Antiviral Targets and Known Antivirals (HAART)

Abstract

In 2021, the number of HIV-positive people worldwide was estimated to be 38.4 million. Since its discovery four decades ago, the scope of the HIV infection has outstripped all predictions, necessitating the urgent need to develop novel antivirals against the virus that target crucial stages in the virus’ life cycle. New antiviral drug classes that were developed in response to the HIV epidemic were coupled to offer very highly active antiretroviral treatment. These novel highly active antiretroviral therapies (HAART) were developed as a result of the emergence of drug-resistant strains of the virus. By inhibiting these enzymes, reverse transcriptase, integrase, and protease that are essential for viral attachment, entry, integration, and maturation, antiretroviral therapy (ART) strategies can suppress the virus, lower the viral load, boost CD4 count, and ultimately halt the progression of the disease. Advances in research on the biology of both the immature and the mature forms of the HIV capsid in terms of its structure and function have made it possible to discover and/or design small molecules and peptides that interfere with the virus’s assembly and maturation. This article presents and reviews HAART’s current state and strategies as a very active antiviral.

1. Introduction

A virus is composed of a protective capsid that surrounds a core of genetic material, either DNA or RNA. The envelope protein is what makes up the capsid. Each virion is made up of a viral envelope, a matrix, and a capsid, which contains two copies of the single-stranded RNA genome as well as a number of enzymes (Figure 1). A small number of viral proteins, encoded by the HIV genome, always form cooperative relationships with host proteins and other HIV proteins in order to enter host cells and take over their internal machinery. The structure of HIV is distinct from that of other retroviruses. The HIV virion has a diameter of about 100 nm. Its innermost portion is made up of a cone-shaped core that contains [1] the principal core protein, some minor proteins, reverse transcriptase, integrase, and protease enzymes, as well as two copies of the (positive sense) ssRNA genome. Eight viral proteins critical to the HIV life cycle are encoded in the HIV genome, according to research [2].

Figure 1.

Human immuno virus. Source: [3].

Viruses and their hosts have similar metabolic processes because they are obligatory cellular parasites, that is, they depend on a living host for existence, viruses can either encode proteins that are closely related to and similar to host proteins to carry out crucial metabolic and survival activities, or they may have evolved the capacity to directly integrate their genome into that of the host, co-opting the host’s cellular genetic factors [4].

Although many of these viral infections can be successfully averted through vaccination [4], there is currently an inadequate understanding of vaccinations against several significant pathogens that have been linked to numerous human viral disorders like HIV and hepatitis C virus (HCV). Interest in the creation of novel antiviral chemicals is typically fueled by two crucial aspects and considerations: the prerequisites for a specific antiviral medicine against a particular viral infection are essential in assuming that the proposed antiviral would be able to reasonably control the particular viral infection. Second, what antiviral treatments or preventative measures are now available for the specific viral infection, as well as what antiviral tactics might be used to address this demand [5]?

In 2019, there were roughly 38 million HIV infections worldwide, according to U.S. statistics [6]. Although combination antiretroviral therapy (cART) has improved the success rate being recorded in the management of HIV infection, there is currently no known cure for the illness. Data show that cART has been able to suppress the viral load to the point where it is undetectable, and carriers frequently lead nearly normal lives with measurable increased average life expectancies compared to historical data [7]. Though highly active antiretroviral therapy (cART), also known as HAART (highly active anti-retroviral therapy), has been successful in treating HIV-1 infections, the rise of resistance forces the development of new antiviral medicines that would be more effective and efficient in viral suppression.

Despite this success in treating HIV-1 infections that have been attributed to cART, also known as HAART (highly active antiretroviral therapy), the emergence of resistance drives a pressing need to create new antiviral agents that are more effective and efficient in viral suppression. Furthermore, cross-resistance (a phenomenon in which the emergence of resistance to one therapeutic agent simultaneously results in the emergence of resistance to other agents in that class) has prompted the need to develop novel compounds as well as ones that are active against novel targets [8].

In some cases where HIV infection becomes chronic, the specifics of a pattern of its progression in a patient may completely differ from the average, thereby manifesting differently in different patients, suggesting complexity in the pattern of infection in different individuals. A lot of factors ranging from genetics to environment have been implicated in such a scenario. This variability is of great public health concern and has huge clinical importance, as well as raising the question of whether or not an average treatment regimen is optimal for a given patient [7].

