Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors

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Discovery and development of

AIDS epidemic hit Western societies. NRTIs inhibit the reverse transcriptase (RT), an enzyme that controls the replication of the genetic material of the human immunodeficiency virus (HIV). The first NRTI was zidovudine, approved by the U.S. Food and Drug Administration (FDA) in 1987, which was the first step towards treatment of HIV. Six NRTI agents and one NtRTI have followed. The NRTIs and the NtRTI are analogues of endogenous 2´-deoxy-nucleoside and nucleotide. Drug-resistant viruses
are an inevitable consequence of prolonged exposure of HIV-1 to anti-HIV drugs.

History

In the summer of 1981 the acquired immunodeficiency syndrome (AIDS) was first reported.

tenofovir (see table 4).[4][6]

The HIV-1 reverse transcriptase enzyme

Function

Figure 1 A: Mechanism of action of nucleoside analogues reverse transcriptase inhibitors, for example zidovudine. B: Mechanism of action of the nucleotide analogue reverse-transcriptase inhibitor, tenofovir

Most standard HIV drug therapies revolve around inhibiting the reverse transcriptase enzyme (RT), an enzyme that is necessary to the HIV-1 virus and other

retroviruses to complete their life cycle.[5] The RT enzyme serves two key functions. First, it controls the replication of the viruses genetic material via its polymerase activity. It converts the viral single-stranded RNA into an integration competent double stranded DNA. Subsequently, the generated DNA is translocated into the nucleus of the host cell where it is integrated in its genome by the retroviral integrase. The other role of the RT is its ribonuclease H activity that degrades RNA only when it is in a heteroduplex with DNA.[7][8]

Structure

HIV-1 RT is an asymmetric heterodimer which is 1000

tRNA primer. The p66 subunit has the two active sites: polymerase and ribonuclease H. The polymerase has four subdomains that have been named “fingers“, “thumb“, “connection“ and “palm“ for it has been compared to the right hand.[7][8][9]

Mechanism of action

Activation of nucleoside and nucleotide reverse-transcriptase inhibitors is primarily dependent on cellular entry by

hydrophilic
and have limited membrane permeability and therefore this step is very important. NRTIs are analogues of
endogenous 2´-deoxy-nucleoside and nucleotide. They are inactive in their parent forms and require successive phosphorylation.[6]

Nucleosides must be triphosphorylated, while nucleotides, which possess one phosphonated group, must be diphosphorylated.

triphosphate analogues.[6]
In their respective triphosphate forms, NRTIs and the only NtRTI available compete with their corresponding endogenous deoxynucleotide triphosphate (dNTPs) for incorporation into the nascent DNA chain (see figure 1).
phosphoester bond with the next nucleic acid, blocks further extension of the DNA by RT, and they act as chain terminators.[10][12]

Discovery and development

First step towards treatment of HIV- zidovudine

In 1964 zidovudine (AZT) was synthesized by Horwitz at the Michigan Cancer Foundation. The 3´hydroxyl group in the deoxyribose ring of thymidine is replaced by an

toxic since it is converted into the triphosphate by the cellular enzymes and therefore it is activated in uninfected cells.[14]

Further development of nucleoside analogues

Dideoxynucleosides

Table 1 Comparison of chemical structures:
Dideoxyadenosine and didanosine
Dideoxyadenosine Didanosine
Chemical

structure

Dideoxynucleosides are analogues of nucleoside where the sugar ring lacks both 2´ and 3´-hydroxyl groups.[9] Three years after the synthesis of zidovudine, Jerome Horwitz and his colleagues in Chicago prepared another dideoxynucleoside now known as zalcitabine (ddC).[16] Zalcitabine is a synthetic pyrimidine nucleoside analogue, structurally related to deoxycytidine, in which the 3´-hydroxyl group of the ribose sugar moiety is substituted with hydrogen.[17] Zalcitabine was approved by the FDA for the treatment of HIV-1 in June 1992.[3][18]

2´,3´-dideoxyinosine or didanosine is converted into dideoxyadenosine in vivo. Its development has a long history.[19] In 1964 dideoxyadenosine, the corresponding adenosine analogue of zalcitabine was synthesised. Dideoxyadenosine caused kidney damage so didanosine was prepared from dideoxyadenosine by enzymatic oxidation (see table 1). It was found to be active against HIV without causing kidney damage.[16] Didanosine was approved by the FDA for the treatment of HIV-1 in October 1991.[18] Zalcitabine and didanosine are both obligate chain terminators, that have been developed for anti-HIV treatment. Unfortunately, both drugs lack

side-effects.[14]

