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HIV and effectiveness of inhibitor cocktail over single inhibitor

HIV and effectiveness of inhibitor cocktail over single inhibitor


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I'm looking for clarification on the answer to this question. It's in my biochemistry class but I figured this is more Biology than Chemistry, so I'm asking it here.

The question is:

One of the more effective treatments for HIV-positive individuals has been for them to take protease inhibitor cocktails. Certain proteases are required for the virus to mature and these inhibitors prevent them from functioning. A cocktail of inhibits means that numerous inhibitors are used at once. Why would a cocktail be more effective than a single inhibitor?

My answer to this is that there could be more than one mutation of the virus, and with each mutation a variation in specific protease. So, the protease cocktail would defend against a wider range of the mutated virus.

Is this correct? Is there an answer that might involve describing epitopes?


Your answer is correct. HIV-1 encodes a single homodimeric aspartic protease, with each monomer containing the classic Asp-Thr-Gly motif, and the dimer's active site being formed with the two monomeric active sites creating a cleft where the proteolysis takes place. In it, water acts as a nucleophile in conjunction with the aspartic acid residue to hydrolyze the peptide bond in the protein's target.

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WikiMedia: HIV-replication-cycle.svg

Protease inhibitors act by "sticking" in the binding cleft, obscuring the aspartate and preventing binding of the target proteins. However, these small-molecule inhibitors are very specific to HIV-1 and the amino acid residues that compose the binding cleft, otherwise they could potentially inhibit one or more of the many aspartyl proteases our body makes naturally. While it is not very likely that a destructive mutation in the Asp-Thr-Gly motif would result in a replication-competent virus, other more conservative mutations may occur in the binding cleft that still allow the target to bind and be cleaved. However, depending on the exact protease inhibitor being used, a single mutation, even if it's conservative, may still be enough to dramatically decrease the inhibitor's binding efficiency and allow protease to maintain some or all of its functional capacity. This is why cocktails of inhibitors are used: they each depend on different amino acids for their binding, so if mutations arise at some point that do affect the activity of one inhibitor, others may still be unaffected. Evolutionarily-speaking, the inhibitors put a tremendous selection pressure on the virus, which combined with HIV-1's naturally "sloppy" replication process, leads to mutants appearing in relatively short periods of time.

There are two kinds of epitopes in the adaptive immune system: those recognized by antibodies and B cells, and those recognized by T cells when presented in the context of MHC. Antibody epitopes are generally found on the surface of a pathogen or a pathogen-infected cell (when speaking in the context of infectious diseases), as proteins or other compounds capable of raising an immune response that are only located completely inside of the pathogen or infected cell are not available for binding. T cell epitopes, on the other hand, are linear peptide fragments (and sometimes other molecules, like glycolipids) generated by internal processing in the antigen-presenting cell, and are usually pretty representative of the complete contents of the cell, native and foreign. Native epitopes generally don't produce immune responses - when they do, autoimmunity occurs. Foreign epitopes are recognized by circulating T cells, and help prime an immune response against the infected cells. (As a side note, since HIV-1 infects a subset of T cells, it is a way for them to escape immune detection). Mutations in the protease protein may affect some of the epitopes it produces, but those mutations are just as likely to increase visibility to the immune system as decrease it, so overall there's no net gain or loss.


According to wikipedia:

HIV protease inhibitors are peptide-like chemicals that competitively inhibit the action of the virus aspartyl protease. These drugs prevent proteolytic cleavage of HIV Gag and Pol polyproteins that include essential structural and enzymatic components of the virus. This prevents the conversion of HIV particles into their mature infectious form.

Competitive inhibition means that it just slows down the conversion, but it does not stop it. In the case of building a virus we are talking about a multi step process. By using multiple inhibitors we slow down multiple steps in this process. So it is much more effective than just using a single inhibitor for slowing down only a single step in the process…


How Do Protease Inhibitors Work?

Protease inhibitors are antiviral drugs. They interrupt the way HIV uses a healthy cell to make more virus. When HIV enters a healthy cell, its only goal is to make more viruses to infect other healthy cells. It does this by making the cell produce certain proteins the virus can use to copy itself. Two of the proteins used by the virus are reverse transcriptase and protease. The goal of the protease inhibitor is to stop the protease from helping to assemble a new virus.

The diagram above shows the virus entering the cell (1), the cell making new proteins (2-3), the proteins forming a new virus (4) and the cell releasing the new virus to infect other cells (5). It also shows some steps in the process that can be interrupted by protease inhibitors and other antiviral drugs (reverse transcriptase inhibitors) that are taken along with protease inhibitors.

What can protease inhibitors do?

Protease inhibitors are the most powerful anti-HIV drugs available so far. Although many different factors affect how well any drug will work for an individual, some people who have taken protease inhibitors have had the following benefits:

increase in CD4 (t cell) counts, which can help fight infections

decrease in the amount of virus in the blood (viral load), which may slow down the disease process

feeling of improved overall health and ability to do more of their usual activities (ie: work, travel, socialize)

Researchers are not sure how long protease inhibitors will work in a person infected with HIV, but they have seen promising results in studies. They are hopeful that people will live longer, healthier lives because of the benefits of these new drugs.

Is this a cure?

Protease inhibitors are not called a cure because researchers do not yet know how well they will work in different people. Some people have had their viral load drop to a level that is too low for current tests to measure. Even though the virus cannot be found in their blood, doctors believe HIV is still in their bodies and that it would reproduce quickly if they stop taking the protease inhibitor. In other people, protease inhibitors may not work as well or the benefits might not last. Clinical trials are going on to help answer questions about where HIV "hides" and why people have different results with protease inhibitors.

Which protease inhibitor will I take?

There are five approved protease inhibitors: ritonavir (Norvir) and nelfinavir (Viracept) are for use by adults and children, while indinavir (Crixivan) and the two forms of saquinavir (Invirase and Fortovase) are approved for adults only. Invirase was approved in 1995, and Fortovase, a new, stronger form of saquinavir was approved in 1997. The company that makes both drugs will continue to make Invirase available to people who already take that form of saquinavir through Spring 1998. After that, it will be available to them under a limited distribution program. A decision about which drug to take should be made with a doctor who knows your individual condition, and has medical knowledge of HIV disease. By finding out about the available treatment options, you can talk to your doctor about the risks and benefits of different drug combinations.