Recent research has shown that the mechanism of action as well as the pathogenesis of HIV-1 is complex and dynamic being characterized by the interplay and modulation of both viral and host cellular factors. Since the identification of HIV as the causative agent of AIDS, a lot of efforts and resources have been committed to keeping the disease under control. A lot of comprehensive research into understanding the individual steps of the viral replication/life cycle and the dynamics during infection has led to the development of a wide range of antiviral compounds exploiting different molecular strategies. Every stage in the viral life cycle/replication stage and every gene product of the virus remains a potential target for therapeutics and prevention [9].

The pathophysiology and mode of action of HIV-1 are complicated and dynamic, as evidenced by the interaction and modification of both viral and host cellular components, according to a recent study. Many resources and efforts have been devoted to controlling the disease ever since HIV was discovered to be the primary cause of AIDS. A variety of antiviral drugs utilizing various molecular methods have been developed as a result of extensive research into the dynamics of infection as well as the individual steps of viral replication and life cycle. Every stage of the viral life cycle/replication as well as every virus gene product continue to be potential targets for treatment and averting disease [9].

The primary molecular-based strategies against viruses, with a focus on HIV-I, that have been described in the literature are outlined in this review, along with their potential to lead to the creation of stronger and more potent antiviral drugs for clinical use.

2. HIV-1 infection and therapeutic targets

The ability of HIV to propagate and cause pathogenesis in patients is essentially determined by the efficacy of HIV entry into a host cell. Prior to HIV-1 entering its target cells (CD4-expressing T-lymphocytes, macrophages, and monocytes), the viral envelope glycoprotein (Env) gp120 fuses with the host cell membrane via the primary cellular receptor CD4 [10]. This causes changes in Env conformation that make the interaction between the gp120 V3 loop and coreceptor CCR5 or CXCR4 easier. The trimeric spikes that make up the HIV-1 envelope are each composed of three gp120 surface subunits and three gp41 trans-membrane subunits. The CD4 receptor and either the CCR5 or the CXCR4 co-receptor are often needed for viral binding and entrance into the cell [11]. The machinery that drives the fusing of the viral and host cell membranes is set off by coreceptor binding, which also causes the six-helix bundle of Env to form. Effectively hindering HIV entry and dissemination is possible by functionally blocking receptors, coreceptors, or HIV Env [10].

The viral reverse transcriptase is used to convert the viral RNA genome to double-stranded DNA following receptor-mediated binding and internalization. Following the integration of the viral genomic DNA into the host genome in the nucleus, the full 9.2-kb mRNA transcript is produced from the imported viral genomic DNA. This transcript can be doubly spliced to produce 1.8-kb mRNAs that code for the proteins Tat, Rev, and Nef. Tat and Rev move to the nucleus where Rev mediates the export of mRNAs during the late phase while Tat triggers transcription.

Env, Vpu, Vpr, and Vif proteins are encoded by single-spliced 4.0 kb transcripts, while 9.2 kb unspliced mRNA transcripts are responsible for Rev. is required for the nuclear export of Gag and Gag-Pol. These unspliced mRNAs serve as the genomic RNA for virus progeny as well. A number of Gag molecules bind to the viral DNA before being transported to lipid rafts on the host cell’s plasma membrane. Viral proteins are used to assemble the viral genome as a dimer, which then suddenly leaves the cell [11]. Once the viral particles have reached maturity, they are expelled from the host cell. The above-described processes offer several options for various therapeutic treatments, interventions and tactics.

3. Virus-receptor interaction and entry

The ability of HIV to propagate and cause pathogenesis in patients is essentially determined by the efficacy of HIV entry into a host cell. Prior to HIV-1 entering its target cells (CD4-expressing T-lymphocytes, macrophages, and monocytes), the viral envelope glycoprotein (Env) gp120 fuses with the host cell membrane via the primary cellular receptor CD4 [10]. This causes changes in Env conformation that make the interaction between the gp120 V3 loop and coreceptor CCR5 or CXCR4 easier. The trimeric spikes that make up the HIV-1 envelope are each composed of three gp120 surface subunits and three gp41 trans-membrane subunits. The CD4 receptor and either the CCR5 or the CXCR4 co-receptor are often needed for viral binding and entrance into the cell [11].

