Docking Study of HIV-1 Reverse Transcriptase (HIV-1 RT) with Well-Known Nucleoside Reverse Transcriptase Inhibitors (NRTIs)

The nucleic acid-binding cleft in HIV-1 reverse transcriptase (HIV-1 RT) is essential because of its interactions with the polymerase and RNase H active sites. Studying its binding sites for nucleoside reverse transcriptase inhibitors (NRTIs) is crucial for understanding how to effectively select drug compounds, properly administer drugs, and prevent drug resistance. Materials and Methods: We conducted docking studies using Molegro Visual Docker


INTRODUCTION
Reverse transcriptase inhibitors (RTIs) play a significant role in the treatment of HIV and, in some cases, hepatitis B. These crucial antiretroviral drugs primarily target reverse transcriptase, a key viral DNA polymerase responsible for the replication of HIV and other retroviruses.This class of drugs can be divided into four main forms: Nucleoside analog reverse transcriptase inhibitors (NARTIs or NRTIs), Nucleotide analog reverse transcriptase inhibitors (NtARTIs or NtRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), and Nucleoside reverse transcriptase translocation inhibitors (NRTTIs) 1 .

The HIV-1 Reverse Transcriptase (RT)
The reverse transcriptase (RT) of HIV-1 is a crucial enzyme in the virus's life cycle, responsible for converting the single-stranded RNA genome found in visions into double-stranded DNA.This DNA is then inserted into the host genome by the integrase (IN) enzyme.The RT enzyme is an asymmetric heterodimer composed of two related subunits, p66 and p51, which are derived from the cleavage of the Gag-Pol polyprotein by the viral protease (PR).The p66 subunit has 560 amino acids, and the p51 subunit has 440 amino acids, sharing a common amino terminus 2 .
The enzymatic functions of RT, DNA polymerase, and RNase H, are essential for the replication process.The larger p66 subunit contains the active sites for both enzymatic activities, while the smaller p51 subunit plays a structural role.The RT heterodimer consists of two distinct domains in p66: polymerase and RNase H.The polymerase domain comprises four subdomains, namely fingers, palm, thumb, and connection, which are also present in p51.However, the positions of these subdomains differ between p66 and p51, contributing to their distinct roles in the RT enzyme 3,4 .

The nucleic-acid binding cleft in HIV-1 RT
The nucleic-acid binding cleft in HIV-1 RT is primarily formed by the p66 subunit's fingers, palm, thumb, connection, and RNase H subdomains.The connection and thumb subdomains of p51 contribute to the floor of this cleft.The binding cleft accommodates the nucleic acid, interacting with both the polymerase and RNase H active sites, which are approximately 17-18 base pairs apart on the substrate.The αH and αI helices of the p66 thumb help position the nucleic acid by interacting with both the primer and template strands 2 .
The nucleic-acid binding cleft in HIV-1 reverse transcriptase (RT) is primarily formed by the p66 subunit's fingers, palm, thumb, connection, and RNase H subdomains.The connection and thumb subdomains of p51 contribute to the floor of this cleft.This binding cleft allows the nucleic acid to interact with both the polymerase and RNase H active sites, which are approximately 17-18 base pairs apart on the substrate.The αH and αI helices of the p66 thumb play a crucial role in positioning the nucleic acid correctly by interacting with both the primer and template strands .The polymerase active site, essential for DNA synthesis, is found in the p66 subunit and consists of three carboxylate residues (D110, D185, and D186) in the palm subdomain.These residues bind two divalent ions, typically magnesium ions in vivo, which are crucial for catalysis.The YXDD motif, highly conserved in retroviral RTs, includes the carboxylate residues D185 and D186.Other conserved residues contribute to the dNTP binding site, such as R72 and K65, which bind the β-and γ-phosphates of the incoming dNTP, and Y115, which helps bind the deoxyribose ring and acts as a steric gate that differentiates between deoxy and ribonucleoside triphosphates.Additionally, Q151 interacts directly with the 3'-OH of the incoming dNTP 2,5 .
The highly efficient dNTP binding process in HIV-1 RT, as observed in kinetic studies, is more efficient when using DNA/DNA substrates compared to RNA/DNA substrates.The rate of nucleotide incorporation (kpol) is also found to be higher when using RNA/DNA substrates.Transient kinetics experiments have identified the conformational change in the "fingers" subdomain of RT as the rate-limiting step in single nucleotide incorporation.Steady-state kinetic experiments suggest that the overall reaction is limited by RT's dissociation from the nucleic acid substrate.Nucleoside reverse transcriptase inhibitors (NRTIs), a class of antiretroviral drugs, target HIV-1 RT and function as chain terminators upon incorporation into viral DNA.They can form stable dead-end complexes with RT, potentially hindering viral replication progression.The chemical step of nucleotide incorporation requires two divalent metal ions, primarily magnesium ions, which facilitate the reaction by coordinating the incoming dNTP's phosphates and the three catalytic aspartate residues (D110, D185, and D186) in the polymerase active site.This coordination allows for the 3'-OH group's attack on the alphaphosphate of the incoming nucleotide and stabilizes reaction intermediates.Structural studies suggest that the YMDD loop in the polymerase active site may contribute to unfavorable interactions with the extended primer terminus, implying that translocation occurs after dNMP incorporation.The pyrophosphate product is likely released before the translocation of RT 2,[5][6][7] .

