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Ernesto Freire

Publications and source records attributed to Ernesto Freire.

35 records · Page 2Linked to original sources
PubMed2004

Design of inhibitors against HIV, HTLV-I, and Plasmodium falciparum aspartic proteases.

Aspartic proteases have emerged as targets for substrate-based inhibitor design due to their vital roles in the life cycles of the organisms that cause AIDS, malaria, leukemia, and other infectious diseases. Based on the concept of mimicking the substrate transition-state, we designed and synthesized a novel class of aspartic protease inhibitors containing the hydroxymethylcarbonyl (HMC) isostere. An unnatural amino acid, allophenylnorstatine [Apns; (2 S ,3 S )-3-amino-2-hydroxy-4-phenylbutyric acid], was incorporated at the P1 site in a series of peptidomimetic compounds that mimic the natural substrates of the HIV, HTLV-I, and malarial aspartic proteases. From extensive structure-activity relationship studies, we were able to identify a series of highly potent peptidomimetic inhibitors of HIV protease. One highly potent inhibitor of the malarial aspartic protease (plasmepsin II) was identified. Finally, a promising lead compound against the HTLV-I protease was identified.

Animals↗
PubMed2004

Interactions of HIV-1 proteins gp120 and Nef with cellular partners define a novel allosteric paradigm.

During the course of infection, a subset of HIV-1 proteins interacts with multiple cellular partners, sometimes in a hierarchical or sequential way. These proteins include those associated with the initial infection event, with the preparation of the cell for the replicative cycle of the virus and with the exit of new virions from the infected cell. It appears that the interactions of viral proteins with multiple cellular partners are mediated by the occurrence of ligand-induced conformational changes that direct the binding of these proteins to subsequent partners. Two of the most studied HIV-1 proteins that are known to interact with different cellular partners are gp120 and Nef. Here we discuss the interactions of these two proteins with their cellular partners and present new results indicating that the conformational changes undergone by these proteins define a novel allosteric paradigm. In the traditional view, conformational changes are thought to occur between well defined structural conformations of a protein. In gp120 and Nef, those changes involve conformations characterized by the presence of large regions devoid of stable secondary or tertiary structure. Those unstructured regions contain the binding determinants for subsequent partners and only become functionally competent by ligand-induced structuring or un-structuring of those regions. By switching binding epitopes between structured and unstructured conformations the binding affinity can be modulated by several orders of magnitude, thus effectively precluding binding against unwanted partners. A better understanding of these interactions would lead to improved strategies for inhibitor design against these viral targets.

Allosteric Regulation↗
PubMed2003

Multidrug resistance to HIV-1 protease inhibition requires cooperative coupling between distal mutations.

The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/AIDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir. This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the K(m) of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than 1 order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggesting that the mutations at the dimer interface (L10I/L90M) play a major role in eliciting a cooperative response. These studies demonstrate that cooperative interactions contribute an average of 1.2 +/- 0.7 kcal/mol to the overall resistance, most of the cooperative effect (0.8 +/- 0.7 kcal/mol) being mediated by the mutations at the dimerization interface. Not all inhibitors in clinical use are affected the same by long-range cooperative interactions between mutations. These interactions can amplify the effects of individual mutations by factors ranging between 2 and 40 depending on the inhibitor. Dissection of the energetics of drug resistance into enthalpic and entropic components provides a quantitative account of the inhibitor response and a set of thermodynamic guidelines for the design of inhibitors with a lower susceptibility to this type of mutations.

Amino Acid Substitution↗
PubMed2003

High-affinity inhibition of a family of Plasmodium falciparum proteases by a designed adaptive inhibitor.

Drug development against viral or microbial targets is often compounded by the existence of naturally occurring polymorphisms or drug resistant mutations. In the case of Plasmodium falciparum, the etiological agent of malaria, four related and essential proteases, plasmepsin I, II, and IV and the histo-aspartyl protease (HAP), have been identified in the food vacuole of the parasite. Since all of these enzymes are involved in the hemoglobin degradation of infected victims, the simultaneous inhibition of the four enzymes can be expected to lead to a faster starvation of the parasite and to delay the onset of drug resistance, since four enzymes will need to mutate in a concerted fashion. This study describes the design of an adaptive inhibitor intended to inhibit the entire plasmepsin family. Adaptive inhibitors bind with extremely high affinity to a primary target within the family and maintain significant affinity against the remaining members. This objective is accomplished by engineering the strongest and most specific interactions of the inhibitor against conserved regions of the binding site and by accommodating target variations by means of flexible asymmetric functional groups. Using this approach, we have designed an inhibitor with subnanomolar affinity (0.5 nM) against the primary target, plasmepsin II, and with no loss or a very small loss of affinity against plasmepsin IV, I, and HAP (K(i) ratios of 0.4, 7.1, and 17.7, respectively). The core of the inhibitor is defined by an allophenylnorstatine scaffold. Adaptability is provided by an asymmetric amino indanol functional group facing one of the key variable regions in the binding site. Adaptive inhibitors, which display high affinity against several variations of a primary target, are expected to play an important role in the chemotherapy of infectious diseases.

Amino Acid Sequence↗
PubMed2003

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance.

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.

Binding Sites↗
PubMed2003

The binding of HIV-1 protease inhibitors to human serum proteins.