The virion gp120 surface subunit (SU protein) binds to the CD4 receptor to start the HIV-1 infection. A non-covalent bond between the SU protein and the gp41 transmembrane subunit (TM protein) holds the SU protein to the virus. A cellular convertase called furin located in the endoplasmic reticulum (ER) proteolytically separates SU and TM from the Envelope (Env) precursor protein. Both continue to be noncovalently linked and are transported by vesicles to the host plasma membrane. The TM protein facilitates the fusion of the viral membrane with the host cell membrane, while the SU protein is in charge of receptor recognition on CD4+ T-lymphocytes [12].

A structural change in SU brought on by binding to the CD4 receptor makes the binding site for a co-chemokine family receptor visible. Chemokine receptors CCR-5 (R5 HIV-1 isolates) and CXCR-4 (X4 HIV-1 isolates), which are employed by HIV-1 viruses that are monocytes/macrophage- and T-cell-tropic, respectively, are the two main co-receptors needed for HIV-1 entry [13]. Another structural change that results from the SU protein’s binding to the co-receptor exposes the N-terminal portion of TM. This component also referred to as the fusion peptide, facilitates the fusing of the host and viral membranes. Through a process called syncytium formation, the Env protein is also able to mediate the union of infected and uninfected cells [14, 15]. The CD4-SU interaction, SU-chemokine co-receptor interaction, and the TM-mediated virus-cell membrane fusion process are the three main targets of current methods.

4. Antiretrovirals

Antiretroviral (ARV) drugs, as defined by the World Health Organization, are drugs used to treat HIV. Antiretroviral therapy, or ART, is the term used to describe the use of three or more ARV medications in combination to treat HIV infection. ART requires ongoing care. Combination ART and highly active ART are synonyms [16]. Currently, over twenty-four antiretroviral medications that have been licensed by the FDA are being used to treat HIV infection [17]. These are divided into many classes based on how they work, particularly when it comes to virus replication [18].

They include:

1. Specific CD4-directed post-attachment inhibitors bind to chemokines co-receptors, thereby preventing HIV from attaching to and entering host cells;

2. Chemokine receptor antagonists (CRAs) selectively block interactions between the human CCR5 receptor and the HIV-1 gp120 protein which, in turn, prevents HIV entry into cells;

3. Fusion inhibitors (FIs) disrupt HIV binding and, ultimately, fusion with host cells;

4. The transcription of viral RNA into double-stranded DNA can be prevented with nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs);

5. Targeting the same process, the activity of the key enzyme HIV-1 reverse transcriptase may be reduced or blocked with non-nucleotide reverse transcriptase inhibitors (NNRTIs);

6. Integrase inhibitors (IIs) hinder the transport and attachment of pro-viral DNA to host-cell chromosomes;

7. HIV replication and the formation of mature, infectious viral particles can be prevented with protease inhibitors (PIs);

8. Pharmacokinetic enhancers do not directly interfere with viral replication but rather boost the concentration of antiretrovirals in the blood to make them more effective. Numerous variations of the cart have been approved.

The initial therapy usually starts with a combination of three antiretrovirals, including two NRTIs plus an NNRTI, or two NRTIs plus a protease inhibitor. The use of four antiretrovirals was shown not to improve outcomes over a combination of three compounds [19].

The ability of the virus to maintain latency or persist in dormant memory CD4+ T cells (reservoirs) and other cell types at various sites throughout the body presents the biggest challenge for cART or HAART [17, 20]. When treatment is stopped or instructions are not followed as directed, the virus can re-emerge and become active. Its mechanism, viral integration location, pro-viral orientation, genomic architecture, and stochastic gene expression have all been suggested as significant factors [17, 21, 22] despite the fact that there is a dearth of knowledge and understanding of it.

Reactivation and subsequent depletion (“shock and kill”) of the virus in latent reservoirs has been one therapeutic strategy [23, 24, 25], while manipulation of the signaling pathways necessary for latency establishment has been another strategy [26]. It is important to highlight that despite the use of all these techniques, cART has not yet completely eliminated or eradicated the virus [27]. As a result, cART must be continued for the entirety of a carrier’s life.