Table 2 Comparison of chemical structures:
Zalcitabine and lamivudine
Zalcitabine Lamivudine
Chemical

structure

Further modification of the dideoxy framework led to the development of 2´,3´-didehydro-3´-deoxythymidine (stavudine, d4T). Activity of stavudine was shown to be similar to that of zidovudine, although their phosphorylation patterns differ; the

affinity for zidovudine to thymidine kinase (the enzyme responsible for the first phosphorylation) is similar to that of thymidine, whereas the affinity
for stavudine is 700-fold weaker.[9]

2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) was discovered by Bernard Belleau. The history

of lamivudine can be traced back to the mid-1970s while Bernard Belleau was investigating sugar

enantiomers of BCH-189 (2',3'-dideoxy-3'-thiacytidine) had in vitro activity against HIV. Lamivudine is the negative enantiomer and is a pyrimidine nucleoside analogue. The 3' carbon of the ribose ring of 2'-deoxycytidine has been replaced by a sulfur atom because it had greater anti-HIV activity and is less toxic than the positive enantiomer.[16][20][21]

Next in line was 2',3'-dideoxy-5-fluoro-3'-thiacytidine (Emtricitabine, FTC) which is a structural homologue of lamivudine. The structural difference is the 5-fluoro-modification of the base moiety of lamivudine. It is similar in many ways to lamivudine and is active against both HIV-1 and hepatitis B virus (HBV).[21][22]

Carbocyclic nucleoside

Carbocyclic analogues of dideoxyadenosine were investigated for their anti-HIV activity. Minimal activity was first observed. Many nucleoside analogues were prepared and examined but only one had significant activity and satisfied the requirements for

cyclopropyl group on its 6-amino nitrogen of the adenine ring increased lipophilicity and thus enhanced brain penetration. The resulting compound is known as abacavir (see table 3).[16] Abacavir was approved by the FDA for use in therapy of HIV-1 infections in December 1998.[20]

This drug is the only approved antiretroviral that is active as a guanosine analogue in vivo. First it is monophosphorylated by adenosine phosphotransferase and then the monophosphate is converted to carbovir 3´-monophosphate. Subsequently, it is fully phosphorylated and the carbovir is incorporated by the RT into the DNA chain and acts as a chain terminator. Carbovir is a related guanosine analogue that had poor oral bioavailability and thus was withdrawn from clinical development.[19]

Table 3 Comparison of chemical structures: Dideoxyadenosine, didanosine and abacavir
Dideoxyadenosine Didanosine Abacavir
Chemical structure

Acyclic nucleotide – the only approved NtRTI

Nucleotide analogues require only two phosphorylation steps whereas nucleoside analogues require three steps. Reduction in the phosphorylation requirement may allow more rapid and complete conversion of drugs to their active metabolites. Such considerations have led to the development of phosphonate nucleotide analogues such as tenofovir. Tenofovir disoproxil fumarate (Tenofovir DF) is the

broad spectrum antiviral activity of 2,3-dihydroxypropyladenine.[24] Tenofovir DF was the first nucleotide reverse-transcriptase inhibitor approved by the FDA for the treatment of HIV-1 infection in October 2001.[18][23]

Table 4 A schematic overview of the approved NRTIs and NtRTI and their corresponding endogenous deoxynucleosides and deoxynucleotide.
Nucleotide analogue Nucleoside analogues

Purine analogues

Pyrimidine analogues
Purine analogues
N

u
c
l
e
o
s
i
d
e


Adenosine 		Chemical structure of thymidine
Deoxythymidine 		Chemical structure of deoxycytidine
Deoxycytidine
Adenosine
Guanosine
D

r
u
g


Tenofovir

({[(2R)-1-(6-amino-9H- purin-9-yl)propan-2-yl]oxy}methyl) phosphonic acid

Chemical structure of zidovudine
Zidovudine

3´Azido-2´,3´-dideoxythymidine, azidothymidine (AZT)

Chemical structure of Stavudin
Stavudine

2´,3´-Didehydro-2´,3´-dideoxythymidine (d4T)

Chemical structure of Emticitabine
Emtricitabine

(-)-ß-L-3´-thia-2´,3´-dideoxy-5-fluorocytidine ((-)FTC)

Chemical structure of Lamivudine
Lamivudine

2´,3´-Dideoxy-3´-thiacytidine (3TC)

Chemical structure of Zalcitabine
Zalcitabine

2´,3´-Dideoxycytidine (ddC)


Didanosine

2´,3´-Dideoxyinosine (ddI)


Abacavir

(4-(2-amino-6-(cyclopropylamino)- 9H-purin-9yl) cyclopent-2enyl)methanol(ABC)


why does the table eat the next section heading if nothing is written here?

