Multi-mechanism antiretroviral approach targeting macrophage derived viral sanctuaries Pubblico

Abstract

Abstract

Macrophages are a significant viral reservoir for HIV-1, and eradication cannot occur without elimination of virus from these cells. Current HAART cannot achieve eradication, and understanding how HIV-1 ensconces current therapies and hides in viral reservoirs is necessary to achieve systemic eradication. The goal of this thesis is to understand these mechanisms by assessing the following parameters in activated and resting macrophages versus lymphocytes: 1) antiviral potency and intracellular concentrations of nucleoside analogs, 2) potency of clinically relevant nucleoside analogs, protease inhibitors (PI), and Raltegravir (RAL; integrase inhibitor), 3) levels/ratios of dNTP/rNTP, 4) targeted inhibition of pro-HIV pathways. Additionally, drug-based methods to systemically eliminate macrophages will be explored, with the goal of employing the optimized therapy in rhesus macaques to better understand the role of macrophages in HIV-1 infection. The hypothesis of this thesis is 1) nucleoside analogs, PI, and RAL may not efficiently eliminate HIV-1 in macrophages, 2) different levels/ratios of dNTP/rNTP in macrophages versus lymphocytes could alter potency of nucleoside analogs, and 3) targeted inhibition of pro-HIV pathways will markedly reduce HIV-1 replication in macrophages and lymphocytes. AZT, TDF, and 3TC display the most favorable intracellular concentration/potency profile, and should be considered for all cocktails in treatment of HIV-1 infection. Potency of HIV-1 PI is not altered by activation state of lymphocytes or macrophages, and should be considered for cocktails aimed at targeting HIV-1 replication across both cell types. Jak inhibitors Tofacitinib (RS-1294) and Jakafi (RS-1374) are potent, safe, sub-micromolar inhibitors of HIV-1 replication and should be considered for combination studies in both lymphocytes and macrophages. Ratios of rNTP:dNTP are similar in macrophages, but not lymphocytes, conferring preferential incorporation of rNTP into the growing viral DNA strand in macrophages only, and ribonucleosides represent a novel class of antiretroviral agents that specifically targets HIV-1 replication in macrophages. Fosamax and Boniva were identified as specific, potent, sub-micromolar inducers of cell death in macrophages. These data demonstrate that cocktails containing a PI + AZT, TDF, or 3TC, + ribonucleoside inhibitor + Jak inhibitor + Fosamax or Boniva may represent a multifaceted approach to target HIV-1 replication not only in lymphocytes, but also macrophages.

Table of Contents

Table of Contents

Chapter 1: Introduction...1-27

Overall goal of thesis...1
Human immunodeficiency virus type 1...1
HIV-1 genomic organization...4-5
HIV-1 replication cycle...6
Targets of HIV-1 infection...8
Antiretroviral therapy...10-12

Entry inhibitors...11
Reverse transcriptase inhibitors...10-10
Integrase inhibitors...11
Protease inhibitors...11-12

Obstacles in achieving eradication...15-16

Latency...16-17
Resistance...17

Pro HIV events during infection...17-23

Dysregulation of the inflammatory response and activation state of HIV-1 target cells...17-18
Pro-HIV pathways...18-19

Activation state of macrophages and relationship to HIV-1 infection...22-23
Functional cure versus systemic eradication...25
Macrophage depleting agents: Mechanism and potential therapeutic agent for treatment of HIV-1 infection...25-26
Summary: Why elimination of HIV-1 from macrophages is sentinel to achieving eradication...26-27

Chapter 2: Methodology...30-38

Preparation of macrophages for cellular pharmacology...30
Culture of activated or resting macrophages...30
Cellular pharmacology...30-31
Preparation of macrophages for antiviral studies...31-32
Preparation and culture of lymphocytes...32
Cellular pharmacology studies in lymphocytes...32
Antiviral potency studies in lymphocytes...32-33
Statistical methods...33
Assessment of proportionality...33-34
Toxicity studies...34-35
Viability and proliferation assays in lymphocytes...35
Viability assays in macrophages...35
Assessment of inhibition of Jak or Tyk2 by kinase inhibitors...35-36
Combination studies assessing antiviral potency...36
Antiviral potency in rhesus macaque lymphocytes and macrophages...36
Reactivation of latent HIV-1 in primary human lymphocytes...36-37
Extraction of intracellular nucleotide fraction and LC-MS/MS analysis...37
RT purification...37
Assay for rNTP incorporation during DNA synthesis of HIV-1 RT using ³²P-UTP...37-38

Chapter 3: Results...39-78

Cellular pharmacology...39-44
Proportionality between extracellular [nucleoside analog] and nucleoside analog-TP per 106 cells...45-47
Antiviral potency and toxicity of HIV-1 PI in macrophages and lymphocytes...48
Antiviral potency of HIV-1 PI in macrophages with varying extracellular serum concentrations...49
Antiviral potency and toxicity of cellular factor inhibitors...52-53
Antiviral potency and toxicity of various Jak inhibitors...53-54
Viability of macrophages exposed to various concentrations of Jak inhibitors...54
Viability and proliferation assays in lymphocytes...54
Antiviral potency of combination of Tofacitinib+Jakafi...54
Antiviral potency in rhesus macaque lymphocytes and macrophages...55
Inhibition of reactivation of latent HIV-1 in primary human lymphocytes...64
Levels and ratios of dNTP/rNTP in activated and resting lymphocytes and macrophages...66
DNA synthesis profiles of HIV-1 RT in cellular dNTP pools of lymphocytes or macrophages...69
Antiviral potency of ribonucleoside inhibitors in lymphocytes and macrophages...72
Synthesis of triphosphorylated RS-1285 and RS-1292 for use in cell free biochemical assays...74
DNA synthesis profiles of HIV-1 RT in cellular dNTP pools of macrophages in the presence of RS-1292-TP...76
Antiviral potency and toxicity of Fosamax, Boniva, or investigational MDA provided by collaborator...78

Chapter 4: Discussion...85-109

Discussion about intracellular drug concentrations and antiviral potency of nucleoside analogs and RAL...85-90
Discussion about antiviral potency of HIV-1 PI in acute or chronic infection of resting or activated macrophages...90-91
Discussion about targeted inhibition of pro-HIV cellular factors...91-97

Discussion about levels/ratios of dNTP:rNTP and discovery of a novel class of ART that specifically targets HIV-1 replication in macrophages...98-102
Discussion about identification of macrophage depleting agents for use in a rhesus macaque model...103-104
Summary conclusions...105-108

References...109-113

Appendices

List of published and submitted publications to date
Hard copy of all published and submitted publications to date

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Pharmacology
Parola chiave	HIV-1 Human Immunodeficiency Virus Type 1
Committee Chair / Thesis Advisor	Schinazi, Raymond F. , Emory /VA Medical Center
Committee Members	Liotta, Dennis C, Emory University Brown, Lou Ann, Emory University Shim, Hyunsuk, Emory University

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