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Abstract

The advent of antiretroviral combination therapy has significantly impacted the HIV/AIDS epidemic. No longer a death sentence, HIV infection can be controlled and suppressed using cocktail therapies that contain two or more small molecule drugs. This review aims to highlight the discovery, development, and impact of one such molecule, namely, emtricitabine (FTC, emtriva), which is one of the most successful drugs in the fight against HIV/AIDS and has been taken by over 94% of individuals infected with HIV in the USA. We also pay tribute to Dr. John C. Martin, former CEO and Chairman of Gilead Sciences, who unexpectedly passed away in 2021. A true visionary, he was instrumental in delivering FTC, as part of combination therapy with TDF (tenofovir, viread) to the global stage. As the fight to eradicate HIV marches on, we honor Dr. Martin’s legacy of collaboration, achievement, and hope.

Part 1: Introduction

In the early eighties, when the first patients came down with what would later be described as AIDS, no one could have predicted how much this illness would change the face of infectious disease research and drug discovery and development. At that time, topical and oral acyclovir was the only recently approved for treatment of herpesvirus infections as the first true selective antiviral agent. The fact is that a handful of small molecules that were to be discovered in the next two decades changed the face of HIV/AIDS forever and illuminated our path toward the development of additional antiviral agents for other infectious diseases, including hepatitis B, hepatitis C, and even coronaviruses.

With these small molecule discoveries, concepts in drug resistance and combination therapy were re-discovered, developed, and subsequently revolutionized antiviral treatment, especially for HIV and hepatitis C. These small molecules targeted viral enzymes such as the reverse transcriptase, integrase, and protease which carry out essential functions for virus replication. Initially, there was a lot of hesitancy to use combination therapy in patients since the standard approach was to use monotherapy and the expected end point was death. Emergence of resistant viruses to monotherapy in culture and in humans was common and confirmed the viral target of the drug. But it was only with the advent of combination chemotherapy and early sequencing studies that the tide of treatment failure began to change. In this way, cocktail therapy, also referred to as highly active antiretroviral therapy (HAART), which consists of combining multiple antiviral agents to drive down viral load and prevent the emergence of resistance, proved to be highly effective and became the standard of care. Initially, the combined use of an oxathiolane nucleoside such as lamivudine (3TC) plus 3^′-azido-3^′deoxythymidine (AZT) was shown to delay the emergence of resistant viruses and increase survival in infected persons. As more agents became available, three drug combinations were established as the norm and survival was increased to 95% or higher for those with access and adherence to treatment. The possibility of reducing transmission among treatment-experienced individuals, prevention of mother-to-child-transmission, pre-exposure prophylaxis, prolonging life expectancy, and increased quality of life for individuals living with HIV became a reality.

This review aims to highlight the discovery, development, and impact of one such small molecule—emtricitabine (FTC, emtriva, FDA-approved on July 2, 2003). FTC is one of the most successful drugs in the fight against HIV/AIDS. FTC is the backbone of truvada (FDA-approved on 08/02/2004), a cocktail therapy saving many lives across the globe. The other antiviral agent in this cocktail therapy, TDF (viread), was approved on October 24, 2001 thanks to the work done by Dr. Antónín Hóly in the Czeck Republic with assistance from Drs. Erik DeClercq and Jan Balzarini in Leuven, Belgium [1]. This international effort and the marriage of two critically important anti-HIV drugs to create truvada resulted in one of the most formidable drug combinations for HIV prevention and treatment. This review tracks the history of FTC, from discovery to FDA-approval, and highlights the unique interplay between biochemical properties of FTC, combination therapy, and curbing drug resistance. We also pay tribute to the scientists, innovators, and visionaries who recognized the potential of this small molecule and ushered its delivery to the global stage. One such person was Dr. John Martin, former CEO and Chairman of Gilead Sciences, who unexpectedly passed away on March 30, 2021, leaving behind a legacy of collaboration, achievement, and hope.

