Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors

Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors (NRTIs and NtRTIs) began in the 1980s when the 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.structurestructureucleosiderug({oxy}methyl) phosphonic acid3´Azido-2´,3´-dideoxythymidine, azidothymidine (AZT)2´,3´-Didehydro-2´,3´-dideoxythymidine (d4T)(-)-ß-L-3´-thia-2´,3´-dideoxy-5-fluorocytidine ((-)FTC)2´,3´-Dideoxy-3´-thiacytidine (3TC)2´,3´-Dideoxycytidine (ddC)2´,3´-Dideoxyinosine (ddI)(4-(2-amino-6-(cyclopropylamino)- 9H-purin-9yl) cyclopent-2enyl)methanol(ABC) Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors (NRTIs and NtRTIs) began in the 1980s when the 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. In the summer of 1981 the acquired immunodeficiency syndrome (AIDS) was first reported. Two years later the etiological link to AIDS, the human immunodeficiency virus (HIV) was identified. Since the identification of HIV the development of effective antiretroviral drugs and the scientific achievements in HIV research has been enormous. Antiretroviral drugs for the treatment of HIV infections belong to six categories: Nucleoside and nucleotide reverse-transcriptase inhibitors, Non-nucleoside reverse-transcriptase inhibitors, protease inhibitors, entry inhibitors, co-receptor inhibitors and integrase inhibitors. The reverse transcriptase of HIV-1 has been the main foundation for the development of anti-HIV drugs. The first nucleoside reverse-transcriptase inhibitor with in vitro anti-HIV activity was zidovudine. Since zidovudine was approved in 1987, six nucleosides and one nucleotide reverse-transcriptase inhibitor (NRTI) have been approved by FDA. NRTIs approved by the FDA are zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine and the only nucleotide reverse-transcriptase inhibitor (NtRTI) approved is tenofovir (see table 4). 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. 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. 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. 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. Activation of nucleoside and nucleotide reverse-transcriptase inhibitors is primarily dependent on cellular entry by passive diffusion or carrier-mediated transport. NRTIs are highly 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. Nucleosides must be triphosphorylated, while nucleotides, which possess one phosphonated group, must be diphosphorylated. This stepwise activation process occurs inside the cell and is mediated by a coordinated series of enzymes. The first, and often rate limiting, phosphorylation step (for nucleoside analogues) are most commonly catalyzed by deoxynucleoside kinases. Addition of the second phosphate group to nucleoside monophosphate analogues is completed by the nucleoside monophosphate kinases (NMP kinases). A variety of enzymes are able to catalyze the final phosphorylation step for NRTIs, including nucleoside diphosphate kinase (NDP kinase), phosphoglycerate kinase, pyruvate kinase and creatine kinase, resulting in formation of respective antivirally active triphosphate analogues.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). Unlike dNTPs substrate, NRTIs lack a 3´-hydroxyl group on the deoxyribose moiety. Once incorporated into the DNA chain, the absence of a 3´-hydroxyl group, which normally forms the 5´- to 3´- phosphoester bond with the next nucleic acid, blocks further extension of the DNA by RT, and they act as chain terminators. 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. 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. This compound had been prepared in 1964 as a potential anti-cancer agent but was shown to be ineffective. 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 themid 1980‘s. However, ziduvodine is relatively toxic since it is converted into thetriphosphate by the cellular enzymes and therefore it is activated in uninfected cells. Dideoxynucleosides are analogues of nucleoside where the sugar ring lacks both 2´ and 3´-hydroxyl groups. Three years after the synthesis of zidovudine, Jerome Horwitz and his colleagues in Chicago prepared another dideoxynucleoside now known as zalcitabine (ddC). 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. Zalcitabine was approved by the FDA for the treatmentof HIV-1 in June 1992. 2´,3´-dideoxyinosine or didanosine is converted into dideoxyadenosine in vivo. Its development has a long history. 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. Didanosine was approved by the FDA for the treatmentof HIV-1 in October 1991.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.