Nucleoside analog reverse transcriptase inhibitors (NARTIs or NRTIs)
NRTIs and NtRTIs work in a similar way, competing with natural deoxynucleotides for incorporation into the viral DNA chain.By doing so, they effectively cause chain termination, which halts viral DNA synthesis.This mechanism is crucial for preventing the virus from replicating and spreading within the host.However, NRTIs undergo further metabolism in the body, converting into NRTI triphosphates.These metabolites can lead to mitochondrial impairment, resulting in side effects such as lactic acidosis.Some well-known NRTIs include Zidovudine (Retrovir, AZT, or azidothymidine), the first FDA-approved antiretroviral drug used to treat HIV infections and prevent mother-to-child transmission of the virus.Another prominent NRTI is Didanosine (Videx and Videx EC), an adenosine analog also used for treating HIV 8 .
Zalcitabine (Hivid) is a discontinued dideoxycytidine drug once used for treating HIV but later withdrawn from the market due to side effects.Stavudine (Zerit and Zerit XR) is a d4T NRTI used in combination with other antiretroviral drugs for HIV treatment.Lamivudine (Zefix and Epivir) is another NRTI approved for treating both HIV and hepatitis B. This dual-purpose drug effectively inhibits the reverse transcriptase enzyme in both viruses, making it a valuable addition to antiviral therapy.Abacavir (Ziagen) is an analog of guanosine used to treat HIV infections, either alone or in combination with other antiretroviral drugs 8,9 .
Emtricitabine (Emtriva and FTC) is structurally similar to lamivudine and is approved for treating both HIV and hepatitis B. This NRTI is often used in combination with other antiretroviral drugs to enhance its effectiveness in suppressing viral replication.Entecavir (Baraclude) is a guanosine analog specifically designed for the treatment of hepatitis B. It works by inhibiting the reverse transcriptase enzyme, preventing the virus from replicating within the host's liver cells.Truvada is a combination of emtricitabine and tenofovir disoproxil fumarate, used to treat HIV infections and for pre-exposure prophylaxis (PrEP) to prevent HIV transmission in high-risk individuals.Azvudine (RO-0622) is another NRTI 8,9 .

NRTI Resistance in HIV
Resistance to NRTIs in HIV, which stands for Nucleoside Reverse Transcriptase Inhibitors, occurs through two primary mechanisms.The first mechanism involves the reduced incorporation of nucleotide analogs into DNA during the reverse transcription process.This reduction happens due to specific mutations in the Nterminal polymerase domain of the Reverse Transcriptase (RT) enzyme, responsible for converting viral RNA into DNA.These mutations can impact the enzyme's affinity or ability to bind to the drug, ultimately leading to resistance 10,11 .
For example, the M184V mutation is known to confer resistance to two NRTIs: lamivudine (3TC) and emtricitabine (FTC).These drugs are commonly used in combination therapies to treat HIV infection.As more mutations accumulate in the RT enzyme, the virus becomes less susceptible to the effects of NRTIs, potentially compromising the effectiveness of antiretroviral therapy [11][12][13] .
The second resistance mechanism involves the excision or hydrolytic removal of the incorporated drug.This process essentially reverses the polymerization reaction, allowing the DNA chain to be extended by releasing the triphosphate drug.This resistance development is driven by excision enhancement mutations, such as M41L, D67N, K70R, L210W, T215Y/F, and K219E/Q.These mutations are particularly selected by thymidine analogs like Zidovudine (AZT) and Stavudine (D4T), which are also NRTIs.Additionally, other mutations, including insertions and deletions, can contribute to NRTI resistance by enhancing the excision process.These resistance mechanisms can make HIV less susceptible to NRTIs, requiring the continuous development of new drugs and treatment strategies to effectively combat the virus.As the virus evolves and develops resistance to existing medications, healthcare professionals must stay informed about emerging therapies and adapt treatment plans accordingly to ensure the best possible outcomes for patients living with HIV [10][11][12][13] .