Part 2: Emtricitabine: From discovery to clinical approval

Discovery and development

Nucleoside analog inhibitors are the backbone of cocktail combination therapy against HIV and have been successful in combating viral infections such as herpes, hepatitis B, and hepatitis C. In the context of HIV, these small molecules are referred to as nucleoside analog reverse transcriptase inhibitors (NRTIs) and act as chain terminators of viral RNA-dependent-DNA-synthesis due to the lack of a 3^′-hydroxyl group on the triphosphorylated NRTI. Currently, there are multiple FDA-approved nucleoside analogs for the treatment of HIV. These include zidovudine (AZT, retrovir), emtricitabine (FTC, emtriva), lamivudine (3TC, epivir), zalcitabine (ddC, hivid), abacavir (ABC, zIagen), didanosine (ddI, videx), stavudine (d4T, zerit), and two tenofovir prodrug derivatives, 5^′-disoproxil fumarate (TDF, viread) and 5^′-alafenamide (TAF, vemlidy) (Figure 1).

Figure 1.

List of US Food and Drug Administration (FDA) approved drugs targeting HIV reverse transcriptase: zidovudine (AZT, retrovir), emtricitabine (FTC, emtriva), lamivudine (3TC, epivir), zalcitabine (ddC, hivid), abacavir (ABC, zIagen), didanosine (ddI, videx), stavudine (d4T, zerit), 5^′-disoproxil fumarate (TDF, viread), and 5^′-alafenamide (TAF, vemlidy)

Emtricitabine [(-)-FTC or FTC] was first synthesized as a racemate at Emory University in 1989 by post-doctoral fellow Dr. Woo-Bang Choi under the guidance of Drs. Dennis Liotta and Raymond Schinazi. The Emory group went on to purify the (-)-enantiomer and describe FTC’s antiviral activity against HIV and later HBV. At the time of conception, it was not commonly accepted that nucleosides such as FTC and 3TC would be stable with a pentose ring containing a sulfur and oxygen. In addition, based on treatment results with AZT, the prevailing sentiment was that nucleoside analogs are all toxic (except for acyclovir which was used to treat herpesvirus infections). Further, the common belief was that FTC would be cleaved by a thymidine phosphorylase and deaminated to 5-FU, a known cytotoxic anticancer drug. We now know that both minus and plus-forms of FTC are active and non-toxic and that they are not cleaved by phosphorylases. Lastly, until this discovery nobody expected that the so-called L-nucleoside would demonstrate any antiviral activity. For nucleoside chemists, that was the biggest game changer.

At the time, many other analogs of FTC were also synthesized, but none had the safety profile and potency of FTC itself, especially the (-)-enantiomer. Dr. Schinazi’s group was the first to test and characterize the antiviral properties and mechanism of action of the (-)-enantiomer. This compound was licensed to Wellcome (then Glaxo-Wellcome), and then returned to Emory since Glaxo-Wellcome had acquired lamivudine (3TC). FTC was subsequently relicensed to a company founded by Drs. Schinazi, Karl Hostetler, and Dennis A. Carson called Triangle Pharmaceutical which developed FTC up to phase III before the Gilead Sciences (under the leadership of John Martin) announcement in December 2002 of the acquisition of Triangle Pharmaceuticals for US$464 million (Figure 2). Interestingly, Gilead’s net income for the 6 months ending in June 2002 was only US$15.9 million. The acquisition of FTC was transformative for the company and led to Gilead’s dominance of the HIV therapeutic market for the next two decades. Globally, FTC has saved millions of lives thanks to the foresight of Drs. John Martin and David Barry (then CEO of Triangle Pharmaceutical), who had the courage to take an almost abandoned drug and drive it to FDA approval for the benefit of millions of HIV-infected persons.

Figure 2.

