project shows what an asset the screening lab is to the community,” said Pablo Sobrado, a professor of biochemistry in the College of Agriculture and Life Sciences and director of the screening laboratory. “Without the robots and chemical libraries available at the screening lab, this work would not have been possible. We are very fortunate at Virginia Tech to have this facility.” Aspergillus fumigatus is common and is typically found in soil and decaying organic matter. Most people are exposed to it daily with little consequence, but it can cause lung damage in people with compromised immune systems, such as organ transplant recipients and people with AIDS or leukemia. The mortality rate of this population, when exposed to the fungus, is more than 50 percent, according to the authors. "Growing antibiotic resistance is demanding the development of target-directed therapies," said Julia S. Martin del Campo, a postdoctoral research scientist in Sobrado's lab. "This approach requires the discovery of enzyme inhibitors that block essential pathogen pathways. The discovery of Celastrol as a SidA inhibitor represents the first building block in the development of drugs against A. fumigatus and related pathogens.” The Virginia Tech Center for Drug Discovery was established in 2012 and is an interdisciplinary group committed to continuing the growth and advancing the stature of the existing drug discovery and development programs at Virginia Tech. The center is housed in the College of Science, with support from the College of Science, the Fralin Life Science Institute, the Institute for Critical Technology and Applied Science, and the College of Agriculture and Life Sciences.

Zhang R.,New York University | Bloch N.,New York University | Nguyen L.A.,University of Rochester | Kim B.,Center for Drug Discovery | Landau N.R.,New York University
PLoS ONE | Year: 2014

SAMHD1 restricts the replication of HIV-1 and other retroviruses in human myeloid and resting CD4+ T cells and that is counteracted in SIV and HIV-2 by the Vpx accessory protein. The protein is a phosphohydrolase that lowers the concentration of deoxynucleoside triphosphates (dNTP), blocking reverse transcription of the viral RNA genome. Polymorphisms in the gene encoding SAMHD1 are associated with Aicardi-Goutières Syndrome, a neurological disorder characterized by increased type-I interferon production. SAMHD1 is conserved in mammals but its role in restricting virus replication and controlling interferon production in non-primate species is not well understood. We show that SAMHD1 is catalytically active and expressed at high levels in mouse spleen, lymph nodes, thymus and lung. siRNA knock-down of SAMHD1 in bone marrow-derived macrophages increased their susceptibility to HIV-1 infection. shRNA knock-down of SAMHD1 in the murine monocytic cell-line RAW264.7 increased its susceptibility to HIV-1 and murine leukemia virus and increased the levels of the dNTP pool. In addition, SAMHD1 knock-down in RAW264.7 cells induced the production of type-I interferon and several interferon-stimulated genes, modeling the situation in Aicardi-Goutières Syndrome. Our findings suggest that the role of SAMHD1 in restricting viruses is conserved in the mouse. The RAW264.7 cell-line serves as a useful tool to study the antiviral and innate immune response functions of SAMHD1. © 2014 Zhang et al.

PubMed | University of Texas Health Science Center at San Antonio, Center for Drug Discovery and Yeshiva University
Type: | Journal: Scientific reports | Year: 2016

SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.

Ryoo J.,Seoul National University | Choi J.,Seoul National University | Oh C.,Seoul National University | Kim S.,Seoul National University | And 15 more authors.
Nature Medicine | Year: 2014

The HIV-1 restriction factor SAM domain-and HD domain-containing protein 1 (SAMHD1) is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool. However, phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1 for defects in RNase or dNTPase activity, we identify SAMHD1 point mutants that cause loss of one or both functions. The RNase-positive and dNTPase-negative SAMHD1 D137N mutant is able to restrict HIV-1 infection, whereas the RNase-negative and dNTPase-positive SAMHD1 Q548A mutant is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of cell infection. SAMHD1 silencing in macrophages and CD4 + T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in cells and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA. © 2014 Nature America, Inc.