The HIV-1 genome is composed of nine viral genes (gag, pol, vif, vpr, tat, rev, vpu, env, and nef) that are required for all processes and stages of the viral life cycle, including viral assembly, viral entry and receptor binding, membrane fusion, reverse transcription, integration, and proteolytic protein processing [11]. HIV-1 is diploid and has two plus-stranded RNA copies of its genome (Figure 2) [28]. It is now necessary to develop alternative therapeutic strategies that are safer, more effective, and more resistant to viral escape because the current cART regimens are unable to cure HIV-1 patients, as these drugs cannot eradicate latent viral reservoirs, may also fall short in completely suppressing viral replication despite drug intensification, and also because there is a possibility that resistance will develop and that there will be toxic side effects. Gene-based and nucleic acid-based therapeutics based on gene editing, ribozymes, and RNA interference (RNAi) [11] are examples of such cutting-edge therapeutic approaches.

Figure 2.

The HIV-1 genome and strategies for antiviral targeting. Source: [11].

5. Interfering strategies against HIV-1

Over the past 30 years, antiviral agents that target viral proteins or host factors have been successfully developed. Based on their inhibitory mechanisms, antiviral reagents can be divided into two groups:

1. Inhibitors that target the viruses themselves.

2. Inhibitors that target host cell factors.

Virus-targeting antivirals (VTAs) can function directly (DVTAs) or indirectly (InDVTAs) to inhibit biological functions of viral proteins, mostly enzymatic activities, or they can also prevent the proper development of the viral replication machinery or protein(s), depending on the situation.

Host-targeting antivirals (HTAs) include reagents that target the host proteins that are involved in the viral life cycle (Figure 2), regulating the function of the immune system or other biological/cellular processes in host cells [28].

6. Molecular strategies to inhibit HIV replication in host cells

6.1 Direct virus-targeting antivirals

Attachment inhibitors: The first stage of viral invasion is the attachment to host cells by contact with the functional receptor(s). For enveloped viruses, receptor identification and host cell attachment are carried out by viral proteins found on the virion’s outer envelope. The envelope proteins gp120 and gp41, which are organized on the viral membrane as a trimer of three trans-membrane gp41 and three noncovalently linked gp120 surface subunits, mediate the invasion and entry of HIV, a typical enveloped virus that belongs to the family of Retroviridae (Figure 2). In response to the CD4 receptor being recognized, gp120 initiates conformational changes that reveal the binding sites for the co-receptors (CCR5 and CXCR4). As a result, anti-HIV medicines have been created that function as antagonists to prevent interactions between HIV and its receptor and co-receptors [28].

6.2 Entry inhibitors

Following direct membrane fusion or endocytosis, a virus releases its genome into the cytoplasm of its host cells after being attached to host cells. Entry inhibitors have been successfully developed for antiviral medicines due to viral entry being one of the crucial early stages in the viral life cycle. When gp120 binds to the receptor, it activates gp41, which then aids HIV-1 in entering cells as an enveloped virus. Two heptad repeat domains (HR1 and HR2) plus a hydrophobic fusion peptide (FP) at the N terminus make up the extracellular component of gp41. A meta-stable prefusion intermediate is created when the FP of gp41 is incorporated into the host cell membrane and HR1 forms a triple-stranded coiled-coil structure. After then, HR2 folds into the hydrophobic grooves of HR1’s coiled-coil to create a stable six-helix bundle that positions the viral and cellular membranes next to each other in preparation for fusion. Inhibitors of membrane fusion are created to prevent the conformational alterations necessary for membrane fusion. The first and only clinically licensed fusion inhibitor is the T20 peptide (Enfuvirtide), which is a peptidic mimic of HR2 and functions by competitively binding to HR1 [29]. T20 can block a wide variety of HIV strains at the nanomolar level, but due to the medication’s poor bioavailability (its plasma half-life is 4 hours), make the clinical application of this drug difficult [28].

6.3 Fusion inhibitors

Theywork to prevent the conformational alterations necessary for membrane fusion. The first and only clinically licensed fusion inhibitor is the T20 peptide (Enfuvirtide), which is a peptidic mimic of HR2 and functions by competitively binding to HR1 [29].

1. Structure of the HIV-1 genome. The genome contains nine genes and two long terminal repeats (LTRs) that can be targeted by RNA interference (RNAi). Certain genomic regions are more conserved than others, making them better targets. In addition, many of the genes are alternatively spliced, requiring careful target design.