Reverse Transcriptase Model Preparation
The model of HIV-1 reverse transcriptase (RT) (6ASW) was obtained from the Protein Data Bank database at https://www.rcsb.org/structure/6asw.it.It depicts the structure of HIV-1 RT in a ternary complex with double-stranded DNA and an incoming d4TTP (2',3'-didehydro-2',3'-dideoxythymidine-5'-triphosphate) at pH 9.0.They aimed to understand the interaction between HIV-sensitive RT and d4TTP, one of the first chain-terminating nucleoside analogs used to treat HIV infection.The 6ASW structure was obtained using X-ray diffraction at a resolution of 2.60 Å, with R-values for free, work, and observed being 0.220, 0.179, and 0.180, respectively.The HIV-1 RT complex was expressed in Escherichia coli, and the structure contains mutations.For model preparation, we used Molegro Visual Docker software version 5.0 14,15 .

Predicting Physicochemical Properties and Pharmacokinetics
The SwissADME web tool is an essential resource for researchers involved in drug development.It provides a convenient platform to forecast key parameters such as physicochemical properties and pharmacokinetics.This valuable tool can be accessed without registration at http://www.swissadme.ch,making it easily accessible to a wide range of users.With an intuitive interface and reliable predictive models like BOILED-Egg and iLOGP, SwissADME ensures a seamless experience for users, enhancing their work in drug development [17][18][19][20][21] .

RESULTS
We present the ligand efficiency values (LE1) of studied compounds in their interaction with HIV-1 reverse transcriptase (RT).The highest ligand efficiency for zalcitabine (CID Number 24066) was observed to be -10.081kcal/mol, while the lowest LE1 for abacavir (CID Number 441300) was -7.460 kcal/mol.Abacavir has the highest interaction energy of -182.209kcal/mol and the lowest interaction energy is observed for Emtricitabine with -134.314kcal/mol.All the studied compounds interact with amino acids from both the p51 and p66 subunits of HIV-1 RT.The Additionally, the compounds bind to sites containing specific amino acids from both the p66 and p51 subunits.In the p66 subunit, these amino acids include Gln

Physicochemical and Pharmaceutical Properties of Studied Compounds
The molecular weight of the compounds studied ranges from 211 to 286 daltons, with 15 to 20 heavy atoms.Among these compounds, zalcitabine has the highest solubility in physiological fluids.They exhibit low hydrophobicity, with abacavir having the highest level at iLOGP=2.12.These compounds are highly absorbed in the gastrointestinal tract, do not penetrate the blood-brain barrier, and do not inhibit cytochrome P450 enzymes.Their skin penetration is minimal, except for abacavir, which is actively transported into cells by Pgp.The other compounds are not substrates for Pgp (refer to Table 3 and Table 4).