Resistance

Currently, appearance of

drug resistant viruses is an inevitable consequence of prolonged exposure of HIV-1 to antiretroviral therapy. Drug resistance is a serious clinical concern in treatment of viral infection, and it is a particularly difficult problem in treatment of HIV.[25] Resistance mutations are known for all approved NRTIs.[26]

Two main mechanisms are known that cause NRTI drug resistance: Interference with the incorporation of NRTIs and excision of incorporated NRTIs.

steric hindrance that can exclude certain drugs, for example lamivudine, from being incorporated during reverse transcription. In case of excision of incorporated NRTIs the resistant enzymes readily accept the inhibitor as a substrate for incorporation into the DNA chain.[27] Subsequently, the RT enzyme can remove the incorporated NRTI by reversing the polymerization step. The excision reaction requires a pyrophosphate donor which RT joins to the NRTI at the 3´primer terminus, excising it from the primer DNA.[27]
To achieve efficient inhibition of HIV-1 replication in patients, and to delay or prevent appearance of drug resistant viruses, drug combinations are used.
HAART, also known as highly active antiretroviral therapy consists of combinations of antiviral drugs which include NRTIs, NtRTI, non-nucleoside reverse-transcriptase inhibitors and protease inhibitors.[28]

Current status

Currently, there are several NRTIs in various stages of clinical and

preclinical development. The main reasons for continuing the search for new NRTIs against HIV-1 are to decrease toxicity, increase efficiency against resistant viruses, and simplify anti-HIV-1 treatment.[6][26][29]

Apricitabine (ATC)

Apricitabine is a deoxycytidine analogue. It is structurally related to lamivudine where the positions of the oxygen and the sulfur are essentially reversed.[21] Even though apricitabine is a little less potent in vitro compared to some other NRTIs, it maintains its activity against a broad spectrum of HIV-1 variants with NRTI resistance mutations. Apricitabine is in the final stage of clinical development for the treatment of NRTI-experienced patients.[6]

Elvucitabine (L-d4FC)

CD4+ cell numbers dropping as early as two days after initiation of dosing.[22][29]

Amdoxovir (DAPD)

Amdoxovir is a guanosine analogue NRTI prodrug that has good bioavailability.[6][22][29] It is deaminated intracellularly by adenosine deaminase to dioxolane guanine (DXG). DXG-triphosphate, the active form of the drug, has greater activity than DAPD-triphosphate.[22] Amdoxovir is currently in phasa II clinical trials.[24][29]

Racivir (RCV)

Racivir is a racemic mixture of the two β-enantiomers of emtricitabine (FTC), (-)-FTC and (+)-FTC. Racivir has excellent oral bioavailability and has the advantage of needing to be taken only once a day. Racivir can be considered to be used in combination of two NRTIs and has shown promising antiviral activity when used in combination. Racivir is currently in phase II clinical trials.[6][22][29]

Table 5 Several drug candidates that are undergoing clinical development
Drug candidate Apricitabine Elvucitabine Amdoxovir Racivir
Chemical structure
Phase of development Final stage of clinical development On hold phase II phase II

There are several more NRTIs in development. Either the sponsors have filed for an Investigational New Drug (IND) application, the application has been approved by the FDA or the drugs are in different phases of clinical trials. Some of the NRTIs that are in development exhibit various attractive pharmacological properties that could make them desirable for the treatment of patients in need of new agents.[6][22][29]

See also

References

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  29. ^ a b c d e f Agrawala, R.K.; Krishnan, P.N.; Raman, S.; Ravichandran, S.; Veerasamy, R. (2008), "An overview on HIV-1 reverse transcriptase inhibitors" (PDF), Digest Journal of Nanomaterials and Biostructures, 3 (4): 171–187, archived from the original (PDF) on 2011-07-20
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