2. HIV-1 targeting. Several steps of the HIV-1 viral replication cycle can be targeted by RNAi. Current drug targets are in parentheses [1]. The first step is receptor binding and membrane fusion by the HIV envelope glycoproteins gp120 and gp41 to host receptors CD4 and either CCR5 or CXCR4. This step can be inhibited by knocking down the HIV-1 co-receptors, CCR5 or CXCR4 [2]. Next, the viral genome must be reverse transcribed by the viral reverse transcriptase (RT) and [3] integrated into the cellular genome which is mediated by the viral integrase protein and host factors LEDGF, Importin, and Chaperonin. After integration [4]. The virus is transcribed, which is mediated by viral (TAR and tat) and host (pTEFb, tat-SF1, SPT5, cyclin T1) factors, [5] exported to the cytoplasm (dependent on DDX3 and Rev) and then translated and [6] subjected to post-translational processing by the viral protease [7]. Finally, the proteins are processed and [8] packaged into new viral particles.

Over the past few decades, research has focused heavily on the development of potential means of inhibiting the enzymes implicated in the replication of HIV-1, thereby inhibiting the corresponding pathway leading to a delay in progression or complete elimination of the infection in infected individuals. It’s important to keep in mind that HIV-1 uses the host cell’s replication apparatus, which reduces the number of potential viral targets. The evolutionary freedom for some virus components that interact with host cell molecules has been constrained as a result of the intimate host-virus relationship [30].

6.3.1 Targeting the viral enzymes

6.3.1.1 HIV-1 reverse transcriptase (RT)

Early 1980s revelation that HIV-1, a human retrovirus, causes AIDS sparked retroviral research and drew attention to viral enzymes, which are now the main targets of anti-AIDS medications [31]. Of the 26 medications now approved to treat HIV-1 infections, 14 are RT inhibitors, including the first anti-HIV medication, AZT, which targets RT [32]. Reverse transcription is carried out by the two enzymes DNA Polymerase and RNase, which are both present in RT. Reverse transcription is influenced by other viral and cellular variables, it is important to note [31]. Of the 26 medications now approved to treat HIV-1 infections, 14 are RT inhibitors, including the first anti-HIV medication, AZT, which targets RT [32]. Reverse transcription is carried out by the two enzymes DNA polymerase and RNase, which are both present in RT. Reverse transcription is influenced by other viral and cellular variables, it is important to note [31]. Transcribed backwards [31]. By inserting mutations into the viral genome, HIV-1 reverse transcriptase (RT), which is created from a Gag-Pol polyprotein by cleavage with the viral protease (PR), aids in the emergence of drug resistance to all anti-AIDS medications. The Reverse Transcription complex, or RTC, has been shown to contain a number of viral proteins, including MA, CA, NC, IN, and Vpr. RNA- and DNA-dependent DNA synthesis, RNase H activity, strand transfer, and strand displacement synthesis are the primary functions of RT and are all critical to the retrotranscription process [33]. RT is primarily responsible for several distinct activities that are all indispensable for the retrotranscription process: RNA- and DNA-dependent DNA synthesis, RNase H activity, strand transfer, and strand displacement synthesis [33].

Mutations in RT cause resistance to RT inhibitors at the molecular level. HIV-1 medications include five non-nucleoside inhibitors (NNRTIs) and eight nucleoside/nucleotide analogs (NRTIs). Different conformational and functional states of the enzyme have been revealed by the structures of RT that have been identified in complexes with substrates and/or inhibitors [32]. Two kinds of RTIs that target the viral enzyme with two separate modes of action are part of the approved combination therapies for HIV-1. The second class consists of substances known as nonnucleoside RT inhibitors (NNRTIs), while the first class consists of substances known as nucleoside/nucleotide RT inhibitors (NRTIs/NtRTIs). When supplied as prodrugs, NRTIs and NtRTIs can be integrated into viral DNA by RT after being taken up by cells and phosphorylated by the host cell enzymes. Since NRTIs do not include a 30 hydroxyl group, their inclusion prevents the synthesis of viral DNA (Figure 3).

Figure 3.

HIV reverse transcriptase with a short piece of DNA bound in the active sites. Source: [34].

The common NRTI resistance mutations cause resistance by two general mechanisms:

1. Mutations that reduce the incorporation of the NRTITP relative to the normal dNTPs.

2. Mutations that lead to a selective excision of the incorporated NRTIs by RT, unblocking the viral DNA. NNRTIs bind to RT and block the chemical step of DNA synthesis [31]. RT enzyme inhibitors such as NRTIs and NNRTIs block its enzymatic function and prevent the completion of synthesis of the double-stranded viral DNA, thus preventing HIV from proliferating [35].

