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Nouri Neamati

Publications and source records attributed to Nouri Neamati.

49 records · Page 3Linked to original sources
PubMed2004

Codon-optimized cloning, expression and characertization of the C-terminal region of human apoptotic protein GADD34 in Escherichia coli.

The human GADD34 (Growth Arrest and DNA Damage-inducible 34) is the product of an apoptosis- and DNA-damage-inducible gene. The C-terminus domain of GADD34 is highly homologous to HSV-1 gamma-1 34.5, HSV-2 and the African swine fever virus virulence-associated factor NL-S. Among these viral proteins, HSV-1 gamma 34.5 protein is known to prevent apoptosis of viral-infected cells. Because of the difficulty in expressing GADD34 protein or any of its fragments, including the C-terminus (amino acids 533-632) in E. coli, partially due to sub-optimal expression of eukaryotic codons in prokaryotic E. coli, we used a codon-optimized cloning scheme to construct the eukaryotic gene that codes for GADD34(533-632). We derived a novel PCR protocol to assemble 20 oligonucleotides into the synthetic GADD34(533-632) gene. The clear advantage of using this protocol is that the assembled gene is without the mutation and deletion that are usually of a major problem in constructing synthetic genes. The synthetic GADD34(533-632) gene was cloned, expressed, and purified in large quantity. We obtained approximately 50 mg of GADD34(533-632) protein per liter minimum-medium culture. To our knowledge, this is the first report of a large-scale production of the C-terminus of GADD34. The production and purification of GADD34(533-632) in large quantity are essential for structure determination as well as for understanding its interactions with other proteins such as phosphatase 1-alpha using NMR spectroscopy and other biophysical methods.

Amino Acid Motifs↗
PubMed2003

Small-molecule HIV-1 integrase inhibitors: the 2001-2002 update.

Integration of viral DNA into host cell chromosomal DNA to form a provirus is an essential step in the viral life cycle. This process is mediated by integrase (IN), a 32 KDa viral enzyme. The unique properties of IN makes it an ideal target for drug design. First, there are no cellular homologues to IN and the reactions catalyzed by IN are unique. Second, IN is absolutely required for viral replication and mutations in a number of key residues dramatically block viral replication. Third, IN has been validated as a legitimate target and the results from S-1360 (1) the only available IN inhibitor under clinical trials suggest synergistic effect with reverse transcriptase (RT) and protease (PR) inhibitors. During the past 10 years a plethora of inhibitors have been identified and some were shown to be selective against IN and block viral replication. The two most predominant classes of inhibitors have been the catechol containing hydroxylated aromatics and more recently the diketoacid containing aromatics. Herein, we review all small molecule compounds reported to inhibit recombinant HIV-1 IN with IC(50) values < 20 M during the past two years. It is important to bear in mind that the true mechanism of action and antiviral activities of many of the compounds are currently not established. However, based on the growing body of literature certain classes of compounds can be easily excluded as bona fide IN inhibitors.

Drug Design↗
PubMed2002

Metal-dependent inhibition of HIV-1 integrase.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202-3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN-DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg(2+) at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg(2+)-based assays, while IN inhibition by salicylhydrazides is strictly Mn(2+)-dependent. Additionally, in target and cell-based assays, the MSHs have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerases I and II at concentrations that effectively inhibit IN. These data suggest that MSHs are selective inhibitors of HIV-1 IN and may serve as leads for antiviral therapeutics.

Antiviral Agents↗
PubMed2002

Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cisplatin based chemoresistance marker in ovarian carcinoma: comparative analysis with expression of MDR1, MRP1, MRP2, LRP and BCRP.

Intrinsic or acquired resistance to chemotherapy is the major obstacle to overcome in the treatment of patients with solid carcinoma. Cisplatin is one of the most effective chemotherapeutic agents for treating ovarian carcinoma. Recently, copper-transporting P-type adenosine triphosphatase (ATP7B) has been demonstrated as one of the genes responsible for cisplatin resistance in vitro. We hypothesized that the expression of ATP7B gene increases resistance to cisplatin in ovarian carcinoma and a priori knowledge of its expression is important for the choice of therapy. The aim of our study was to assess the role of ATP7B gene in ovarian carcinoma and compare its expression with those of multidrug resistance-related transporters such as MDR1, MRP1, MRP2, LRP and BCRP genes. The transporters' gene expression profiles from 82 patients treated with cisplatin-based chemotherapy after surgery were assessed by RT-PCR. We did not observe any significant correlation between ATP7B gene expression and those of MDR1, MRP1, MRP2, LRP or BCRP. The expression level of ATP7B gene was significantly increased (p < 0.05) in patients with moderately-/poorly-differentiated ovarian carcinomas treated with cisplatin-based chemotherapy, thus ATP7B may serve as an independent prognostic factor in these patients. In contrast, the expression level of MDR1, MRP1, MRP2, LRP and BCRP genes were not prognostic indicators of disease. These findings suggest that ATP7B gene may be considered as a novel chemoresistance marker and that inhibitor(s) of ATP7B might be useful, in patients with ovarian carcinoma treated with cisplatin-based chemotherapy.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2002

Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Acetoacetates↗
PubMed2002

Structural determinants for HIV-1 integrase inhibition by beta-diketo acids.

