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Mechanism of HIV drug resistance .Rodrigo Brindeiro / Amilcar Tanuri

Laboratório de Virologia MolecularUFRJ

2-Asso ciate Research Scientist, In ternatio nal Center fo r Aids Care and Treatment Programs-ICAP, Mailman Scho ol of Public Health, Co lumbia Univ ersity, NY, USA.

Sic Transit Gloria Mundi.Ad Majorem Dei GloriamIn Hoc Signo Vinces.

HIV virus and infected CD4+ T cells

Nobel 2008

HPVHIV

Harald zur HausenFrançoise Barré-SinoussiLuc Montagnier

Ciclo replicativo do HIV

Fusion Inhibitors

CCR5 Inhibitors

NRTI and NNRTI Inhibitors

Integrase Inhibitors

Protease Inhibitors

HIV life cycle and Classes of ARV Available

25 drugs approved to treat HIV/AIDS

Hunt R at all 2002 Microbiol & Immun

Mortality Rate Tendency Related to Aids Brazil, 1990-1999.

R 2 = 0,8156Year

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

90 91 92 93 94 95 96 97 98 99

(x 1

00

.00

0h

ab

.)

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Nucleoside/Nucleotide AnaloguesAbacavir (Ziagen, ABC)Didanosine (Videx, ddI)Emtricitabine (Emtriva, FTC)Lamivudine (Epivir, 3TC)Stavudine (Zerit, d4T)Tenofovir (Viread, TDF)Zalcitabine (Hivid, ddC) Discontinued by manufacturer 12/31/06Zidovudine (Retrovir, AZT, ZDV)

Nonnucleoside Reverse Transcriptase InhibitorsDelavirdine (Rescriptor, DLV)Efavirenz (Sustiva, Stocrin, EFV)Etravirine (Intelence, TMC 125)Nevirapine (Viramune, NVP)Rilpivirine (Edurant, TMC 278)

Protease InhibitorsAmprenavir (Agenerase, APV) Discontinued by manufacturer 10/07Atazanavir (Reyataz, ATV)Darunavir (Prezista, DRV, TMC 114)Fosamprenavir (Lexiva, Telzir, FPV)Indinavir (Crixivan, IDV)Lopinavir/Ritonavir (Kaletra)Nelfinavir (Viracept, NFV)Ritonavir (Norvir, RTV)Saquinavir (Invirase, SQV)Tipranavir (Aptivus, TPV)

Fusion InhibitorsEnfuvirtide (Fuzeon, ENF, T-20)

Chemokine Coreceptor AntagonistsMaraviroc (Selzentry, Celsentri, MVC)

Integrase InhibitorsRaltegravir (Isentress, RAL)

List of ARV FDA Approved

HIV Genetic Diversity

• HIV displays large genetic variability and two viruses can be causative agent of AIDS (HIV-1 and HIV-2). RT has no proofreading mechanisms and poses a mutations rate of 1 error per 10000 nt synthesized.

• Every day in a infected individual we have a huge replication burden (1010 particles daily) and all kind mutations appears. Most of them are lost and few are fixed in the viral population.

• HIV-2 can differ from HIV-1 in 50% of the aa of the main genes (gag, pol, and env).

• HIV-2 is less pathogenic and less transmissible by horizontal and vertical routes.

HIV quasispecies

Why HIV get resistant to ARV?

• Low aderance to treatment.• On-and-off ARV therapy.• Infection with a isolate carrying DRM.

The main message here is: “ HIV needs to replicate to accumulate mutations.” The best way to prevent DR is keeping the VL undetectable during ARV treatment .

The drug does not produce or induce DR it just select pre-existing mutant viruses

WT

MUT

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3TC

Days in culture7 14 21 28 35 42 49 56 63 70 77 84 91 98

HIV

vira

l loa

d

105

106

107

108

109

1010

1011

RT CRT B

0.02 0.04 0.08 0.16 0.32 0.64 1.2 2.4 4.8 9.6 19.2 38.4 76.8 3TC concentrations (µM)

M184IM184I

3TC

Days in culture7 14 21 28 35 42 49 56 63 70 77 84 91 98

HIV

vira

l loa

d

105

106

107

108

109

1010

1011

RT CRT B

0.02 0.04 0.08 0.16 0.32 0.64 1.2 2.4 4.8 9.6 19.2 38.4 76.8 3TC concentrations (µM)

3TC

Days in culture7 14 21 28 35 42 49 56 63 70 77 84 91 98

HIV

vira

l loa

d

105

106

107

108

109

1010

1011

RT CRT B

0.02 0.04 0.08 0.16 0.32 0.64 1.2 2.4 4.8 9.6 19.2 38.4 76.8 3TC concentrations (µM)

M184IM184I

In vitro selection B and C virus with 3TC .

M184VM184V

M184VVM184VV

Types of HIV Resistance

• Primary or transmitted drug resistance: Drug resistance in previously untreated persons. Primary drug resistance implies that a virus with drug resistance mutations was transmitted either directly, or through one or more intermediates, from a person with acquired drug resistance. In the first case, ‘primary’ is used to describe persons who have recently been infected. In the second case, ‘primary’ is used to describe persons with transmitted resistance.