Warning Signs Over Effectiveness of HIV “Wonder Drug” in Sub-Saharan Africa Due to Genetic Mutations

Dolutegravir, the current first-line treatment for HIV, may not be as effective as hoped in sub-Saharan Africa, suggests new research published on World AIDS Day. The study finds that this so-called ‘wonder drug’ may be less effective in patients resistant to older drugs.

As HIV copies itself and replicates, it can develop errors, or ‘mutations’, in its genetic code (its RNA). While a drug may initially be able to suppress or even kill the virus, certain mutations can allow the virus to develop resistance to its effects. If a mutated strain begins to spread within a population, it can mean once-effective drugs are no longer able to treat people.

HIV treatment usually consists of a cocktail of drugs that includes a type of drug known as a non-nucleoside reverse-transcriptase inhibitor (NNRTI). However, in recent years, HIV has begun to develop resistance to NNRTIs. Between 10% and 15% of patients in much of sub-Saharan Africa are infected by a strain of HIV resistant to these drugs. If a patient is infected with an NNRTI-resistant strain, they are at a two- to three-fold increased risk of the drug regimen failing.

In 2019, the World Health Organization began to recommend dolutegravir as the preferred first-line treatment for HIV in most populations. Dolutegravir was dubbed a ‘wonder drug’ because it was safe, potent, and cost-effective and scientists had seen no drug resistance against it in clinical trials. However, there is little data on the success of dolutegravir against circulating strains of HIV in sub-Saharan Africa.

In a study published today (December 1, 2020) in Nature Communications, an international team of researchers from South Africa, the UK and the USA examined the genetic code of HIV to determine if drug resistance mutations in 874 volunteers living with HIV affected their treatment success. The individuals were enrolled in a clinical trial for people initiating HIV treatment to compare two drug regimens: efavirenz, an NNRTI and prior first-line therapy in the region, and dolutegravir.

The goal of this study was to determine whether drug resistance to efavirenz prior to starting treatment affected treatment success (suppression of the virus in the blood) over the first two years of therapy with both of these two regimens.

As expected, the presence of drug resistance substantially reduced the chances of treatment success in people taking efavirenz, successfully suppressing the virus over 96-weeks in 65% of participants compared to 85% of non-resistant individuals. However, unexpectedly, the same pattern was true for individuals taking dolutegravir-based treatments: 66% of those with efavirenz resistance mutations remained suppressed over 96-weeeks compared to 84% of those without the mutations. These relationships held true after accounting for other factors, such as treatment adherence.

“We fully expected efavirenz to be less effective among patients HIV strains resistant to NNRTIs,” said Dr. Mark Siedner, faculty member at the Africa Health Research Institute in KwaZulu-Natal, South Africa and Massachusetts General Hospital in Boston, Massachusetts. “What took us completely by surprise was that dolutegravir — a different class of drug which is generally effective in the face of drug resistance — would also be less effective in people with these resistant strains.

“We are working now to tease out if this was due to the virus or the participants — for instance, if people with resistance are less likely to take their pills regularly. Either way, if this pattern holds true, it could have far reaching impacts on our predictions of long-term treatment control for millions of people taking dolutegravir in the region.”

Professor Ravi Gupta from the Department of Medicine at the University of Cambridge said: “This a huge concern. Dolutegravir was very much seen as a ‘wonder drug’, but our study suggests it might not be as effective in a significant number of patients who are resistant to another important class of antiretroviral drugs.”

The researchers say it is not clear why efavirenz-resistant mutations should affect susceptibility of dolutegravir, though one hypothesis is that integrase inhibitors such as dolutegravir push the virus to replicate and mutate faster, in turn developing resistance to the new drug in an evolutionary arms race. Alternatively, it could be due to poor adherence to treatment regimens, even though the analysis accounted for adherence by two independent methods. Further research is needed to find out why.

Professor Gupta added: “What this shows is that we urgently need to prioritize point of care tests to identify people with drug resistance HIV, particularly against efavirenz, and to more closely and accurately monitor treatment adherence. The development of such tests is at an advanced stage, but there a lack of investment from funders and philanthropic donors. We urgently need agencies and individuals to step forward and help support these programs.

“In addition, we need to provide widespread access to viral load monitoring so that we can find those who are struggling, get them on more appropriate regimens, and limit the emergence of resistance when patients are failing therapy.”

Reference: “Reduced efficacy of HIV-1 integrase inhibitors in patients with drug resistance mutations in reverse transcriptase” by Mark J. Siedner, Michelle A. Moorhouse, Bryony Simmons, Tulio de Oliveira, Richard Lessells, Jennifer Giandhari, Stephen A. Kemp, Benjamin Chimukangara, Godspower Akpomiemie, Celicia M. Serenata, Willem D. F. Venter, Andrew Hill and Ravindra K. Gupta, 1 December 2020, Nature Communications.
DOI: 10.1038/s41467-020-19801-x

The study was carried out by researchers at: the Africa Health Research Institute, University of KwaZulu-Natal, University of Witwatersrand, KwaZulu-Natal Research Innovation and Sequencing Platform, and the Centre for the AIDS Programme of Research in South Africa (CAPRISA), in South Africa the University of Cambridge, University of Liverpool, and Imperial College London in the UK and Massachusetts General Hospital and Harvard Medical School, USA.

The research was supported by USAID, Unitaid, the South African Medical Research Council (SAMRC), with investigational drug donated by ViiV Healthcare and Gilead Sciences, and by Wellcome and the National Institutes of Health.


What types of HIV medications are there?

Treatment for HIV involves taking medication that reduces the amount of the virus in the body. This is called antiretroviral therapy. Two other options, PEP and PrEP, can prevent HIV.

HIV is a type of virus called a retrovirus. In a person with HIV, antiretroviral therapy reduces the amount of the virus in the body to very low levels. When levels are so low that doctors consider them undetectable, the virus can no longer damage the body or transmit to others.