Among all the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3'-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3'-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3'-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.

Acetoacetates↗
PubMed2002

Copper-transporting P-type adenosine triphosphatase (ATP7B) is expressed in human breast carcinoma.

This is the first report to show that a copper-transporting P-type adenosine triphosphatase, ATP7B, is expressed in certain breast carcinomas, and a priori knowledge of its expression is important for the choice of therapy. We investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain breast carcinomas. To test this hypothesis, ATP7B expression and protein level were examined in 41 breast carcinomas using RT-PCR and immunohistochemistry. ATP7B gene / protein could be detected in 22.0% (9 / 41) of breast carcinomas and ATP7B gene expression was correlated well with the protein expression. In nine ATP7B-positive tumors, adjacent normal breast tissue was similarly analyzed, revealing that ATP7B is upregulated in breast carcinoma. ATP7B gene expression in poorly differentiated carcinoma was significantly higher than that in well- / moderately differentiated carcinoma (P = 0.012). Furthermore, we found no association between the ATP7B gene / protein expression and that of MDR1, MRP1, LRP and BCRP. These findings suggested that ATP7B gene expression might be a chemoresistance marker for cisplatin in patients with poorly differentiated breast carcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 integration by diketo derivatives.

A series of diketo derivatives was found to inhibit human immunodeficiency virus type 1 (HIV-1) integrase activity. Only L-708,906 inhibited the replication of HIV-1(III(B)) (50% effective concentration, 12 micro M), HIV-1 clinical strains, HIV-1 strains resistant to reverse transcriptase or fusion inhibitors, HIV-2 (ROD strain) and simian immunodeficiency virus (MAC(251)). The combinations of L-708,906 with zidovudine, nevirapine, or nelfinavir proved to be subsynergistic. In cell culture, addition of L-708,906 could be postponed for 7 h after infection, a moment coinciding with HIV integration. Inhibition of integration in cell culture was confirmed by quantitative Alu-PCR.

Acetoacetates↗
PubMed2002

Structure-based inhibitor design targeting HIV-1 integrase.

HIV integrase (IN) is a viral-encoded protein that catalyzes the breaking and joining reactions that mediate integration of viral DNA into the host genome. Therefore, IN offers a unique target for the development of novel anti-HIV and anti-AIDS therapeutics. To take advantage of this potential, drug discovery efforts via structure-based design approaches have been undertaken. Presented is a review of computer-aided drug design efforts targeting HIV IN. Included is an overview of the life-cycle of HIV, with emphasis on the mechanism of action of IN, biological assays for measuring IN activity and identifying IN inhibitors, and the appropriate cell-based assays required for determining the antiviral activity of IN inhibitors. This is followed by a review of the available three-dimensional structures of HIV IN. Structure-based drug design efforts are then critiqued, including both ligand-based (e.g. pharmacophore) and target-based (e.g. docking) methods. Results from recent computational chemistry studies of IN are also discussed.

Catalysis↗
PubMed2002

New paradigms in drug design and discovery.

The new millennium has ushered in an era of science that will revolutionize a great majority of our daily activities. That revolution is being experienced by a growing number of the population who are pushing the average life expectancy closer to the 80-year mark. The primary reason for this increase is the changes we have made in the last 2-3 decades both in how we live our lives as well as how we treat our maladies when they arise. The advent of new techniques in diagnostics and surgery have allowed many to survive debilitating illnesses when their chances would have been slim only a few years ago. In addition, several new therapeutic agents have been developed in the latter part of the 20th century that have improved our quality of life and increased our overall survival time. New medicines to treat cardiovascular, degenerative, infectious, and neoplastic diseases are rapidly being discovered in an effort to further lengthen our lifetimes. The processes used by academic and industrial scientist to discover new drugs has recently experienced a true renaissance with many new and exciting techniques being developed in only the past 5-10 years. In this review, we will attempt to outline these latest protocols that chemists and biomedical scientist are currently employing to rapidly bring new drugs to the clinic.

Animals↗
PubMed2001

Effect of HIV integrase inhibitors on the RAG1/2 recombinase.

Assembly of functional Ig and T cell receptor genes by V(D)J recombination depends on site-specific cleavage of chromosomal DNA by the RAG1/2 recombinase. As RAG1/2 action has mechanistic similarities to DNA transposases and integrases such as HIV-1 integrase, we sought to determine how integrase inhibitors of the diketo acid type would affect the various activities of RAG1/2. Both of the inhibitors we tested interfered with DNA cleavage and disintegration activities of RAG1/2, apparently by disrupting interaction with the DNA motifs bound specifically by the recombinase. The inhibitors did not ablate RAG1/2's transposition activity or capture of nonspecific transpositional target DNA, suggesting this DNA occupies a site on the recombinase different from that used for specific binding. These results further underscore the similarities between RAG1/2 and integrase and suggest that certain integrase inhibitors may have the potential to interfere with aspects of B and T cell development.

Amino Acid Motifs↗