News Article | October 11, 2016

A popular antibiotic called rifampicin, used to treat tuberculosis, leprosy, and Legionnaire's disease, is becoming less effective as the bacteria that cause the diseases develop more resistance. One of the mechanisms leading to rifampicin's resistance is the action of the enzyme Rifampicin monooxygenase. Pablo Sobrado, a professor of biochemistry in the College of Agriculture and Life Sciences, and his team used a special technique called X-ray crystallography to describe the structure of this enzyme. They also reported the biochemical studies that allow them to determine the mechanisms by which the enzyme deactivates this important antibiotic. The results were published in the Journal of Biological Chemistry and PLOS One, respectively. "In collaboration with Professor Jack Tanner at the University of Missouri and his postdoc, Dr. Li-Kai Liu, we have solved the structure of the enzyme bound to the antibiotic," said Sobrado, who is affiliated with the Fralin Life Science Institute and the Virginia Tech Center for Drug Discovery. "The work by Heba, a visiting graduate student from Egypt, has provided detailed information about the mechanism of action and about the family of enzymes that this enzyme belongs to. This is all-important for drug design." Heba Adbelwahab, of Damietta, Egypt, a graduate student in Sobrado's lab, was a key player in the research and first author of the PLOS One paper. "Antibiotic resistance is one of the major problems in modern medicine," said Adbelwahab. "Our studies have shown how this enzyme deactivates rifampicin. We now have a blueprint to inhibit this enzyme and prevent antibiotic resistance." Rifampicin, also known as Rifampin, has been used to treat bacterial infections for more than 40 years. It works by preventing the bacteria from making RNA, a step necessary for growth. The enzyme, Rifampicin monooxygenase, is a flavoenzyme -- a family of enzymes that catalyze chemical reactions that are essential for microbial survival. These latest findings represent the first detailed biochemical characterization of a flavoenzyme involved in antibiotic resistance, according to the authors. Tuberculosis, leprosy, and Legionnaire's disease are infections caused by different species of bacteria. While treatable, the diseases pose a threat to children, the elderly, people in developing countries without access to adequate health care, and people with compromised immune systems.

News Article | March 3, 2017

Picking up a quest abandoned by Big Pharma, academic labs are using new technology to develop contraceptive drugs for men. Somewhere in Martin Matzuk’s collection of two billion chemicals, he hopes, is one that might safely make a man temporarily sterile—the elusive “male pill.” Right now, male contraception means a condom or a vasectomy. But Matzuk, who is director of the Center for Drug Discovery at Baylor College of Medicine, is among a handful of scientists who are renewing the search for a better option—an easy-to-take pill that’s safe, fast-acting, and reversible. Big drug companies long ago dropped out of the search for a male contraceptive able to chemically intercept millions of sperm before they reach a woman’s egg. But Matzuk’s lab shares in $600,000 worth of awards that the Bill and Melinda Gates Foundation gave out last year to “test the feasibility” of “disruptive and high-risk approaches” to male birth control. That sum is pocket change next to the $147.9 million the same foundation spent in 2015 on family planning efforts aimed at women—efforts that it says reduce poverty. Scientists like Matzuk also think excessive population growth is a cause of scarcity and environmental degradation. “We just can’t sustain the population at the rate we’re going,” he says. A male pill could reduce the number of unintended pregnancies, which by one account make up 40 percent of all pregnancies worldwide. “Right now the chemical burden for contraception relies solely on the female. That’s an unfair balance in the equation,” says Charles Easley, an assistant professor at the University of Georgia, who is also involved in the Gates-backed hunt for a male pill. “I think there’s not much activity in this field because we have an effective solution on the female side.” To restart the search for a pill, Matzuk is beginning with lists of genes active in the testes and then creating mice that lack those genes. To do that, he’s working with researchers in Japan to use the gene-editing technology called CRISPR to snip out the genes one by one. Matzuk has so far made more than 75 of these “knockout” mice and says CRISPR makes the work much faster than it would be otherwise. These mice are allowed to mate, and if their female partners don't get pregnant after three to six months, it means the gene might be a target for a contraceptive. Of 2,300 genes that are particularly active in the testes of mice, Matzuk has zeroed in on 30. His next step, he says, will be a novel screening approach to test whether any of about two billion chemicals can disable these genes in a test tube. Promising chemicals could then be fed to male mice to see if they cause infertility.

Vishnumurthy K.,Center for Drug Discovery | Makriyannis A.,Center for Drug Discovery
Journal of Combinatorial Chemistry | Year: 2010

Microwave-promoted novel and efficient one-step parallel synthesis of dibenzopyranones and heterocyclic analogues from bromo arylcarboxylates and o-hydroxyarylboronic acids via Suzuki-Miyaura cross coupling reaction is described. Spontaneous lactonization gave dibenzopyranones and heterocyclic analogues bearing electron-donating and -withdrawing groups on both aromatic rings in good to excellent yields. © 2010 American Chemical Society.

News Article | September 22, 2016

Researchers with the Virginia Tech Center for Drug Discovery have identified a compound that blocks the growth of a fungus that causes deadly lung infections and allergic reactions in people with compromised immune systems.

FALLS CHURCH, Va., Dec. 5, 2016 /PRNewswire-USNewswire/ -- Inova announced today that Milton L. Brown, MD, PhD, has joined Inova where he will serve as director of the new Inova Center for Drug Discovery and Development and as deputy director for Drug Discovery for the Inova Schar Cancer...

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