The Centers for Disease Control and Protection (CDC) recommend consistent treatment with antiretroviral therapy for everyone with HIV, regardless of how long they have had it or their current state of health.

To date, the Food and Drug Administration (FDA) have approved more than 20 medications to treat HIV.

Compared with earlier drugs, modern drugs used in antiretroviral therapy are more potent, less toxic, and easier to take as directed. They also produce fewer and less severe side effects.

This article describes the medications that the FDA have approved for treating and preventing HIV, along with their possible side effects. We also look at how healthcare providers choose an appropriate HIV regimen.

The aim of antiretroviral therapy is to reduce a person’s viral load, or the amount of the virus in the blood, to an undetectable level. If the viral load is decreasing, this indicates that the treatment is working.

Undetectable amounts of the virus cannot damage the immune system or pass on to others. To keep HIV levels undetectable, it is crucial to take medications consistently as prescribed and attend regular checkups.

Various classes of antiretroviral drugs target HIV at different stages of its life cycle — the stages at which it replicates and spreads in the body.

Below, learn about the different types of antiretroviral drug that currently have approval from the FDA.

Nucleoside reverse transcriptase inhibitors (NRTIs) prevent HIV from replicating by blocking an enzyme called reverse transcriptase. This reduces the viral load of HIV in a person’s body.


Materials and methods

Protease gene construction

The 3X-protease (L63P, V82T, I84V) gene was constructed using standard site-directed mutagenesis of a synthetic protease variant. The N-terminal encoding sequence of HXB2 was replaced by a synthetic gene sequence having the (neutral) polymorphisms V3I, K14R, and S37N. The protease variant also included an additional substitution of Q7K to prevent autoproteolysis (Rose et al. 1993).

Protease expression and purification

The gene encoding HIV protease was cloned into the plasmid pXC34 (ATCC), which contains a λ PL promoter (Cheng and Patterson 1992). The protease was expressed by heat induction in Escherichia coli TAP 106 cells using this plasmid. Cells from 12 L of fermentation were lysed and the protein was purified from inclusion bodies (Hui et al. 1993). The inclusion body centrifugation pellet was dissolved in 50% acetic acid followed by another round of centrifugation to remove impurities. Size exclusion chromatography was used to separate high molecular weight proteins from the desired protease. This was carried out on a 2.1-L Sephadex G-75 superfine (Sigma Chemical) column equilibrated with 50% acetic acid. The protein was refolded in 10 mM formic acid (Todd et al. 1998). A final purification was performed with a Pharmacia Superdex 75 FPLC column equilibrated with 0.05 M sodium acetate at pH 5.5, 5% ethylene glycol, 10% glycerol, and 5 mM DTT.

Synthesis of inhibitors

The synthesis and preparation of the protease inhibitors used in this study are shown in Figure 1b ▶ . The penultimate piperazines (compound I in Fig. 1b ▶ ) were prepared according to the method published in US patent 5,436,067, assigned to Merck & Co., Inc. T-amylamine was substituted for t-butylamine in the preparation of the piperazine amide for synthesis of the precursor to XN1336�. In the cases of the precursors to XN1336� and XN1336�, (S)-3-phenylbutyric acid (purchased from Fluka) replaced hydrocinnamic acid for the preparation of compound I (Fig. 1b ▶ ). Preparation of the final products was accomplished by reductive alkylation of compound I by either pyridine-3-carboxaldehyde (XN1336�, XN1336�) or piperonal (807�𠄴, XN1336�), in the presence of sodium triacetoxyborohydride, as described (Abdel-Magid et al. 1996). The final products were purified by preparative thin layer chromatography using silica plates and 10% methanol in methylene chloride as the eluent. Structures and purities were confirmed by proton and carbon NMR and mass spectroscopy, with no evidence of hydration.

Crystallization and data collection

Crystals were set up using a three- to fivefold molar excess of inhibitor to protease, which ensures ubiquitous binding. The final concentration of protease was approximately 2 mg/mL in 0.05 M sodium acetate at pH 5.5, 5% ethylene glycol, 10% glycerol, 5 mM DTT. Equal volumes of the inhibitor–protein mixture and the reservoir solution were combined to set up hanging drops of 5 μL. The reservoir solution consisted of 126 mM phosphate buffer at pH 6.2, 63 mM sodium citrate, and ammonium sulfate in a range of 27�% (Silva et al. 1996). Crystals were grown at ambient temperature and were evident within 24� hours. The data collection took place at room temperature on an R-AXIS-IV imaging plate system. The data were reduced and scaled using the programs DENZO and SCALEPACK (Otwinowski 1993), respectively. Crystals of both the indinavir and the indinavir analog protease complexes were of the P212121 space group, with one dimer per asymmetric unit.

Refinement

The program CNS [Crystallography and NMR System (Brunger et al. 1998)] was used to refine the structures. The wild-type HIV-1 protease indinavir complex (1HSG) (Chen et al. 1994) served as a model for solving the 3X-protease indinavir structure by molecular replacement. The 3X-protease indinavir complex was, in turn, used as a model for refining the corresponding 3X-protease XN1336� structure. The model used for refinement of the subsequent indinavir analog 3X-protease 807�𠄴 complex was a high-resolution, well-refined substrate–protease structure recently solved in our laboratory (M. Prabu-Jeyabalan, E. Nalivaika, and C.A. Schiffer, unpubl.), having the same P212121 space group. The structure for 3X-protease 807�𠄴 complex was then used as the model to refine the structure of the 3X-protease XN1336� complex. For each structure, an initial rigid body refinement was performed at 4Å resolution with difference Fourier electron density maps (Fo − Fc and 2Fo − Fc) subsequently computed. The program CHAIN (Sack 1988) was used for model building. Positional and B-factor refinements were carried out, and the difference Fourier electron density map (Fo − Fc) unambiguously revealed the positions of the inhibitors. The resolution limits were gradually extended in equal steps to accomplish the highest possible resolution. Solvent molecules were added manually at positions indicated by the electron density. For cross-validation, R-free values were monitored and simulated annealed omit maps were used to decrease model bias. The stereochemical parameters of the final structures were analyzed using the programs PROCHECK (Laskowski et al. 1993) and WHATIF (Vriend 1990 Hooft et al. 1996). There were no outliers in the Ramachandran maps. The crystallographic and refinement statistics are shown in Table 1 ​ 1. . Complexes were superimposed using the α-carbon backbone of the relatively immobile (Rose et al. 1998) terminal domain of all these structures (residues 1𠄹 and 86�) with the program MIDAS (Ferrin et al. 1988). The coordinates have been deposited in the protein databank, 1K6C, 1K6P, 1K6T, and 1K6V for the 3X-protease indinavir, 807�𠄴, XN 1336� and XN1336� complexes, respectively.