FTC development timeline

Mechanism of action

Emtricitabine is a 2^′-deoxycytidine derivative with sulfur at the 3^′-position on the ribose ring and a fluorine at the 5-position of the base. In its 5^{^′}-triphosphate form, it targets the HIV reverse transcriptase (HIV RT), the viral DNA-dependent RNA polymerase that synthesizes the viral genome. As a C analogue, FTC is incorporated by HIV RT opposite G nucleosides in the template. Due to chemical modifications in its ribose moiety, 3^′-OH bond formation cannot occur when FTC is present at the 3^′ end of the DNA primer, thus resulting in chain-termination. Disruption of this process results in rapid reduction of systemic viral loads to undetectable levels in HIV-infected individuals, allowing for significant rebound of CD4⁺ T-cells, and providing a tandem mechanism resulting in control of systemic viremia and restoration of functional immunity. What makes FTC an outstanding nucleoside analog is its ability to inhibit HIV RT with remarkable efficiency. Anderson et al. demonstrated that the 5-fluorine substitution on FTC causes increased overall efficiency of incorporation during both DNA- and RNA-dependent DNA synthesis by RT [2–4]. Close examination of the binding affinity of NRTIs to RT showed that the binding affinity constant (K_d) of FTC-triphosphate (TP) was lower (signifying enhanced binding to the catalytically competent RT: nucleic acid complex) than its natural counterpart, dCTP [3,5]. Further biochemical analysis showed that the incorporation efficiency (rate of polymerization divided by K_d) of FTC-TP is 2.5 times higher than that of 3TC-TP during DNA-dependent DNA synthesis[3]. This incorporation efficiency is 9 times higher during RNA-dependent DNA synthesis [2]. The enhanced incorporation kinetics of FTC-TP compared to 3TC-TP may in part explain the increased in vitro activity of FTC over 3TC [2,5–7]. In addition, because FTC-TP is a poor substrate for phosphodiesterases, its intracellular half-life is approximately 39 h in human lymphocytes, compared to 22 h for 3TC-TP [8].

Drug resistance

In 1993, the Schinazi team showed for the first time that treatment with FTC or 3TC in vitro and in vivo (for 3TC) resulted in the selection of a mutation in the active site of HIV polymerase, namely, M184V/I, which causes a 500–1000-fold resistance to these compounds [9–11]. Other groups including Dr. Mark Wainberg’s (McGill University) and Dr. Brendan Larder’s (Burroughs-Wellcome) group later confirmed these observations [12–14]. Dr. Schinazi named the M184V mutation “the mother of all mutations” since it is part of the “conserved” YMDD motif, the active site of HIV, which was previously thought to be a highly conserved region of HIV polymerase. Kinetic and structural modeling studies revealed that the beta-branch of the valine residue causes a steric hindrance due to the position of sulfur in the oxathiolane ring. This steric clash causes the oxathiolane ring of 3TC-TP to move from the position it would occupy when bound to the wild-type enzyme, to a new position, thus increasing the distance between the 3^′-OH of the last base of the primer and the α-phosphate group of the incoming NRTI-TP. This, in turn, negatively affects the binding of 3TC-TP and FTC-TP (Figure 3) [2,15–17]. Additionally, the Schinazi group and others showed that the M184V mutation also impacts RT processivity and fidelity, which may lead to reduced viral fitness of mutant strains [18–20].

Figure 3.

Binding mode of FTC-TP (green carbon sticks) complexed with DNA in HIV-RT active site (PDB ID: 6UJX). Two important residues associated with drug resistance, M184 and K65, are shown in magenta carbon sticks. Both residues are in close contact with FTC-TP

Another important mutation in HIV RT, namely, K65R, was first discovered in 1994 by the Wainberg group [21,22]. The K65R mutation is associated with a low to intermediate level of resistance to TDF both in vitro and in vivo [23,24]. The K65R substitution occurs in the conserved IKKK region of HIV-1 RT. Molecular modeling based on crystal structures of HIV-1 RT suggests that during dNTP incorporation, part of this subdomain rotates toward the primer-template and the polymerase active site. As a result of this movement, K65 makes contact with the incoming dNTP and forms a salt bridge between the lysine and the phosphate of the incoming dNTP [25,26]. The change from lysine to arginine at amino acid 65 likely affects the interaction between the enzyme and the triphosphate moieties of dNTPs and NRTIs, altering the nucleotide-binding specificity or the phosphodiester bond formation efficiency of RT [25,27–30]. Interestingly, susceptibility to tenofovir is enhanced in the presence of the M184V mutation [31]. White et al. have demonstrated a decreased replication capacity of viruses with the K65R mutation either alone or in combination with M184V mutation [32]. Although viruses containing both the K65R and M184V mutations are rarely observed clinically [33,34], these mutations within HIV RT exhibit both decreased binding affinity and decreased catalytic rate of incorporation of NRTI-TP in comparison with wild-type RT [35,36]. As such, FTC/TDF is a powerful combination, ideal for repelling the emergence of drug resistant virus.