6.3.1.2 Nucleoside RT inhibitors

There are currently eight NRTIs clinically available, structurally resembling both pyrimidine and purine analogues [33]. These agents, in order to inhibit reverse transcription, have to be phosphorylated by cellular kinases to their triphosphate derivatives. All NRTIs follow the same mechanism of RT inhibition: once activated to their triphosphate form, they are incorporated by RT into the growing primer (Figure 4), competing with the natural dNTPs and terminating DNA synthesis due to their lack of the 3-hydroxyl group (Figure 5). Therefore, once incorporated into dsDNA they prevent the incorporation of the incoming nucleotide. Importantly, while HIV-1 RT uses these NRTIs as substrates, the cellular DNA polymerases do not recognize them with the same affinity [33]. HIV-1 resistance to NRTIs usually involves two general mechanisms: NRTI discrimination, which reduces the NRTI incorporation rate, and NRTI excision which unblocks NRTI-terminated primer [33]. HIV-1 resistance to NRTIs usually involves two general pathways: NRTI discrimination, which reduces the NRTI incorporation rate, and NRTI excision which unblocks NRTI-terminated primers [33].

Figure 4.

Chemical structures and PubChem identification numbers of approved NRTIs: A: Stavudine (PubChem CID 18283), B: Lamivudine (PubChem CID 60825), C: Zalcitabine (PubChem CID 24066), D: Didanosine (PubChem CID 135398739), E: Abacavir (PubChem CID 441300) and F: Emtricitabine (PubChem CID 60877) respectively. Source: https://pubchem.ncbi.nlm.nih.gov.

Figure 5.

Chemical structures and PubChem identification numbers of new NRTIs acting as chain terminators; A: Nevirapine (PubChem CID 4463), B: Delavirdine (PubChem CID 5625), C: Efavirenz (PubChem CID 64139), D: Etravirine (PubChem CID 193962), E: Rilpivirine (PubChem CID 6451164), and F: Doravirine (PubChem CID 58460047) respectively. Source: https://pubchem.ncbi.nlm.nih.gov.

6.3.1.3 Nucleotide RT inhibitors (NtRTIs)

These are substances, such as [R]-9-[2phosphonylmethoxypropyl]-adenine (tenofovir, PMPA), that already have a phosphonate group that is resistant to hydrolysis. Therefore, the intracellular activation pathway is shortened, allowing for a more quick and thorough conversion to the active agent. They only require two phosphorylation steps to transform into their active diphosphate derivatives. Similarly to NRTIs, NtRTIs are phosphorylated to the corresponding diphosphates by cellular enzymes and serve as alternative substrates (competitive inhibitors); once incorporated into the growing viral DNA, they act as obligatory chain terminators. NtRTIs such as tenofovir are taken as prodrugs to facilitate penetration of target cell membranes. Subsequently, endogenous chemolytic enzymes release the original nucleoside monophosphate analogue that exerts its action [33].

6.3.1.4 Nonnucleoside RT inhibitors NNRTIs

Nonnucleoside RT inhibitors, or NNRTIs, are chemically and structurally unrelated substances that bind noncompetitively to a hydrophobic RT pocket near the polymerase active site. This causes the protein to deform and prevents the chemical process of polymerization [36]. When NNRTIs bind to RT, they modify the conformation of certain residues (Y181 and Y188) in the rotamer and stiffen the thumb region, which prevents DNA synthesis. It’s significant to note that NNRTIs, unlike NRTIs, do not depend on intracellular metabolism to function [33]. According to research in docking, molecular modeling, and crystallography, first-generation NNRTIs adopt a butterfly-like conformation [37]. The stabilization of the NNRTI binding in the allosteric site is accomplished through (i) stacking interactions between the NNRTIs aromatic rings and the side chains of Y181, Y188, W229, and Y318 residues in the RT lipophilic pocket; (ii) electrostatic forces (particularly significant for K101, K103, and E138 residues); (iii) van der Waals interactions with L100, V106, V179, Y181, G190, W229, L234, and Y318 residues; (iv) hydrogen bonds between NNRTI and the main chain (carbonyl/amino) peptide bonds of RT.