Protease inhibition assays

Inhibition of the triple-mutant protease by the various inhibitors was determined. Activity assays were performed at 37ଌ with the substrate AcSQNYPVV-NH2 (Sigma). Constant concentrations of protease, 0.05 mg/mL, and substrate, 492 μM, were used for all experiments. Inhibitor concentrations were varied from 0.0𠄰.80 μM. For each concentration of inhibitor, aliquots of the reaction mixture were removed at 15-min intervals, quenched with equal volumes of cold trichloroacetic acid, and assayed by reverse phase HPLC (Moore et al. 1989). The percent of activity remaining for each inhibitor concentration was plotted and IC50s were obtained (Fig. 4 ▶ ).

Thermodynamic binding assays

An isothermal titration calorimeter, a VP-ITC (MicroCal Inc.), was used to measure binding energies of inhibitors. Twenty to twenty-four 10�-μl injections of 0.2 mM inhibitor were made into 29 μM wild-type HIV-1 protease and 22.7 μM 3X-protease HIV-1 protease at 20ଌ. The buffer in which both proteins and the inhibitor were suspended was 10 mM sodium acetate, 2.0% DMSO, and 2mM TCEP at pH 5.0. Heats of dilution were subtracted from the corresponding heats of reaction to obtain the heat due solely to the binding of the ligand to the enzyme. Data was processed and analyzed using the MicroCal Origin software package.


Key Points

So far, inhibitors against HIV-1 have been designed to antagonize the viral reverse transcriptase and protease enzymes. However, there are concerns about both the long-term effects of the protease inhibitors and the ability of HIV-1 to evolve resistance to these drugs.

New attempts to block HIV-1 infection have diversifed to consider many steps in the viral life cycle of HIV-1 that are crucial to infection. These include virus?cell attachment, virus entry and virus uncoating. The reverse transcription of viral cDNA, nuclear import and integration into the host cell's genome are also potential sites of inhibition.

Antagonists of viral entry are now in, or approaching, human clinical trials these inhibitors are directed against both the viral glycoproteins that interact with receptors and co-receptors on the host cell membrane. The design of post-entry inhibitors remains problematic the more advanced inhibitors include agonists of the integrase enzyme, which mediates viral cDNA integration into the host cell's genome.

Design of new viral-entry inhibitors also considers the escape pathways adopted by the evolving HIV-1 virus in response to inhibition of its normal entry route. It is predicted that the most successful therapeutic approach will be a ?cocktail? of inhibitors, which block infection at several points, including the potential escape pathways.


Abstract

The potent new antiviral inhibitor TMC-114 (UIC-94017) of HIV-1 protease (PR) has been studied with three PR variants containing single mutations D30N, I50V, and L90M, which provide resistance to the major clinical inhibitors. The inhibition constants (Ki) of TMC-114 for mutants PRD30N, PRI50V, and PRL90M were 30-, 9-, and 0.14-fold, respectively, relative to wild-type PR. The molecular basis for the inhibition was analyzed using high-resolution (1.22−1.45 Å) crystal structures of PR mutant complexes with TMC-114. In PRD30N, the inhibitor has a water-mediated interaction with the side chain of Asn30 rather than the direct interaction observed in PR, which is consistent with the relative inhibition. Similarly, in PRI50V the inhibitor loses favorable hydrophobic interactions with the side chain of Val50. TMC-114 has additional van der Waals contacts in PRL90M structure compared to the PR structure, leading to a tighter binding of the inhibitor. The observed changes in PR structure and activity are discussed in relation to the potential for development of resistant mutants on exposure to TMC-114.

Department of Biology, Molecular Basis of Disease, Georgia State University.

Department of Chemistry, Molecular Basis of Disease, Georgia State University.

Department of Computer Science, Georgia State University.

Corresponding author. Phone: 404-651-0098. Fax: 404-651-2509. E-mail: [email protected]

Abbreviations: PR, protease HIV-1, human immunodeficiency virus type 1 HAART, highly active antiretroviral therapy AIDS, acquired immunodeficiency syndrome PI, protease inhibitor THF, tetrahydrofuran.


Results and Discussion

Previously, Eckert et al. (8) used mirror-image phage display to discover a first generation of D-peptides that bind specifically to the hydrophobic pocket of the gp41 N-trimer and inhibit HIV-1 entry (IC50 = 11–270 μM, HXB2 strain). Briefly, in mirror-image phage display (31), the desired natural target is made synthetically with D-amino acids and is used to screen for binding of L-peptides displayed on phage. By symmetry, D-peptide versions of the phage peptides will bind to the natural L-target. This phage library contained 10 randomized residues (10-mer library) flanked by cysteines (CX10C). Because of the vast possible sequence diversity of this library, only one in ≈3 × 10 6 possible sequences was screened, and we therefore reasoned that more potent D-peptide inhibitors likely remained to be discovered.

Importantly, a consensus sequence ( C X5 EW X WLC ) was identified from the original phage screen that allowed us to develop a constrained library in which the consensus residues (underlined) were fixed while the other six positions were randomized. This constraint allowed us to construct a comprehensive library that comprised all possible sequences. As expected, phage display screening of this library identified a family of D-peptides with improved average potency over the original D-peptides (≈4-fold data not shown). Surprisingly, one of the most potent D-peptides identified (2K-PIE1) was an 8-mer (i.e., missing two of the randomized residues, CX3EWXWLC). This phage clone (PIE1-ϕ) was not intentionally part of the library and likely arose from a very rare replication error. The selection of this sequence despite its very low prevalence in the initial library suggested that the 8-mer family might be a richer source of tight binders than the 10-mers.