Combination therapy

As demonstrated by the M184V/K65R story, combination nucleoside analog inhibitor therapy has repeatedly been shown to be essential for effective control of HIV replication, reducing the risk of HIV disease progression and death [37–39]. First trials of combination therapy were done with zidovudine (AZT) plus didanosine (ddl) or zalcitabine (ddc) [37–39]. In 1993, Schinazi and colleagues showed that a patient on 3TC who secretly took concomitant AZT did not develop resistance to either AZT or 3TC and survived longer than patients on monotherapy [40,41]. In 1997, the first combination therapy was approved with a fixed dose of the RT inhibitors, lamivudine (3TC), and zidovudine (AZT) with trade name combivir for the treatment of HIV-infected patients [42]. Later, with the development of truvada (FTC+TDF) and atripla [FTC+TDF+efavirenz (EFZ)], FTC was established as the highly effective backbone of combination therapy. These combinations were powerful in blocking the emergence of the 184V/I mutation in the presence of tenofovir, and emergence of K65R mutation in HIV RT did not confer cross-resistance. Further, FTC is practically devoid of any significant side effects (gastrointestinal discomfort, rash, and hyperpigmentation of palms and soles have been rarely reported [43,44]) and both FTC and 3TC have been well tolerated with no significant treatment-limiting adverse effects. Thanks to its excellent safety and favorable resistance profile in the context of combination therapy, it is currently estimated that over 94% of persons living with HIV who are on antiviral therapy have FTC or 3TC as part of their treatment. FTC is currently serving as the backbone of nine different fixed-dose combination drug regimens, including truvada, atripla, biktarvy, complera, descovy, genvoya, odefsey, stribild, and symtuza (Table 1). In 2020, biktarvy ranked as the seventh most prescribed drug in the world with sales of US$7.26 billion [45]. More recent regimens combine FTC with TAF, a new prodrug of tenofovir with an improved safety profile [46,47]. Finally, like 3TC, FTC was found to be active against HBV, as first reported in PNAS in 1991, but FTC was never developed or approved by the US FDA for HBV treatment presumably because it competed with Gilead’s adefovir dipivoxil (hepsera), TDF, TAF, and 3TC (developed at the time by GSK).

Table 1.

Combination therapies for HIV with FTC backbone.

Year of FDA approval	Trade name	Generic name	Host*/viral target(s)	Pharmaceutical developer
2004	Truvada	Emtricitabine (FTC)/Tenofovir disoproxil fumarate (TDF)	RT/RT	Gilead Sciences
2006	Atripla	Efavirenz (EFV)/Emtricitabine (FTC)/Tenofovir disoproxil fumarate (TDF)	RT/RT/RT	Gilead Sciences
2011	Complera	Emtricitabine (FTC)/Rilpivirine hydrochloride (RPV)	RT/RT	Gilead Sciences
2012	Stribild	Cobicistat (COBI)/Elvitegravir (EVG)/Emtricitabine (FTC)/Tenofovir disoproxil fumarate (TDF)	CYP3A*/Integrase/RT/RT	Gilead Sciences
2015	Genvoya	Cobicistat (COBI)/Elvitegravir (EVG)/Emtricitabine (FTC)/Tenofovir alafenamide (TAF)	CYP3A*/Integrase/RT/RT	Gilead Sciences
2016	Descovy	Emtricitabine (FTC)/Tenofovir alafenamide (TAF)	RT/RT	Gilead Sciences
2016	Odefsey	Emtricitabine (FTC)/Rilpivirine hydrochloride (RPV)/Tenofovir alafenamide (TAF)	RT/RT/RT	Gilead Sciences
2018	Biktarvy	Bictegravir (BIC)/Emtricitabine (FTC)/Tenofovir alafenamide (TAF)	Integrase/RT/RT	Gilead Sciences
2018	Symtuza	Darunavir (DRV)/Cobicistat (COBI)/Emtricitabine (FTC)/Tenofovir alafenamide (TAF)	Protease/CYP3A*/RT/RT	Janssen
2020	Generic	Emtricitabine (FTC)/Tenofovir disoproxil fumarate (TDF)	—	Teva, Apotex, Zydus, Cipla, Mylan, and others