6.3.1.5 Protease inhibitors (PIs)

The majority of viruses contain one or more proteases that are essential to the progression of the virus/viral life cycle. In order for the viral proteins to function properly, release functional viral proteins and individually during replication, transcription, and maturation, the viral proteases perform the proteolysis of a polyprotein precursor [28]. Viral proteases can also modify host cell processes including ubiquitination and ISGylation to protect viral proteins in an efficient manner [38]. In contrast to the diversity of viral protease functions and structures, the catalytic active site of viral proteases generates stringent substrate specificity in protein cleavage. Synthetic substrate peptides, which can be designed according to the natural substrates of individual viral proteases, usually generate high-affinity binding, hence, provide potent candidates for further drug discovery. One of the great successes is the HIV-1 protease inhibitors (PIs). There are ten PIs currently approved to treat HIV-1 infection: amprenavir (APV), atazanavir (ATZ), darunavir (TMC114), fosamprenavir (Lexiva), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and tipranavir (TPV). The cross-resistance to PIs occurs in the active site of HIV-1 protease because all HIV-1 PIs have small identity chemical structures derived from their natural peptidic substrate. Because of this, PIs are frequently used in conjunction with other anti-HIV medications to prevent drug resistance. PIs are now the most effective antiviral medication kinds because the majority of their pharmacological drawbacks have been resolved [39].

6.3.1.6 Integrase inhibitors (INIs)

Integrase inhibitors (INIs) are the latest class of antiretroviral drugs approved for the treatment of HIV infection and are becoming ‘standard’ drugs in the treatment of both naïve as well as heavily pretreated individuals with HIV [40]. Integrase enzyme inhibition is an appealing therapeutic target for HIV-1 treatment due to the lack of a homolog in human cells and its critical involvement in HIV-1 replication [41]. INIs have the potential to reduce the likelihood of the virus adapting or becoming resistant due to their distinct mechanism of action, when administered alone or in conjunction with other classes of anti-HIV medications. Their structural complexity and mechanism of action suggest several therapeutic interventions, including interfering with integrase binding to viral cDNA ends, interfering with integrase oligomerization, interfering with strand transfer activity, interfering with 3-P activity, and interfering with IN-protein interactions with cellular cofactors [42].

INIs are classified in 10 scaffolds based on their structures including hydroxylated aromatics, diketo acids, naphthyridinecarboxamides, pyrrolloquinolones, dihydroxypyrimidinecarboxamides, azaindolehydrixamic acids, 2-hydroxyisoquinoline-1,3[2H,4H]-diones, 6,7-dihydroxy-1-oxoisoindolines, quinolone-3-carboxylic acids and carbamoyl pyridines [41]. INIs fall into one of two categories based on their mechanism of action: protein-protein interaction inhibitors (PPIIs) and integrase strand transfer inhibitors (INSTIs). INSTIs bind to Mg²⁺ ions and target the enzyme’s active site. Competitive inhibitors prevent 3′-end processing by engaging in direct competition with viral DNA for binding to integrase [43]. Significant progress has been made since integrase was identified as a potential therapeutic target, leading to the approval of three INIs, notably dolutegravir, elvitegravir, and raltegravir (Figure 6).

Figure 6.

Chemical structures and PubChem identification numbers of INIs approved for the treatment of HIV; A: Dolutegravir (PubChem CID 54726191), B: Elvitegravir (PubChem CID 5277135), and C: Raltegravir (PubChem CID 54671008) respectively. Source: https://pubchem.ncbi.nlm.nih.gov).

INSTIs are all IN inhibitors with FDA approval [42]. PPIIs work by preventing integrase, which is necessary for viral replication, from interacting with the host protein lens epithelial-derived growth factor (LEDGF/p75). Data indicate that LEDGF/p75 instructs integrase to insert viral DNA into transcriptionally active areas in the host genome, despite the fact that the exact mechanism of action is unknown. The inhibitors of this protein are already being created and patented. They are less likely to acquire resistance and are more likely to be highly target selective [44]. Integrase binding inhibitors (INBIs) like V-165, however, might also be categorized as INIs because of their capacity to interfere with integration while having no noticeable impact on viral DNA synthesis. Investigation into the mechanism of action revealed that V-165 prevents the development of viral DNA-IN complexes. Because of its interference activity, it is categorized as an IN binding inhibitor. Similar actions are shared by other substances, such as styrylquinolines, which compete with the LTR substrate for IN binding [45].

