The Structural and Biochemical Effects of Modifying HIV-1 Capsid Assembly by Small-molecules Restricted; Files Only

Abstract

Human immunodeficiency virus type 1 (HIV-1) is a pandemic causing over one million infections annually. For people living with HIV-1, access and adherence to antiretroviral therapies (ART) is critical to prevent acquired immunodeficiency syndrome (AIDS). Current ART formulations are well-tolerated and highly effective at suppressing viral replication, preventing disease progression and transmission. Additionally, antiretrovirals can prevent new infections as pre-exposure prophylaxis (PrEP), a chemical barrier protecting HIV-1–negative individuals at risk of exposure. Lenacapavir (LEN) is a first-in-class long-acting drug approved for ART and PrEP that targets the HIV-1 capsid core. For HIV-1, the capsid is a shuttle for cellular trafficking, a protective shell for replication, and a scaffold for host-factor interactions (see Chapter VI). The capsid lattice is formed by over 1,000 copies of the HIV-1 capsid protein (CA), a viral component required for establishing an infection and producing mature virions. The CA domain is one of the most-conserved regions of HIV-1; impairing capsid maturation, modifying core morphology, and hyper-stabilizing or destabilizing the lattice all negatively impact viral fitness. Thus, CA is an optimal target for antiretroviral inhibitor design.

Chapter I details advances in capsid research and numerous CA-targeting antiviral compounds, referred to as capsid effectors (CEs). It also discusses mechanisms of action (MOA) as ART or PrEP for five CE subclasses that target distinct binding site. Chapter II determines the MOA for ZW-1261, a compound targeting the same pocket as LEN (Site 1). ZW-1261 perturbs mature capsid by modifying pentameric subunits required for genome encapsulation and core integrity. Chapters III and IV describe collaborative efforts that identified small-molecules that covalently attach to HIV-1 CA via “clickable” sulfur(VI) fluoride exchange (SuFEx) reactions. These compounds react with CA (Sites 2 and 3), providing scaffolds for covalent antiviral design or reactive probes. Chapter V outlines “TSAR”, an R package that improves thermal shift assay workflows that can screen for biological interactions. TSAR analysis identified polyanionic metabolites that thermally-stabilize CA in vitro (likely at Site 5); one hit, gallic acid, inhibits HIV-1 replication. Overall, the work here characterizes the molecular mechanism of capsid perturbations for CE subclasses and further identifies novel compounds.

Table of Contents

Abstract - iv

Publications - vi

Acknowledgments - ix

Table of Contents - x

Table of Figures - xvii

Chapter I – Introduction - 1

A. HIV-1 Global Impact - 2

B. HIV-1 Replication and the Role of the Capsid - 4

C. HIV-1 Treatment and Prevention Strategies - 11

i. Antiretroviral Therapy (ART) and Pre-Exposure Prophylaxis (PrEP) - 13

ii. Long-acting antiretroviral therapy (LAART) - 15

D. Capsid as an Antiviral Target - 17

E. HIV-1 Capsid Protein (CA) - 20

F. Site 1 of CA – The FG Binding Pocket - 25

i. PF74 - 27

ii. ZW-1261 - 31

iii. Lenacapavir (GS-6207) and GS-CA1 - 32

G. Site 2 of CA – CAP-1 Binding Site - 35

i. CAP-1 - 36

ii. Benzodiazepines and Benzimidazoles - 37

iii. BBS-103 - 38

H. Site 3 of CA – Two-fold interface - 39

i. CAI - 40

ii. Ebselen - 42

iii. MKN-1A - 43

I. Site 4 of CA – Apical Binding Site - 43

J. Site 5 of CA – Central Pore (IP6) Binding Site - 45

i. Carboxybenzenes - 48

ii. ACAi-028 - 48

K. The importance of categorizing CA-targeting antivirals - 49

L. HIV-1 Capsid and CA Constructs - 56

i. WT CA or CA FL - 57

ii. Capsid-like particles (CLPs) - 60

iii. CA121 - 61

iv. CA121-HIS - 64

v. T = 1 icosahedron - 65

vi. T = 4 icosahedron and CA129 - 68

vii. SUV-templated CA - 68

M. The Hepatitis B Capsid Core - 69

i. Hepatitis B Virus (HBV) Replication and Core Protein (Cp) - 69

ii. Capsid Assembly Modulators (CAMs) - 72

N. Biochemical assays for studying CA - 74

i. BLI - 74

ii. CA in vitro assembly assay - 78

iii. Thermal Shift Assay (TSA) - 80

iv. Electron Microscopy - 80

Chapter II – Mechanism of ZW-1261 impacting HIV-1 capsid morphology at CAPENT. - 81