DISCUSSION
The compounds studied, including Zidovudine, Stavudine, Zalcitabine, Emtricitabine, Abacavir, Entecavir, and Didanosine, have molecular weights ranging from 211 to 286 daltons and exhibit low hydrophobicity.They are highly absorbed in the gastrointestinal tract, do not penetrate the blood-brain barrier, and do not inhibit cytochrome P450 enzymes.Abacavir is an exception, as it is actively transported into cells by Pgp.The compounds show varying degrees of skin penetration, solubility, and interactions with specific enzymes and transporters (refer to Table 3 and Table 4).We calculated the ligand efficiency values of various compounds that interact with the HIV-1 reverse transcriptase (RT) enzyme.Zalcitabine has the highest ligand efficiency at 10.081 kcal/mol, while Abacavir has the lowest LE at -7.4606 kcal/mol.The compounds interact with amino acids from both the p51 and p66 subunits of HIV-1 RT, and their binding sites involve specific amino acids from both subunits.The nucleicacid binding cleft in HIV-1 RT is primarily formed by the p66 subunit's fingers, palm, thumb, connection, and RNase H subdomains, with the p51 subunit's connection and thumb subdomains contributing to the floor of this cleft.
The polymerase active site, essential for DNA synthesis, is found in the p66 subunit and consists of three carboxylate residues that bind two divalent ions, typically magnesium ions in vivo, which are crucial for catalysis.The highly efficient dNTP binding process in HIV-1 RT is more efficient when using DNA/DNA substrates compared to RNA/DNA substrates.The chemical step of nucleotide incorporation requires two divalent metal ions, primarily magnesium ions, which facilitate the reaction by coordinating the incoming dNTP's phosphates and the three catalytic aspartate residues in the polymerase active site.Nucleoside analog reverse-transcriptase inhibitors (NRTIs) function by competing with natural deoxynucleotides for incorporation into the viral DNA chain, effectively causing chain termination and halting viral DNA synthesis.These inhibitors undergo further metabolism in the body, converting into NRTI triphosphates, which can lead to mitochondrial impairment and side effects such as lactic acidosis.NRTIs were the first class of antiretroviral drugs developed to combat HIV and, in some instances, hepatitis B. They operate by mimicking natural nucleosides, which are the building blocks of DNA 22,23 .Mutation of the residue remaining from the NRTI fusion occurs when it enters the DNA strand.As the virus replicates, mutations can occur in the reverse transcriptase active site or nearby regions.These mutations can alter the enzyme's structure, affecting the drug's ability to bind to the active site and reducing its effectiveness.Different NRTIs may result in distinct mutations.Enhanced phosphorolytic removal, also known as "primer unblocking," is a resistance mechanism that involves mutations increasing the removal of incorporated NRTIs from the viral DNA.This mechanism helps overcome chain termination.Thymidine analog mutations (TAMs) such as M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E are selected by thymidine analogs like zidovudine (ZDV) and stavudine (d4T), contributing to resistance against all NRTIs except lamivudine (3TC).The degree of crossresistance depends on the NRTI and the number of TAMs present [22][23][24] .
NRTI-associated resistance mutations can lead to multi-resistance, such as the Q151M mutation, which confers intermediate resistance to ZDV, ddI, d4T, and ABC.When combined with other mutations, Q151M can result in high-level resistance to these NRTIs and decreased susceptibility to tenofovir (TDF).Insertions at position 69, in combination with TAMs, can also cause high-level resistance to ZDV, d4T, ddI, ABC, and TDF.When NRTIs are used as monotherapy, resistance patterns can change significantly.For example, using 3TC or FTC with thymidine analogs like ZDV or d4T can delay the emergence of TAMs, preserving future treatment options.However, combining thymidine analogues with ddI increases the risk of cross-resistance.In the case of ABC/3TC, if virological failure occurs, the resistance profile may still preserve future options, as the impact on susceptibility is moderate for ABC and TDF when L74V is present alone or in combination with M184V.The lowest rate of mutation selection was observed when ABC/3TC was used with a boosted protease inhibitor regimen 22,25 .
Combining TDF with 3TC or FTC results in reduced susceptibility to TDF and most other NRTIs when the M184V/K65R profile is present.However, the impact of K65R on d4T susceptibility is not fully understood, and more data is needed to determine the resistance profile when TDF/FTC is combined with other antiretroviral agents.Understanding these mutational patterns and their interactions is crucial for effective HIV-1 treatment strategies and optimizing the selection of antiretroviral agents.Regular monitoring of treatment response and early intervention in case of virological failure can help preserve treatment options and prevent the development of drug-resistant viral strains.Triple-NRTI therapies like ABC/3TC/ZDV have shown a low rate of resistance mutations at failure, potentially safeguarding future treatment options.However, ABC/3TC/TDF has exhibited high rates of virological failure, with patients developing both M184V and K65R mutations.Other NRTI combinations, such as 3TC/ddI/TDF and CBV/TDF, have also demonstrated high rates of failure 26 .

Figure 2. The five binding sites with the highest binding affinity of the studied compounds for the reverse transcriptase enzyme model [PDB: 6ASW].
The combination of ZDV given twice daily with TDF once daily as an NRTI backbone may have an antagonistic mechanism of resistance, preventing the emergence of the K65R mutation.This combination should be further explored.To prevent the development of drug-resistant viral strains and maintain treatment options, it is crucial to promptly intervene in cases of treatment failure and monitor the response to antiretroviral therapy.Further research is needed to understand the mutational pathways associated with different NRTI-containing regimens and their implications for future treatment strategies.In clinical practice, it is recommended to use 3TC or FTC in combination with thymidine analogues to avoid ongoing viral replication on NRTI-based regimens.The 215Y TAM pathway should be considered, as it confers higherlevel resistance to NRTIs, including ZDV.Maintaining 3TC in a failing regimen is advisable, as it retains partial activity despite the presence of resistance mutations [22][23][24][25][26][27] p51 subunit involves amino acids Trp 88, Glu 89, Gly 93, Ile 94, Lys 154, Pro 157, Ala 158, Tyr 181, Gln 182, Tyr 183, and Met 184, while the p66 subunit involves amino acids Val 90, Gln 91, Leu 92, Gln 161, Ser 162, Thr 165, and Gln 182.

Figure 1 .
Figure 1.The sequence and secondary structure of the p51 (6ASW[A]) and p66 (6ASW[B]) subunits of the reverse transcriptase enzyme in molecular model [PDB: 6ASW].Each amino acid is represented by a different letter and color.The secondary structure is depicted as continuous lines above the amino acid sequence, with red indicating alpha helix and blue indicating beta sheet.

Figure 3 .
Figure 3. Egg-plot of the studied compounds.The yellow areas represent compounds that passively cross the blood-brain barrier, white areas indicate passive absorption in the digestive system, blue dots show entry through P-glycoproteins, and red dots signify compounds that are removed via glycoproteins in the central nervous system.

ISSN: 2357-0547 (Print) Research Article / JAPR / Sec. D ISSN: 2357-0539 (Online) Yousefi, 2024, 8 (3), 176-187 and
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