Crystal Structure of the IQN17:2K-PIE1 Complex.

To more fully understand the interaction of 2K-PIE1 with its target we determined the crystal structure of its complex with the gp41 N-trimer pocket mimic IQN17 (8) (Fig. 2). The structure was solved at 1.7 Å by molecular replacement and contains two IQN17 subunits and two 2K-PIE1 inhibitors in the asymmetric unit. A crystallographic threefold axis generates two trimers from the two independent subunit–inhibitor complexes [see supporting information (SI) Table 3 and SI Text for a description of data collection and refinement statistics]. Electron density clearly shows a number of important features of the inhibitor, including the main pocket-binding residues (dTrp10, dTrp12, and dLeu13) and the disulfide bond between dCys5 and dCys14 (Fig. 2 B).

Structural analysis of the IQN17:2K-PIE1 inhibitor complex. (A) IQN17, consisting of IQ (orange) and gp41 (N17, gray) segments, with inhibitors (green, yellow, and purple) located in the canonical gp41 binding pockets. The purple inhibitor is mostly occluded in this view. (B) Omit map for 2K-PIE1 contoured at 3.0 × rmsd. Five of the eight pocket residues (gray, HXB2 numbering) that make hydrophobic contacts with 2K-PIE1 (green) are shown. Two hydrogen bonds (black) at the binding interface are also shown. (C) Overlay of D10-p1 (slate) and 2K-PIE1 (green) superposed by alignment of the IQN17 trimers. Intramolecular disulfide bonds (solid yellow) are also shown. (D) A slab view through the center of 2K-PIE1 (green) reveals an intact hydrophobic core (black) that excludes water. (E) A similar view of D10-p1 (slate) reveals the presence of several water molecules (red) in its core that nearly form a water channel. (F) End-on view of the complex (same color scheme as A) in which the surface from the last three residues of IQN17 have been removed. This view illustrates the packing of the inhibitor into the deep hydrophobic pocket. dK2 (blue), equivalent to the N-terminal Lys in PIE7 used for cross-linking, is highlighted.

Comparison of our 2K-PIE1 and the previously reported D10-p1 (8) structure, both of which were determined in complex with IQN17, reveals a striking similarity in the pocket-binding interface (Fig. 2 C). The inhibitors' pocket-binding residues dTrp10, dTrp12, and dLeu13 (2K-PIE1 numbering), which contribute ≈60% of the binding surface, are nearly superposable (Fig. 2 C). The essentially identical binding interfaces and buried solvent-accessible surface areas (475 Å 2 for 2K-PIE1 vs. 469 Å 2 for D10-p1) are surprising in light of 2K-PIE1's significantly improved potency over D10-p1 and suggest that binding of these inhibitors depends significantly on factors remote from the direct contact surface.

Overall, the comparison suggests that the improved potency and binding (see below) of 2K-PIE1 is a consequence of its reduced size (10-mer to 8-mer), which creates a more compact D-peptide with better packing while maintaining the pocket-binding interface. One major difference between the inhibitors is the path of the backbone distal to the pocket interface (Fig. 2 C). dPro8 in 2K-PIE1 appears to facilitate the turn required for circularization, possibly allowing other residues to adopt more relaxed conformations. In support of this idea, a Pro in this position appears to be a better solution for 8-mers than other residues (see below). The more compact structure of 2K-PIE1 vs. D10-p1 (volume is 1,556 vs. 1,858 Å 3 , excluding N-terminal Lys residues) allows it to form a better-packed hydrophobic core (Fig. 2 D and E) that excludes the water molecules seen in the core of D10-p1 (Fig. 2 E).

Phage Display of an 8-mer Library.

The surprising emergence of 2K-PIE1 from a 10-mer library and its apparent structural advantages motivated us to perform a dedicated screen of 8-mer sequences. We generated a comprehensive 1.5 × 10 8 member 8-mer phage library of the form CX4WXWLC (3.4 × 10 7 possible sequences). Our mirror-image target was the second-generation trimeric pocket mimic IZN17 (12).

For this screen, we used solution-phase phage display (32) combined with a soluble competitor to increase selection pressure (see SI Text for additional details). Several sequences were identified after six rounds of phage display and characterized in a phage clone binding assay (SI Fig. 5).

Potency of D-Peptides Against HXB2 Entry.

D-peptide versions of the best phage clones (PIE2, PIE7, and PIE8-ϕ) were synthesized and tested against the standard HIV-1 laboratory strain HXB2 in a single-cycle viral infectivity assay (Table 1 and Fig. 3 A). As expected from the phage binding data, PIE7 is the most potent inhibitor (IC50 = 620 nM) and is ≈15-fold more potent than the best first-generation D-peptide (D10-p5).

D-peptide binding and neutralization

Representative viral entry inhibition data. Each point represents the average of quadruplicate measurements normalized to uninhibited control. Error bars represent the SEM. (A) IC50 curves for various inhibitors against HXB2. (B) IC50 curves for PIE7 and (PIE7)3 against JRFL, BaL, and HXB2.

The importance of optimizing residues that do not directly contact the pocket is highlighted by several pairwise comparisons (using 2K-PIE1 numbering) between peptides in Table 1 and SI Fig. 5. For example, PIE7 differs from PIE2 only at residue 11, for which Gln is preferred over Arg. Similarly, PIE7 differs from PIE8 only at residue 8, where Pro is preferred.

It was previously noted that introduction of Lys residues at the N terminus of D-peptides, required for solubility, adversely affects potency (8). 2K-PIE2 is ≈2-fold less potent than PIE2 (Table 1). Because 1K versions of our second-generation D-peptides have good solubility and improved potency, we decided to make 1K the standard N terminus of our second-generation D-peptides (all second-generation peptides have a single N-terminal Lys unless otherwise labeled).

Crystal Structure of the IQN17:PIE7 Complex.