A. Abstract - 83

B. Introduction - 84

C. Results - 88

D. Discussion/Conclusions - 93

E. Materials - 95

F. Methods - 96

i. Expression and Purification of HIV-1 Capsid (CA) - 96

ii. A350 in vitro CA Assembly Assay - 97

v. Thermal Shift Assay (TSA) - 98

iv. BBS-103 treatment - 98

v. Capsid Assemblies & Negative Stain Transmission Electron Microscopy (TEM) - 98

vi. Cryo-EM grid preparation - 99

vii. Single Particle Cryo-EM - 99

viii. Native Mass Spectrometry (nMS) - 100

G. Acknowledgments - 100

Chapter III – Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition - 117

A. Summary - 118

B. Introduction - 118

C. Design - 119

D. Results - 120

i. Screening the library for interactions with the HIV-1 capsid protein: - 120

ii. Identifying a site of covalent attachment for BBS-103: - 122

iii. Mechanism of HIV-1 inhibition by BBS-103: - 124

E. Discussion - 126

i. Limitations of the Study - 127

ii. Significance - 128

F. Acknowledgments - 128

G. Methods - 129

i. Cell Culture and Virus Production - 129

ii. Key resources table - 131

iii. Compounds - 136

iv. General Methods for Chemical Synthesis of hit compounds. - 136

v. Synthesis and Characterization Data - 137

vi. BBS-103 X-Ray Crystallographic Data Collection - 138

vii. Expression and Purification of HIV-1 Capsid (CA) - 139

viii. Thermal Shift Assay (TSA) - 139

ix. CA Assembly Assay - 140

x. Negative stain Transmission Electron Microscopy (TEM) images of in vitro assembled capsid-like particles - 140

xi. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry - 141

xii. Testing Antiviral Activity of SuFEx and Control Compounds - 142

xiii. Testing for Compound Cytotoxicity - 142

xiv. Producer Cell Assay - 143

xv. Enzyme-Linked Immunosorbent Assay (ELISA) - 143

xvi. Endogenous Reverse Transcription (ERT) Assay Using Virions Produced in the Presence of BBS-103 - 144

xvii. Endogenous Reverse Transcription (ERT) Assay Using Virions Produced in the Absence of BBS-103 - 14

xviii. Covalent Docking Simulations - 145

xix. Quantification and Statistical Analysis - 146

Chapter IV – Biochemical analysis of in vitro SuFEx reactions with HIV-1 CA - 168

A. Introduction - 169

B. Methods and Results - 171

i. Computational and Biochemical Screening - 171

ii. CA assembly and antiviral testing - 173

iii. Testing for covalent attachment - 174

C. Discussion and Conclusions - 175

Chapter V – Thermal Shift Analysis in R, identifies endogenous molecules that interact with HIV-1 CAHEX - 187

A. Abstract - 188

B. Importance - 189

C. Introduction - 189

D. Results - 193

i. TSAR Package Accuracy - 193

ii. Vitamin library exemplifies TSAR output types - 194

iii. Multiple acidic metabolites cause a ∆Tm for CAHEX - 194

iv. Gallic acid binds CAHEX and has antiviral activity - 196

E. Discussion - 197

F. Methods - 200

i. Distribution and Package Information - 200

ii. Package Structure - 200

iii. Statistics and Analysis - 200

iv. Protein Purification - 201

v. Compounds - 202

vi. Thermal Shift Assay (TSA) - 203

vii. Biolayer Interferometry (BLI) - 204

viii. Antiviral Activity and Cytotoxicity - 204

ix. Endogenous Reverse Transcription (ERT) Assay - 205

x. Reverse Transcriptase Primer Extension - 206

xi. CA assembly assay and Transmission Electron Microscopy (TEM) - 206

xii. Downloading TSAR - 207

G. Acknowledgments - 207

Chapter VI – The HIV-1 Capsid is an Interaction Hub for Numerous Unstructured Host Proteins - 225

A. Abstract - 226

B. Introduction - 22

C. Results and Discussion - 230

D. Methods - 232

i. Sequences - 232

ii. Software - 233

iii. Predictions of Intrinsic Disorder - 233

iv. Predictions of Liquid-Liquid Phase Separation (LLPS) - 234

Chapter VII – Additional Contributions - 245

A. Development of HIV-1 antivirals to overcome reported resistance-associated mutations in site 1 of CA - 246

B. Cyclized peptides interact with HIV-1 CAHEX. - 253

Chapter VIII – Conclusions and Future Outlook - 260

A. Current and Future HIV-1 Inhibitors - 260

B. Elucidating the structural mechanisms of HIV-1 capsid defects by targeting a clinically-relevant site of CA - 264

C. Discovery and activity of novel small molecules that target HIV-1 CA - 266

D. Concluding Remarks - 270

Chapter IX – References - 272

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Virology Biology, Microbiology Biology, Molecular
Keyword	Capsid Inhibitor HIV-1 Capsid Effector Lenacapavir sulfur(VI) fluoride exchange (SuFEx) Intrinsically Disordered Proteins (IDPs) antiretroviral therapies pre-exposure prophylaxis Human immunodeficiency virus type 1 Capsid Molecular Mechanism Biochemistry
Committee Chair / Thesis Advisor	Anita H. Corbett, Ph.D., Emory University Stefan G. Sarafianos, Ph.D., Emory University
Committee Members	Leila E. Rieder, Ph.D., M.A., Emory University Gregory B. Melikian, Ph.D., Emory University Bo Liang, Ph.D., Emory University

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