Inhibition and resistance mechanisms of HIV targeting antivirals Restricted; Files Only

Cilento, Maria (Summer 2022)

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The current therapy for HIV-1 is known as highly active antiretroviral therapy (HAART). Reverse transcriptase (RT) inhibitors such as tenofovir (TFV), emtricitabine (FTC), lamivudine (3TC), and zidovudine have been the main components of HAART because of the essential role reverse transcriptase plays in the replication cycle. While HAART is effective at suppressing HIV‑1 replication, lack of patient adherence to current once-daily dosing regimens leads to breakthrough resistance mutations. Therefore, long-acting (LA) regimens with less frequent dosing are necessary to overcome this challenge. 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, islatravir) is a promising LA nucleoside reverse transcriptase translocation inhibitor. EFdA can inhibit RT translocation by two mechanisms: immediate chain termination, where DNA synthesis is halted at the site of EFdA-monophosphate incorporation in the nascent DNA chain, or delayed chain termination, where DNA synthesis is arrested after the incorporation of an additional nucleotide. We worked to identify the mechanisms of resistance and inhibition of EFdA and EFdA‑based regimens. We studied EFdA resistance patterns as they may emerge in naïve, TFV, or FTC/3TC treated patients. In viral passaging experiments, we identified the A114S/M184V RT mutations that impart EFdA resistance (~25-fold), but significantly decrease viral fitness. Importantly, these mutations confer up to 40-fold enhanced sensitivity to TFV compared to wildtype (WT) HIV-1. As HAART typically comprises two nucleoside or nucleotide reverse transcriptase inhibitors, we performed passaging experiments with WT HIV or TFV- and FTC/3TC-resistant viruses in the presence of EFdA/TFV. Our results highlight the high barrier to resistance with EFdA/TFV. We also demonstrate that EFdA and TFV remain potent against diverse HIV isolates with and without clinical resistance mutations. In addition to efficacy, we studied the suitability of various drug combinations as potential LA regimens. Our findings predict additive interactions between the studied drug combinations. These studies demonstrate that   EFdA and EFdA/TFV regimens have the qualities to be effective therapeutics. 

Table of Contents

Table of Contents

Chapter 1: Introduction to Dissertation. 1

1.     Human Immunodeficiency Virus Type 1. 2

1.1       HIV-1 Disease and Epidemiology. 2

1.2       HIV-1 Groups, Subtypes, and Circulating Recombinant Forms. 2

1.3       Highly Active Antiretroviral Therapy and Pre-Exposure Prophylaxis. 3

1.4       Overview of HIV-1 Genome and Replication Cycle. 3

1.5       HIV-1 Reverse Transcriptase Structure and Function. 5

1.6       HIV-1 Reverse Transcription. 6

1.6.1       RT Initiation Complex (RTIC) 8

1.6.2     Role of Reverse Transcriptase in HIV Genetic Diversity and Drug Resistance. 9

1.6       HIV-1 Capsid Role in HIV-1 Reverse Transcription. 10

1.7       HIV-1 Reverse Transcriptase Targeting Inhibitors. 11

1.7.1        Nucleoside Reverse Transcriptase Inhibitors (NRTIs) 12

1.7.2        Nucleoside Reverse Transcriptase Translocation Inhibitors. 12

1.7.3        Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) 13

1.8        HIV-1 Mechanisms of Reverse Transcriptase Inhibitor Resistance. 15

1.8.1        Nucleoside Reverse Transcriptase Inhibitor Resistance Mechanisms. 15

1.8.2        NNRTI resistance. 17

1.9     HIV-1 Capsid Targeting Compounds. 19

1.10    Long-Acting Anti-HIV Regimens. 20

1.11    Questions Addressed in This Work. 21

Figures. 24

Figure 1-1. HIV-1 Replication Cycle. 24

Figure 1-2. Structure of HIV-1 RT in complex with dsDNA and dTTP substrates and binding locations of FDA-approved drugs targeting RT. 25

Figure 1-3. Structure of the HIV-1 RT polymerase active site. 26

Figure 1-4. HIV-1 Reverse Transcription. 27

Figure 1-5. Chemical structures of approved NRTIs and NNRTIs. 29

Figure 1-6. Chemical Structures of 2’deoxyadenosine and 4’-ethynyl-2-fluoro-2’deoxyadenosine 30

Figure 1-7. Interactions of EFdA-TP at the N-site of the HIV-1 RT polymerase active site. 31

Figure 1-8. Stopping Patterns of EFdA.. 32

Figure 1-9. Structure of the HIV-1 RT NNIBP. 33

Figure 1-10. Structural changes occur in HIV-1 RT upon NNRTI binding, which affect the position of nucleic acid binding. 34

Figure 1-11. Mutation K65R in HIV-1 RT imparts resistance to NRTIs through discrimination. 36

Figure 1-12. Structural basis for excision-based AZT resistance: interactions of excision product with TAMs RT. 37

Chapter 2: Development of Human Immunodeficiency Virus Type 1 Resistance to 4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine (EFdA) Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor Resistant-Strains. 40

