New Peptide-Based Pharmacophore Activates 20S Proteasome

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs’ pharmacophore consists of an N-terminal basic pocket-docking “activation anchor” connected via a β turn inducer to a C-terminal “specificity clamp” that binds on the proteasome α surface. By allosteric effects—including destabilization of the proteasomal gate—the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

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Functional Differences between Human and Bovine Immunodeficiency Virus Tat Transcription Factors

Journal of Virology ◽

10.1128/jvi.74.10.4666-4671.2000 ◽

2000 ◽

Vol 74 (10) ◽

pp. 4666-4671 ◽

Cited By ~ 15

Author(s):

Hal P. Bogerd ◽

Heather L. Wiegand ◽

Paul D. Bieniasz ◽

Bryan R. Cullen

Keyword(s):

Cooperative Interaction ◽

Bovine Immunodeficiency Virus ◽

Viral Gene ◽

Tat Protein ◽

Cyclin T1 ◽

Immunodeficiency Virus ◽

Cellular Cofactor ◽

Ltr Promoter ◽

Hiv 1

ABSTRACT Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

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Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro

Biochemical Journal ◽

10.1042/bj3200681 ◽

1996 ◽

Vol 320 (2) ◽

pp. 681-686 ◽

Cited By ~ 40

Author(s):

Fiona McPHEE ◽

Patricia S. CALDERA ◽

Guy W. BEMIS ◽

Antony F. McDONAGH ◽

Irwin D. KUNTZ ◽

...

Keyword(s):

Protease Inhibitors ◽

Cultured Cells ◽

Dimethyl Ester ◽

Molecular Shape ◽

Viral Assembly ◽

Bile Pigments ◽

Viral Maturation ◽

Viral Polyprotein ◽

Hiv 1

Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 µM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with Ki values of approx. 1 µM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 µM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.

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Non-Competitive Protein Phosphatase-1 Inhibitors Prevent Sickling of SS RBCs.

Blood ◽

10.1182/blood.v114.22.4038.4038 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4038-4038

Author(s):

Jerod Hairston ◽

Keon Combi ◽

Altreisha Foster ◽

Bak Kim ◽

Victor R. Gordeuk ◽

...

Keyword(s):

Protein Phosphatase ◽

Cultured Cells ◽

Conflicts Of Interest ◽

Protein Phosphatase 1 ◽

Cell Protein ◽

Trypan Blue Exclusion ◽

Future Design ◽

Small Molecular Inhibitors ◽

Hiv 1

Abstract Abstract 4038 Poster Board III-974 Protein phosphatase-1 (PP1) has been implicated in the regulation of KCC (K:Cl) transporters, which transport K+ and Cl- ions from red blood cells (RBCs) and in the setting of sickle cell disease may contribute to RBC dehydration and sickling. We have studied host cell protein phosphatase-1 (PP1) in the context of HIV-1 replication and designed novel small molecule non-competitive inhibitors of PP1 that are efficient in the inhibition of HIV-1 but not toxic for cultured cells. We analyzed the effect of our novel non-competitive PP1 inhibitors and the conventional competitive PP1 inhibitor, ocadaic acid, on the sickling of hemoglobin SS RBCs in vitro. We cultured hemoglobin SS RBCs overnight at 1% O2 in the presence of the PP1 inhibitors and then photographed the RBCs and counted the percentage of sickled RBCs. We found that the non-competitive PP1 inhibitor, 1E7-04 prevented RBC sickling by 40% at 10 mM concentration. The 1E7-04 was not toxic at 10 mM concentration for cultured CEM T cells as determined by trypan blue exclusion assay using an automatic cell counter. Our study suggests that small molecular inhibitors of PP1 might be candidates for the future design of anti-sickling drugs. Acknowledgments. This work was supported by NHLBI grant U54HL090508-02; NHLBI grant R25 HL003679-08 from the National Institute of Helath and The Office of Research on Minority Health and by U.S. Civilian Research & Development Foundation grant. Disclosures: No relevant conflicts of interest to declare.

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4′-C-Methyl-2′-Deoxyadenosine and 4′-C-Ethyl-2′-Deoxyadenosine Inhibit HIV-1 Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01290-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2379-2389 ◽

Cited By ~ 6

Author(s):

B. Christie Vu ◽

Paul L. Boyer ◽

Maqbool A. Siddiqui ◽

Victor E. Marquez ◽

Stephen H. Hughes

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Cultured Cells ◽

Nucleoside Analogs ◽

Transcriptase Inhibitor ◽

Resistance Mutations ◽

Viral Dna ◽

Infected Cells ◽

Hiv 1

ABSTRACTIt is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine.In vitroexperiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.

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The HIV-1 Tat Protein Stimulates Reverse Transcription In Vitro

Current HIV Research ◽

10.2174/157016207781662443 ◽

2007 ◽

Vol 5 (5) ◽

pp. 474-483 ◽

Cited By ~ 21

Author(s):

Ann Apolloni ◽

Luke Meredith ◽

Andreas Suhrbier ◽

Rosemary Kiernan ◽

David Harrich

Keyword(s):

Reverse Transcription ◽

Tat Protein ◽

Transcription In Vitro ◽

Hiv 1

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Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.22.8985 ◽

1990 ◽

Vol 87 (22) ◽

pp. 8985-8989 ◽

Cited By ~ 92

Author(s):

M. G. Cordingley ◽

R. L. LaFemina ◽

P. L. Callahan ◽

J. H. Condra ◽

V. V. Sardana ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Specific Interaction ◽

Tat Protein ◽

Sequence Element ◽

Tat Peptides ◽

Immunodeficiency Virus

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Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.2970-2983.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 2970-2983 ◽

Cited By ~ 141

Author(s):

Dmitri Ivanov ◽

Youn Tae Kwak ◽

Jun Guo ◽

Richard B. Gaynor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Transcriptional Elongation ◽

Tat Protein ◽

C Terminus ◽

Heptad Repeats ◽

And Function ◽

Hiv 1

ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

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