Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors

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]

HIV-1 RT is an asymmetric heterodimer which is 1000 amino acid long and is composed of two subunits. The larger subunit, p66, is 560 amino acid long and it exhibits all the enzymatic activities of the RT.[8] The smaller subunit, called p51, is 440 amino acid long and it is considered to stabilize the heterodimer but also it may take part in the binding of the 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]

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 azido group which gives us zidovudine.[13] The lack of the 3´hydroxyl group which provides the attachment point for the next nucleotide in the growing DNA chain during the reverse transcription makes it an obligate chain terminator. Ziduvodine is incorporated in place of thymidine and is an extremely potent inhibitor of HIV replication.[14] This compound had been prepared in 1964 as a potential anti-cancer agent but was shown to be ineffective.[15] In 1974 zidovudine was reported to have activity against retroviruses and was subsequently re-screened as an antiviral when the AIDS epidemic hit Western societies during the mid 1980s.[13][15] However, zidovudine is relatively toxic since it is converted into the triphosphate by the cellular enzymes and therefore it is activated in uninfected cells.[14]

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 selectivity and therefore cause 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 derivatives. Lamivudine was developed as the sulfur analogue of zalcitabine (see table 2).[16] It was initially synthesized as a racemic mixture (BCH-189) and analysis showed that both positive and negative 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 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 clinical use. That was 2´,3´-didehydro analogue of dideoxyadenosine. Insertion of a 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

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 prodrug of tenofovir. Tenofovir is an acyclic adenosine derivative. The acyclic nature of the compound and its phosphonate moiety are unique structural features among the approved NRTIs.[21] Tenofovir DF is hydrolyzed enzymatically to tenofovir which exhibits anti-HIV activity.[23][24] It was developed by the synthesis and 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	Deoxythymidine		Deoxycytidine			Adenosine	Guanosine
D r u g	Tenofovir ({[(2R)-1-(6-amino-9H- purin-9-yl)propan-2-yl]oxy}methyl) phosphonic acid	Zidovudine 3´Azido-2´,3´-dideoxythymidine, azidothymidine (AZT)	Stavudine 2´,3´-Didehydro-2´,3´-dideoxythymidine (d4T)	Emtricitabine (-)-ß-L-3´-thia-2´,3´-dideoxy-5-fluorocytidine ((-)FTC)	Lamivudine 2´,3´-Dideoxy-3´-thiacytidine (3TC)	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?

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 is a deoxycytidine analogue with activity against HIV resistant to several other nucleoside analogues, including zidovudine and lamivudine.[22] This is partly because of high intracellular levels of its triphosphate metabolite reached in cells.[6] Clinical trials of elvucitabine are on hold, because it has shown bone marrow suppression in some patients, with CD4+ cell numbers dropping as early as two days after initiation of dosing.[22][29]

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 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]