In an attempt to understand the source of PIE7's improved affinity compared with 2K-PIE1, we determined two independent crystal structures of PIE7 in complex with IQN17 at 2.0-Å and 1.66-Å resolution (see SI Table 3 and SI Text for a description of data collection and refinement statistics). A comparison of 2K-PIE1 and PIE7 reveals several interesting differences (Fig. 4). First, an intramolecular polar contact between the hydroxyl of dSer7 and the carbonyl of dGly3 in 2K-PIE1 is lost in PIE7 but is replaced with a new interaction between the side chain carboxylate of dAsp6 and the amide of dGly3. Second, new hydrophobic interactions are created in PIE7 between the ring carbons of dTyr7 and the pocket residue Trp-571 (SI Fig. 6A ). Third, the carbonyl of dLys2 of PIE7, although somewhat flexible in orientation, forms a direct hydrogen bond with the ε nitrogen of Trp-571 in some of the structures. This interaction is water-mediated in 2K-PIE1. Fourth, in some of the structures the hydroxyl of dTyr7 in PIE7 forms a new water-mediated hydrogen bond with the pocket residue Gln-575. This interaction cannot be formed in the 2K-PIE1 structure. These subtle changes expand PIE7's pocket binding interface and may account for the enhanced potency of PIE7.

Structural analysis of the IQN17:2K-PIE1 and IQN17:PIE7 inhibitor complexes. Shown is a comparison of unique polar contacts observed in the 2K-PIE1 (A) and PIE7 (B) costructures (described in the text).

Multimeric D-Peptides.

Based on the trimeric nature of gp41, we predicted that multimeric D-peptides would have significantly improved affinity for the N-trimer and enhanced antiviral potency. To test this idea, we used a bis(NHS ester)PEG cross-linker to dimerize PIE7 via its unique primary amine (N-terminal Lys) (Fig. 2 F). The length of the PEG spacer (35 Å) was chosen to cover, with slack, the distance between the N-termini of neighboring D-peptides in the crystal structures. To construct the PIE7 trimer, we used two of the same cross-linker to connect a central 2K-PIE7 to two flanking PIE7s. PEG is an ideal material for cross-linking because it is highly flexible, very soluble, nonimmunogenic, and has been used in several approved therapeutic peptides and proteins (33).

The resulting dimeric and trimeric inhibitors, (PIE7)2 and (PIE7)3, have IC50 values of 1.9 nM and 250 pM (Table 1 and Fig. 3 A) against HXB2, respectively. This dramatic gain in potency is a 325- and 2,500-fold improvement over the PIE7 monomer, respectively. To control for possible nonspecific effects of the PEG moiety, we reacted mono(NHS ester)PEG with PIE7 to generate PEG-PIE7. Addition of this PEG group caused a modest ≈1.5-fold reduction in potency against HXB2. Therefore, the improved potency of the oligomers cannot be attributed to an interaction of the PEG with virus, cells, or the D-peptide but is a genuine avidity effect caused by multiple D-peptides binding to the N-trimer.

Surface Plasmon Resonance (SPR) Characterization.

To determine whether the improved potency of our second-generation D-peptides stems from optimization of affinity for the pocket, we characterized the binding properties of the D-peptides to an immobilized N-trimer mimic (IZN36) using SPR (Table 1). The rank order of measured K D values correlated well with antiviral IC50 values, indicating that D-peptide binding to a pocket mimic in vitro is a good predictor of antiviral potency.

The PIE7 monomer and multimers had similar rapid association rates, but the dimer and trimer (data not shown) showed dramatically slowed dissociation rates compared with the monomer (SI Fig. 7). The trimer's binding to the pocket was too tight (low to mid pM) to measure accurately by SPR (the value reported in Table 1 is approximate and likely underestimates the trimer's true affinity). Interestingly, the trimer's potency against HXB2 did not improve as much as expected from its K D, suggesting that trimer potency against HXB2 may have reached a potency limit imposed by association kinetics, as has been reported for another entry inhibitor, 5-helix (34). This kinetic limitation is expected because the exposed N-trimer has an ≈10- to 20-min lifetime in the gp41 prehairpin intermediate (4–6), similar to the time required for binding of our peptides at mid to high pM concentrations.

D-Peptide Inhibitors Are Also Active Against Primary HIV-1 Strains.

HXB2 is a widely used laboratory-adapted HIV-1 strain that is typically more sensitive to entry inhibitors than primary (clinical) HIV-1 strains. Here we report D-peptide inhibitory data against primary strains (standard clade B strains BaL and JRFL). The most potent first-generation D-peptide, D10-p5, showed little or no inhibitory activity against JRFL and modest activity against BaL (Table 1). In contrast, PIE7 inhibits both JRFL (IC50 = 24 μM) and BaL (IC50 = 2.2 μM) entry, although ≈40- and ≈4-fold less potently than HXB2 entry, respectively (Table 1). Similar differences in potency between JRFL and HXB2 have been reported for other entry inhibitors that target the pocket region (18) (e.g., the C-peptide inhibitor C37) (Table 1). Interestingly, Fuzeon, which does not bind to the pocket, shows only a small loss of activity against JRFL (Table 1). Despite the relative insensitivity of JRFL to the PIE7 monomer, the PIE7 trimer is a moderately potent inhibitor of this strain (IC50 = 220 nM) and an extremely potent inhibitor against BaL (IC50 = 650 pM).

Possible Sources of JRFL's Relative Insensitivity to Inhibition by PIE7.

Compared with the sequence of the BaL and HXB2 pocket region (N17), JRFL has the conservative L565M substitution (Table 2, highlighted). All other pocket residues that contact our D-peptides are >97% identical in the >5,000 clade A, B, and C HIV-1 strains from the Los Alamos National Laboratory HIV sequence database (www.hiv.lanl.gov). Residue 565 is Leu or Met in ≈99% of these strains. Our crystal structures show that the D-peptide C-terminal Ala interacts with the L565 equivalent position of IQN17 in each of the available crystal structures (e.g., Fig. 2 B ). Residue 580 (Table 2, highlighted) does not contact the pocket.