PART 3: Small molecules usher in big change

The past several decades in HIV research are a testament to the power of innovation and drug discovery. An HIV diagnosis was once a death sentence. Today, with the discovery of so many effective drugs, HIV infection is treated as a chronic, but manageable condition, that does not need to be the cause of death. In fact, some argue that HIV-related deaths, at least in settings where access to treatment is possible, should be viewed as a “breakdown in the HIV care-continuum,” rather than an inevitable conclusion [48]. It is now well documented that effective treatment with antiviral combination therapy can reduce viral load to levels that prevent transmission [49,50] leading to the undetectable = untransmittable adage [51,52]. What this means is that combination antiviral treatment, such as FTC-containing regimens, are extremely effective tools in the control, elimination, and eradication of the HIV/AIDS epidemic. The powerful impact of combination therapy can be further recognized in the success of treatment, control, and prevention in specific contexts such mother to child transmission [53]. Notably, from 2010 to 2020, new HIV infections among children have been reduced by 35%, largely thanks to measures implemented in medical settings including pre-birth HIV testing of pregnant women, access to combination treatments during childbirth, and adherence to WHO guidelines for breastfeeding [54]. Other high-risk conditions such as viral transmission among men who have sex with men (MSM), injectable drug use, and during blood transfusion have also greatly benefited from combination therapy. Prevention tools, centered around combination therapy, have been essential for epidemic control, even if eradication has remained an unreached goal. Namely, early treatment with antiretroviral drugs (ARV) [49] and effective implementation of pre-exposure prophylaxis (PrEP) have proven critical for preventing transmission among at-risk persons [48,55–58]. FTC/TDF and FTC/TAF combinations used as PrEP are shown to be effective, safe, and impactful for reducing incidence of new infections [59,60].

Part 4: Honoring Dr. John Martin and looking to the future

When truvada (FTC/TDF) became generic on September 30, 2020 (Teva/Gilead deal), treatment prices fell to $210 – $720 a year (depending on the supplier), greatly increasing access to this life-saving drug combination. Generic truvada used in PrEP has had a significant impact in controlling the HIV epidemic and will likely remain a staple of PrEP until TAF also becomes generic [61]. Recent exciting developments in long-acting formulations [62] suggest that we are once again at the cusp of improving PrEP and HIV treatment [62]. It remains to be seen what role FTC or 3TC will play in this new chapter. Studies are being done to develop FTC in long-acting formulations, [63] especially for FTC/TAF, [64] as well as long-acting FTC prodrugs [65].

Considering the undeniable success of combination antiviral therapy for HIV, the United Nations declared the goal of ending the HIV/AIDS epidemic by 2030 [66]. To this end, the 90–90–90 goals aim for 90% of individuals infected with HIV to be aware of their status, 90% of diagnosed individuals to start combination antiretroviral therapy, and for 90% of those on combination therapy to have their viral loads suppressed below the level of detection. The aim of the 90–90–90 goal was to achieve this tremendous feat by 2020 [67,68]. Although progress has been made, the 2021 UNAIDS Fact Sheet shows that the numbers fall short (Table 2) and the goal post has now been moved to 2025 [69]. These efforts have been further challenged due to the COVID-19 pandemic which has impacted access to HIV diagnosis, ARV therapy, and treatment adherence [70].