Types of HIV Resistance

• Acquired or secondary drug resistance: Drug resistance developing in a person who has received antiretroviral therapy. Acquired drug resistance results from the generation of genetic variation in the population of viruses within a person followed by the selection of drug-resistant variants during HAART therapy.

Types of HIV Resistance

• Polymorphism: Polymorphisms are mutations occurring frequently in viruses not exposed to selective drug pressure. A nonpolymorphicmutation is one that does rarely occur in the absence of therapy.

How ARV drugs work?NRTI

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HIV-1 RT linked with RNA, DNA and primer

p51

p66

Active site

Nucleotide

How NRTI are activated ?

Mechanism of action of AZT How RT becomes resistant to NRTI inhibitors?

We have two distinct mechanisms:

Discrimination of the NRTI

Excision of the incorporated NRTI

NRTI discriminationSome RT mutations augment its capacity to

discriminate between the natural nucleotides and the analogs.

Mutations in RT leading to NRTI discrimination

NNRTI MDR Mutation

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Other multidrug resistant mutations in RT

Two or more amino acid insertion S-A, S-S or S-G at HIV-1 RT codon 69(S) in conjunction with other RT mutations are associated with phenotypic resistance to multiple NRTIs

T69 Pos

TAM mutations

• Resistance to zidovudine (ZDV) results from thymidine analogue resistance mutations (TAMs) at HIV-1 reverse transcriptase (RT) codons 41, 67, 70, 210, 215 and 219. Two mutations are possible at codon 215: Y or F. Whereas 215Y occurs alone or with 41L and 210W (TAM-1), 215F rarely occurs with these mutations or by itself; it is usually found with 67N, 70R and 219Q (TAM-2). The 210W mutation most often occurs with 41L and 215Y and rarely occurs with 215F or TAM-2 mutations. We previously demonstrated that the 215F mutant is less fit than T215 wild-type or 215Y (Hu et al., Antiviral Therapy 2004; 9:S68). In the present study, we explored the virological basis for clustering of 210W with other TAM-1 but not TAM-2 mutants, and the clustering of 215F, but not 215Y, with TAM-2 mutants.

What is excision?What is pyrophosphorolisis?

How NRTI excision mechanism works?

For isolates carrying TAM

How TAM works in excision mechanism ? Nucleotide inhibitor (Tenofovir,

Gilead)• Tenofovir was approved by FDA in 2001.

This modif ication enables TDF to jump the 1st phosphorylation step making a favorable PK profile .

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Main TDF Resistance Mutation

Does K65R accumulatemore often in subtype C?

NRTI mutation list

NNRTI

NNRTI NNRTI mechanism of action.HIV-1 RT Linked with Nevirapine (NNRTI)

Active site

Nevirapine

HIV-1 RT linked with Nevirapine with mutations related with NNRTI mutations

Active sites

Nevirapine

NNRTI DR Mutations

Entravirine and its capability to inhibit NNRTI resistant strains specifically K103N

In Brazil The prevalence of 13 specific ETR RAMs in subtype B samples was V179T 2%, G190S 3.7%, Y181V 0,5%, V106I 6%, V179D 2.6%, K101P 3.0%, K101E 5.3%, Y181C 12%, A98G 6.9%, V90I 6.9%,Y181I 3.6%, G190A 15% and L100I 6.1%.

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NNRTI mutation list HIV GAG Maturation process

How protease inhibitors work

Protease inhibitor

HIV ProteaseMajor Mutations Positions 30, 32, 33, 46, 48, 50,

54, 76, 82, 84, 88, 90

HIV Drug Resistance Database, Los Alamos National Laboratoryhttp://resdb.lanl.gov/Resist_DB

Sampling of Minor PI Mutations commonly present in non-B isolates as natural polymorphism

Mutations Positions 20, 36, 63, 71, 77, 93

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New PIs

Atazanavir Tipranavir Darunavir

DRV interacts with more aa in protease active site

Ritonavir enhances the blood levels of

other protease inhibitors

Kempf, et al, 1997

With ritonavirWithout ritonavir

(mg

/ml)

.01

.1

1

10

0 1 2 3 4 5 6 7 8Hours

S

NelfinavirAmprenavirIndinaviraquinavir

.01

.1

10

1

0 1 2 3 4 5 6 7 8

Hours

(mg

/ml)

Genetic Barrier

NRTI PI RTV/PI

Genotipic Res

How a MDR protease looks like

PI mutation list New classes of ARV drugs

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Integrase inhibitors

Mutations N155H, alone and combinations withQ148K/H/R and Y143R/C/H

Maraviroc

Fuseon – T20

Final Remarks • The best way to prevent the spread of HIV DR is to

prevent HIV infection.• The best other way to prevent the spread of HIV DR is

to keep the patients in ARV with undetectable VL.• In RLS the VL is not available in large scale.• 2nd line potent regimens are not available in RLS.• We need to make an effort to implement VL and

make available potent 2nd line regimens to curb the spread of HIV DR.