Pocket region (N17) alignment

The L565M substitution might affect binding of our D-peptides to the JRFL pocket. To test this possibility, we measured binding of PIE7 to JRFL and HXB2 versions of IZN36 by SPR and observed an ≈4-fold increase in K D for binding to the JRFL pocket (data not shown). Another possible contributing factor to JRFL's relative insensitivity is the reduced steric accessibility of JRFL's N-trimer region compared with HXB2 (35), which may explain why PEG-PIE7 has ≈4-fold lower potency than PIE7 against JRFL (vs. a 1.5-fold difference against HXB2) (Table 1). These differences in inhibitor binding caused by the L565M substitution or reduced steric accessibility do not appear to fully account for the ≈40-fold reduction in potency against JRFL. Therefore, an unknown complex factor (e.g., kinetics) is also likely to be involved.

Third Generation D-Peptides.

Although our second-generation multimeric D-peptides are sufficiently potent to begin preclinical studies, the ideal D-peptide for clinical use will require further optimization to improve potency against challenging strains like JRFL. Our current results suggest several straightforward strategies to further improve D-peptide potency and achieve this goal. First, we predict that optimization of the cross-linker length and connectivity in our multimeric D-peptides will strengthen avidity. In our initial strategy, we connected the N termini of monomers (N–N) via the existing N-terminal Lys in PIE7. From the crystal structures, it is apparent that C–C or C–N linkages could be significantly shorter than our current N–N linkage. Second, our structures show that flanking residues (those outside the disulfide bond) present on the phage (and peptides) make important interactions with the pocket. Optimization of these residues in the context of the PIE7 core sequence by phage display will likely provide further improvements in potency.

Third, it is possible that 8-mers are not the most optimal length for these D-peptides. Modeling one or two residue deletions from the 2K-PIE1 structure indicates that 7-mers are still long enough to present the WXWL binding motif to the pocket and maintain the disulfide bond, whereas 6-mers are not (data not shown). Screening of a naïve 7-mer library (CX7C) again identified the WXWL consensus motif and confirmed that 7-mers can bind the pocket and inhibit HIV entry (B.D.W., Y. Shi, and M.S.K., unpublished results). Future phage display screening will ultimately determine which geometry is most optimal for high-affinity pocket binding. Fourth, the crystal structure of PIE7 (our best current monomer) is a valuable platform for rational design using nonnatural amino acid derivatives. For example, PIE7's dTyr7 hydroxyl is not optimally positioned to make direct hydrogen bonds with the pocket. It may be possible to stabilize the complex by extending the position of the dTyr7 hydroxyl by one or two carbons.

Finally, it is important to note that the avidity of our multimers predicts that small improvements in the potency of monomers will result in geometric improvements in the corresponding dimers and trimers, up to the potency limit imposed by association kinetics. We also predict that it will be beneficial to “overengineer” future D-peptides to improve affinity even after reaching this potency limit. Such inhibitors will not show improved potency, but will have a reserve of binding energy that acts as a “resistance capacitor” to defend against potential resistance mutations [i.e., resistance mutations that moderately affect binding would have no effect on potency, as has been reported for the entry inhibitor 5-helix (34)]. Of particular importance, this property will discourage the stepwise accumulation of multiple subtle mutations that combine to confer resistance. Individual mutations would have no effect on inhibitor potency and would not confer a growth advantage in the presence of inhibitors. This resistance capacitor would be especially beneficial for trimeric inhibitors, because resistance mutations would simultaneously affect all three pockets. As a further defense against the development of resistance, our trimeric D-peptides could also be constructed by using three different D-peptide sequences, each with a distinct resistance profile. Such a heterotrimer would present a significant additional barrier to the development of resistance.

Potential Uses of D-Peptides.

Our D-peptides target the highly conserved gp41 hydrophobic pocket region and will likely have an improved resistance profile compared with Fuzeon (25), which targets a less well conserved region of gp41. Further studies of our D-peptides against panels of viruses from diverse HIV-1 clades and in vitro selections for resistance mutations will be required to determine the breadth of their activity and predict susceptibility to resistance mutations. Because the hydrophobic pocket is not targeted by Fuzeon or other entry inhibitors currently in advanced clinical trials (e.g., BMS-378806, PRO 542, Vicriviroc, and Maraviroc), our D-peptides should be additive (or possibly synergistic) with these inhibitors and could form part of a emerging entry inhibitor “cocktail,” similar to the mixtures of HIV-1 protease and reverse transcriptase inhibitors currently used in highly active antiretroviral therapy.

D-peptides represent a new class of drugs that have not been extensively tested in vivo. Because D-peptides are not degraded by proteases they have the potential for oral bioavailability (29, 30), extended persistence in circulation (28), reduced immunogenicity (36), long shelf life, and use in harsh mucosal environments as a topical prophylactic microbicide. The D-peptides reported here are now sufficiently potent for preclinical studies, which will ultimately determine whether these theoretical advantages translate into a valuable new class of agents for the prevention and treatment of HIV/AIDS. These results also suggest that D-peptides may be useful for diverse applications against other therapeutic targets.


Introduction

HIV-1 drug resistance mutations rendering the Highly Active Anti-Retroviral Therapy (HAART) cocktail [1] ineffective have been increasingly reported. Currently, the HAART cocktail consists of reverse transcriptase (RT) inhibitors (RTIs), protease inhibitors, and integrase inhibitors. Together they work to interfere with virus replication, maturation, and viral genome integration, respectively [2]. Of these three enzymatic drug targets, only the RTIs have two classifications based on their modus operandi as nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). The NNRTIs functions non-competitively, binding to an allosteric site to cause structural changes to the RT polymerase active site, whereas the NRTIs directly compete in the active site with nucleotides during the incorporation to terminate the reverse-transcription process [3]. Given that the NRTIs work competitively, NRTI drugs are generally nucleotide analogs, and thus limited in structure. On the other hand, the NNRTI allosteric inhibitors that distantly influence the RT polymerase active site, is open to a wider scope of ligand structures.