Table 2.

2021 status update on the 90–90–90 goal.

	Projected (2020)	Actual 2020–2021 (UNAIDS data)
Individuals infected with HIV to be aware of their status	90	84% [67– >98%]
Diagnosed individuals to start combination antiretroviral therapy	90	73% [56–88%]
Those on combination therapy to have their viral loads suppressed below the level of detection	90	66% [53–79%]

Finally, lessons learned from HIV antiviral drug development markedly impacted other viral diseases. For example, informed by these lessons, hepatitis drug combination strategies have been developed that essentially cure this chronic and life-threatening infection. With the onset of the COVID-19 pandemic, the world is turning once again to small molecule antiviral therapy as an effective solution to preventing infections, reducing morbidity, mortality, and curbing the economic impact of this global health disaster.

Concluding remarks: Legacy of Dr. John Martin

We are still far from having a US and global infrastructure that leads to HIV elimination using drugs like truvada that prevent infections. It is well established now that “dead viruses don’t mutate” [71] and “dead viruses don’t transmit”—hence, we can eliminate this virus with an oral treatment like truvada. To accomplish this, we need routine and even mandated screening for all. Screening is underutilized in the USA and is not being sufficiently implemented. Without this commitment, we will not be able to eliminate HIV in the US. We need full public awareness programs that appeal to people from all walks of life including MSM and injection drug users. Continued existence of stigma against people living with HIV is a hindrance to treatment uptake and adherence and needs to be addressed through social behavior interventions at the community level. We need HIV clinicians that are willing and able to treat everyone including injection drug users. We need persons with HIV (PWH) and on treatment teaching other PWH about therapies, resistance, and adherence. Telehealth can be part of the solution as well as a national network of infrastructure that provides all this including the needed funding and commitment as we have done for COVID-19. Governments should provide free generic truvada to all people who need it to prevent the over 36,800 [72] and 1.7 million [73] new infections in the US and globally, respectively. This will take time and money, but we need to start now. Effective implementation of linkage to care, as was the case with hepatitis C, [74,75] will be critical in reaching the 90–90–90 goal.

Paving the way for broader reach of effective HIV drugs, Dr. John Martin was one of the first to realize the need for implementation of wider use of Gilead’s truvada. He challenged the status quo by providing wide access to Gilead’s top drugs to the underdeveloped world. He also provided manufacture licenses to many generic companies to legally market Gilead’s HIV drugs. John Martin not only produced good science during his lifetime but he was also a scientist with a vision that impacted the entire world when it comes to HIV “treatment as prevention”. He used his scientific and business acumen not for his glory, but to enrich our world and save lives. No patient ever complained about the clinical value of the life-saving medicines developed by Gilead as well as many of the other leading companies who developed effective and safe drugs for HIV and other infectious diseases. In the absence of an HIV vaccine, the potential of truvada to eliminate HIV will be considered John Martin’s main legacy which will be felt by many generations to come all over the world.

Footnotes

Acknowledgments

Dr. Schinazi owns stock in Gilead and GSK. The other authors do not have any conflicts of interest to report. This review is dedicated to the memory of our friend and colleague Dr. John C. Martin.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded in part by NIH grant 1RO1-MH-116695 and by Emory University Center for AIDS Research NIH grant P30-AI-050409.

ORCID iDs

Raymond F Schinazi

Dharmeshkumar Patel

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The best backbone for HIV prevention,treatment,and elimination: Emtricitabine+tenofovir

Abstract

Part 1: Introduction

Part 2: Emtricitabine: From discovery to clinical approval

Discovery and development

Mechanism of action

Drug resistance

Combination therapy

PART 3: Small molecules usher in big change

Part 4: Honoring Dr. John Martin and looking to the future

Concluding remarks: Legacy of Dr. John Martin

Footnotes

Acknowledgments

Declaration of conflicting interests

Funding

ORCID iDs

References