2.1       Abstract 41

2.2       Introduction. 42

2.3        Results. 46

2.3.1          Virus breakthrough during serial passage of viruses in increasing EFdA concentrations 46

2.3.2         Dose response of EFdA-passaged viruses to EFdA.. 46

2.3.3       Development of amino acid mutations in reverse transcriptase during serial passage of viruses with EFdA  47

2.3.4       Validation of EFdA-resistance associated mutations using molecular clones. 48

2.3.5       Susceptibility of select mutants to TDF and FTC.. 48

2.3.6        Replication characteristics of molecular clone viruses. 48

2.3.7       Steady state kinetics and EFdA susceptibility of mutant reverse transcriptases. 49

2.4       Discussion. 50

Figures. 55

Figure 2-1. Selection of resistance to EFdA by serial passage. 55

Figure 2-2. EFdA dose response for viruses selected during serial passage in EFdA. 56

Figure 2-3. Resistance to EFdA of HIV-1 mutants selected during passaging. 57

Figure 2-4. Single-round replication assays using TZM-GFP cells infected with individual mutants. 58

Table 2-1. Amino acid mutations in WT-, K65R-, M184V- and D67N/K70R/T215F/K219Q-derived viruses during serial passage in progressively increasing concentrations of EFdA.. 59

Table 2-2. EC50s Fold Change compared to WT (NL4-3) 60

Table 2-3. Steady state enzyme kinetics for reverse transcriptase mutants. 61

Chapter 3: Identifying Stopping Patterns of EFdA.. 62

3.1       Abstract 63

3.2       Introduction. 63

3.3        Results. 66

3.3.1      Understanding the controls. 66

3.3.2        Stopping Patterns of Untreated and EFdA Treated Cells. 68

3.3.3       Stopping Patterns of RPV Treated Cells. 68

3.4       Discussion. 69

Figures. 73

Figure 3-1. Overview of workflow to identify EFdA stopping patterns. 73

Figure 3-2. Control Oligonucleotides Relative cDNA abundance. 74

Figure 3-3. Control, EFdA-MP, and ddAMP terminated oligonucleotides urea-PAGE gel 75

Figure 3-4. Control, EFdA-MP, ddAMP, and dAMP terminated oligonucleotides. 76

Figure 3-5. Uninhibited and EFdA treated samples. 79

Figure 3-6. Rilpivirine Treated Samples. 80

Supplemental Figures. 81

S3-1. Terminated oligonucleotides urea-PAGE gel 81

Chapter 4: Understanding HIV-1 Resistance to EFdA/TDF combination therapy in WT or NRTI resistant strains and diverse HIV-1 subtypes. 82

4.1       Abstract 83

4.2       Introduction. 84

4.3       Results. 87

4.3.1       Resistance mutations found during serial passages of WT, K65R, M184V viruses in the presence of EFdA and TDF. 87

4.3.2         Validation of mutations found in TDF- and EFdA-passaging using molecular clones and evaluating the specific infectivity. 88

4.3.3        Efficacy of EFdA and TDF in various HIV subtypes. 89

4.3.4        Efficacy of EFdA and TDF in HIV isolates: B, C, CRF_AE, and CRF_AG containing K65R, M184V, and K65R/M184V and evaluating the specific infectivity. 90

4.3.5       Efficacy and specific infectivity of polymorphism Gly68 of CRF_AE K65R/M184V.. 91

4.4       Discussion. 91

Figures. 96

Figure 4-1. Efficacy and Specific Infectivity of mutants identified during EFdA and TDF passaging. 96

Figure 4-2. EFdA inhibits all subtypes with a comparable EC50 to that of NL4-3. 98

Figure 4-3. Efficacy of EFdA and TDF in clinical isolates containing K65R, M184V, K65R/M184V. 99

Figure 4-4. Efficacy of EFdA and TDF and Specific Infectivity of CRF_AE with RT 68 position as Glycine (WT) or Serine. 101

Table 4-1.  Amino acid mutations in wildtype (HXB2), K65R (HXB2) and M184V (LAI) viruses following serial passage in progressively increasing concentrations of 1:1, 10:1 or 100:1 TDF:EFdA combinations 103

Chapter 5: Drug Interactions in Lenacapavir-Based Long-Acting Antiviral Combinations. 104

5.1       Abstract 105

5.2       Introduction. 106

5.3        Results. 107

5.3.1      LEN-EFdA. 107

5.3.2       LEN-RPV. 107

5.3.3       LEN-CAB. 108

5.4       Discussion. 108

Figures. 110

Figure 5-1. Representative Percent Inhibition Dose Response Matrix with LEN and EFdA. 110

Figure 5-2. Synergy scores and matrices of LEN and EFdA, RPV, or CAB. 111

Chapter 6: Additional Research Related to Dissertation. 113

6.1       Abstract and Contributions. 115

Chapter 7: Conclusions and Future Directions. 116

7.1       Conclusions and Future Directions. 117

7.1.1       EFdA has a high barrier to resistance. 117

7.1.2        The future of identifying EFdA stopping patterns. 118

7.1.3        EFdA and TDF have a high barrier to resistance. 119

7.1.4       Combination evaluations of long-acting anti-HIV regimens. 120

7.1.5       Overall Conclusions and Future Directions. 121

Chapter 8: Materials and Methods. 122

8.1       Chapter 2 Materials and Methods. 123

8.2       Chapter 3 Materials and Methods. 128

8.3       Chapter 4 Materials and Methods. 132

8.4      Chapter 5 Materials and Methods. 135

Tables. 136

Table 8-1. Control Oligonucleotides for adapter ligation. 136

References. 138



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