NNRTIs were first discovered [4,5] through multiple compound library screening [6], in which the two derivatives ‘HEPT’ and ‘TIBO’ were found to selectively inhibit HIV-2 replication in vitro. Many NNRTIs were later developed [7] using these two derivatives as study templates. Although the first NNRTI drug (nevirapine or NVP) was approved in 1991 [8], its RT inhibition mechanism of affecting RT flexibility was only recently reported [9]. Another NNRTI, efavirenz (EFZ), binds to the p66 subunit of HIV-RT and restricts the motions and conformational changes of the RT thumb that is necessary for DNA polymerization. In general, the NNRTIs distort the polymerase primer grip, thereby inhibiting the proper positioning of the primer at the 3′-end polymerase active site [10]. Due to the distal effects, the mechanism is deemed to be allostery [9,11]. Further using hydrogen exchange MS, Seckler et al. [9] revealed an allosteric network in EFZ-bound RT structure involving both RT subunits p66 and p51, demonstrating that p51 also underwent substantial conformational changes in addition to p66 in order to trigger allosteric couplings upon NNRTI binding.

To overcome NNRTIs, the viral RT was found to gain mutations that changed the physicochemical properties of the drug-binding pocket [12,13] and/or to disrupt the allosteric mechanism [11]. Given the limited NNRTI options in HAART, there remains a need to extend the effectiveness of the available NNRTIs by delaying the onset of cross-resistance. Further taking advantage of the allosteric coupling, there is a need to identify new druggable pockets, to which future drugs can be designed to synergistically inhibit RT function.

Our paper aims to address these two goals by using the latest reported clinical NNRTI resistant mutations [14] for integration into RT structures as study models. We sought to study the NNRTI drug-resistance mutations in cross-resistance by analyzing structural correlation-based networks of the mutant RT–NNRTI complex structures. In addition, we searched for additional allosteric pockets that influenced the polymerase active site that are comparable with the known NNRTI allosteric pocket. The identification of such new pocket(s) is important for the advent of new NNRTIs, particularly in the context of WHO guidelines on NNRTI drug resistance [15,16].


Warning signs over effectiveness of HIV 'wonder drug' in sub-Saharan Africa

Dolutegravir, the current first-line treatment for HIV, may not be as effective as hoped in sub-Saharan Africa, suggests new research published on World AIDS Day. The study finds that this so-called 'wonder drug' may be less effective in patients resistant to older drugs.

As HIV copies itself and replicates, it can develop errors, or 'mutations', in its genetic code (its RNA). While a drug may initially be able to supress or even kill the virus, certain mutations can allow the virus to develop resistance to its effects. If a mutated strain begins to spread within a population, it can mean once-effective drugs are no longer able to treat people.

HIV treatment usually consists of a cocktail of drugs that includes a type of drug known as a non-nucleoside reverse-transcriptase inhibitor (NNRTI). However, in recent years, HIV has begun to develop resistance to NNRTIs. Between 10% and 15% of patients in much of sub-Saharan Africa are infected by a strain of HIV resistant to these drugs. If a patient is infected with an NNRTI-resistant strain, they are at a two- to three-fold increased risk of the drug regimen failing.

In 2019, the World Health Organization began to recommend dolutegravir as the preferred first-line treatment for HIV in most populations. Dolutegravir was dubbed a 'wonder drug' because it was safe, potent and cost-effective and scientists had seen no drug resistance against it in clinical trials. However, there is little data on the success of dolutegravir against circulating strains of HIV in sub-Saharan Africa.

In a study published today in Nature Communications, an international team of researchers from South Africa, the UK and the USA examined the genetic code of HIV to determine if drug resistance mutations in 874 volunteers living with HIV affected their treatment success. The individuals were enrolled in a clinical trial for people initiating HIV treatment to compare two drug regimens: efavirenz, an NNRTI and prior first-line therapy in the region, and dolutegravir.

The goal of this study was to determine whether drug resistance to efavirenz prior to starting treatment affected treatment success (suppression of the virus in the blood) over the first two years of therapy with both of these two regimens.

As expected, the presence of drug resistance substantially reduced the chances of treatment success in people taking efavirenz, successfully suppressing the virus over 96-weeks in 65% of participants compared to 85% of non-resistant individuals. However, unexpectedly, the same pattern was true for individuals taking dolutegravir-based treatments: 66% of those with efavirenz resistance mutations remained suppressed over 96-weeeks compared to 84% of those without the mutations. These relationships held true after accounting for other factors, such as treatment adherence.

"We fully expected efavirenz to be less effective among patients HIV strains resistant to NNRTIs," said Dr Mark Siedner, faculty member at the Africa Health Research Institute in KwaZulu-Natal, South Africa and Massachusetts General Hospital in Boston, Massachusetts. "What took us completely by surprise was that dolutegravir -- a different class of drug which is generally effective in the face of drug resistance -- would also be less effective in people with these resistant strains.

"We are working now to tease out if this was due to the virus or the participants -- for instance, if people with resistance are less likely to take their pills regularly. Either way, if this pattern holds true, it could have far reaching impacts on our predictions of long-term treatment control for millions of people taking dolutegravir in the region."

Professor Ravi Gupta from the Department of Medicine at the University of Cambridge said: "This a huge concern. Dolutegravir was very much seen as a 'wonder drug', but our study suggests it might not be as effective in a significant number of patients who are resistant to another important class of antiretroviral drugs."

The researchers say it is not clear why efavirenz-resistant mutations should affect susceptibility of dolutegravir, though one hypothesis is that integrase inhibitors such as dolutegravir push the virus to replicate and mutate faster, in turn developing resistance to the new drug in an evolutionary arms race. Alternatively, it could be due to poor adherence to treatment regimens, even though the analysis accounted for adherence by two independent methods. Further research is needed to find out why.

Professor Gupta added: "What this shows is that we urgently need to prioritise point of care tests to identify people with drug resistance HIV, particularly against efavirenz, and to more closely and accurately monitor treatment adherence. The development of such tests is at an advanced stage, but there a lack of investment from funders and philanthropic donors. We urgently need agencies and individuals to step forward and help support these programmes.

"In addition, we need to provide widespread access to viral load monitoring so that we can find those who are struggling, get them on more appropriate regimens, and limit the emergence of resistance when patients are failing therapy."