6.3.1.7 Polymerase inhibitors

Most viruses encode polymerases in the central steps of replication and transcription. Viral-encoded polymerases perform essential enzymatic steps through amplification- or transformation of the viral genome during the viral life cycle [46]. Therefore, viral polymerases are becoming the most attractive and suitable targets for antiviral design and development against many viral infections. Based on the function and structure of viral polymerases, there are two major types of polymerase inhibitors: (i) nucleoside and nucleotide substrate analogs and (ii) allosteric inhibitors. Nucleoside/nucleotide analogs (NAs) play a dominant role in antiviral drugs targeting viral polymerases [28], and were among the first polymerase inhibitors that showed clinical efficacy and are nowadays broadly used to treat hepatitis B-, herpes simplex- and HIV-1 infection [41] and constitute the typical backbone components of modern highly active antiretroviral treatment (HAART). The majority of the time, nucleoside analogs are created as pro-drugs that must undergo intracellular phosphorylation in order to create an analog of [deoxy-] nucleoside-triphosphate (NA-TP) that can be incorporated by the viral polymerase into nascent viral DNA. NA-TP can mimic adenosine, thymidine, guanine, cytosine, or uracil. Nucleoside analogs stop the polymerization process after inclusion because they lack the chemical group required to connect the subsequent incoming nucleotide [46]. The host cell first triphosphates nucleoside analogs to create the active inhibitor, which then competes with the natural nucleoside triphosphates to stop the viral nucleic acids from growing [22]. However, some viral polymerases have the ability to selectively remove or excise incorporated analog, saving the nascent viral DNA and causing a temporary rather than long-lasting method of inhibition. The balance between viral clearance by the immune system and viral replication can be tipped in favor of the immune system by inhibiting the critical step of viral DNA polymerization, which can decrease the likelihood by which circulating virus can successfully infect host cells and the number of viral progeny produced per unit of time [46].

One of the demerits of nucleoside analogs is that the initial phosphorylation step, (production of the monophosphorylated form), required for the activation of a triphosphate may not correctly take in the host cell [22]. Therefore, monophosphate nucleotide analogs were developed as polymerase inhibitors to avoid this problem. To date, most of the approved antiviral drugs for anti-HIV therapy utilize this mechanism, including Zidovudine (AZT, 30-azido-20,30-dideoxythymidine), Didanosine (ddi, 20,30-dideoxyinosine), Zalcitabine (ddC, 20,30-dideoxycytidine), Stavudine (d4T, 20,30-dideoxy-20,30-didehydrothymidine), Lamivudine (3TC, [−]-b-L-30-thia-20,30-dideoxycytidine), and others [22]. Secondly, the inhibition of DNA polymerization by the NAs is not restricted to viral polymerase, but can also affect cellular polymerases, leading to unwanted side effects [46]. The molecular kinetics of the relevant enzymes in relation to a specific inhibitor heavily influences the therapeutic window of NAs. Therefore, for NAs to have a therapeutic benefit, the targeted viral enzyme must be very specific. By altering the kinetics of the viral enzyme, viral resistance development can reverse this selectivity [46].

6.3.2 Prospect for new nucleoside RT inhibitors

There are various variables that restrict the therapeutic usage of NRTIs. First off, interactions between other NRTIs utilized in combination therapies, including the one between AZT and D4T, share the same phosphorylation route and exhibit a less-than-additive effect [33]. Second, interactions between drugs and other molecules, such as those that occur when a protease inhibitor is used in conjunction with the administration of ABC or tenofovir or when ABC is given in combination with ethanol. Thirdly, NRTIs have been connected to a number of negative side effects, including renal dysfunctions, drug hypersensitivity reactions, and mitochondrial toxicity (tied to myopathy, cardiomyopathy, anemia, and lipoatrophy) [47]. Fourthly, given the requirement for ongoing antiviral therapy, the choice of NRTI-resistant strains continues to be the key restriction. Particularly, it has been reported that 6–16% of patients with viremia have viruses resistant to at least one drug, which results in a worse response to therapy and a lower barrier to the selection of additional drug-resistant strains [47]. Additionally, it has been reported that almost 50% of viremic patients actually harbor M184V RT mutant strains. In light of this situation, it is sought that the new NRTIs under research have a favorable resistance profile, diminished side effects, and/or a unique mechanism of action.

7. Conclusion

The increase in knowledge regarding the HIV life cycle and its biochemistry has led to many insights in the field of antiviral design and development. The virus being smart and highly infidel has further grown the quest for more information on the need for effective and efficient antiviral drugs. This has led to the emergence of new strategies and targets against the virus and or the host cell that would ultimately culminate in the development of many novel antiviral agents in the nearest future.