The non-specific binding of a drug to plasma proteins is an important determinant of its biological efficacy since it modulates the availability of the drug to its intended target. In the case of HIV-1 protease inhibitors, binding to human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) appears to be an important modulator of drug bioavailability. From a thermodynamic point of view, the issue of drug availability to the desired target can be formulated as a multiple equilibrium problem in which a ligand is able to bind to different proteins or other macromolecules with different binding affinities. Previously, we have measured the binding thermodynamics of HIV-1 protease inhibitors to their target. In this article, the binding energetics of four inhibitors currently in clinical use (saquinavir, indinavir, ritonavir and nelfinavir) and a second-generation inhibitor (KNI-764) to human HSA and AAG has been studied by isothermal titration calorimetry. All inhibitors exhibited a significant affinity for AAG (K(a) approximately 0.5-10 x 10(5) M(-1)) and a relatively low affinity for HSA (K(a) approximately 5-15 x 10(3) M(-1)). It is shown that under conditions that simulate in vivo concentrations of serum proteins, the inhibitor concentrations required to achieve 95% protease inhibition can be up to 10 times higher than those required in the absence of serum proteins. The effect is compounded in patients infected with drug resistant HIV-1 strains that exhibit a lower affinity for protease inhibitors. In these cases the required inhibitor concentrations can be up to 2000 times higher and beyond the solubility limits of the inhibitors.

Biological Availability↗
PubMed2003

Structural and thermodynamic basis of resistance to HIV-1 protease inhibition: implications for inhibitor design.

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. At the molecular level, resistance to protease inhibition predominantly takes the form of mutations within the protease molecule that preferentially lower the affinity of protease inhibitors with respect to protease substrates, while still maintaining a viable catalytic activity. Mutations associated with drug resistance occur within the active site cavity as well as distal sites. Active site mutations affect directly inhibitor/protease interactions while non-active site mutations affect inhibitor binding through long range cooperative perturbations. The effects of mutations associated with drug resistance are compounded by the presence of naturally occurring polymorphisms, especially those observed in non-B subtypes of HIV-1. The binding thermodynamics of all clinical inhibitors against the wild type protease, drug resistant mutations and non-B subtype HIV-1 proteases has been determined by high sensitivity isothermal titration calorimetry. In conjunction with structural information, these data have provided a precise characterization of the binding mechanism of different inhibitors and their response to mutations. Inhibitors that exhibit extremely high affinity and low susceptibility to the effects of mutations share common features and binding determinants even if they belong to different chemical scaffolds. These binding determinants define a set of rules and constraints for the design of better HIV-1 protease inhibitors.

Drug Design↗
PubMed2002

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

Antibody Affinity↗
PubMed2002

The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors.

The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.

Amino Acid Sequence↗
PubMed2002

Structural parameterization of the binding enthalpy of small ligands.

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long-lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5-7 A of the ligand are included in the calculations. For all seven protein systems considered (HIV-1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c-Src SH2 domain, Hsp90 molecular chaperone, and bovine beta-trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of +/-0.3 kcal x mol(-1).

Animals↗
PubMed2002

Amplification of the effects of drug resistance mutations by background polymorphisms in HIV-1 protease from African subtypes.

The vast majority of HIV-1 infections worldwide are caused by the C and A viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including those identified as targets for antiretroviral therapies. In the case of the HIV-1 protease, we reported earlier [Velazquez-Campoy et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6062-6067] that proteases from the C and A subtypes exhibit a higher biochemical fitness in the presence of widely prescribed protease inhibitors. In this paper we present a complete thermodynamic dissection of the differences between proteases from different subtypes and the effects of the V82F/I84V drug-resistant mutation within the framework of the B, C, and A subtypes. These studies involved four inhibitors in clinical use (indinavir, saquinavir, ritonavir, and nelfinavir) and a second-generation protease inhibitor (KNI-764). Naturally occurring amino acid polymorphisms found in proteases from the C and A subtypes lower the binding affinities of existing clinical inhibitors by factors ranging between 2 and 7.5 which by themselves are not enough to cause drug resistance. The preexisting lower affinity in the C and A subtypes, however, significantly amplifies the effects of the drug-resistant mutation. Relative to the wild-type B subtype protease, the V82F/I84V drug-resistant mutation within the C and A subtypes lowers the binding affinity of inhibitors by factors ranging between 40 and 3000. When the enzyme kinetic properties (k(cat) and K(m)) are included in the analysis, the biochemical fitness of the C and A subtype drug-resistant mutants can be up to 1000-fold higher than that of the wild-type B subtype protease in the presence of the studied inhibitors. From a thermodynamic standpoint, the combined effects of the drug-resistant mutations and the natural amino acid polymorphisms on the Gibbs energy are additive and involve significant alterations in the enthalpy and entropy changes associated with inhibitor binding. At the biochemical level, the combined effects of naturally existing polymorphisms and drug-resistant mutations might have important consequences on the long-term viability of current HIV-1 protease inhibitors.

Africa↗
PubMed2002

Beta-turn Phe in HIV-1 Env binding site of CD4 and CD4 mimetic miniprotein enhances Env binding affinity but is not required for activation of co-receptor/17b site.

HIV-1 enters a host cell after an initial interaction between viral envelope glycoprotein gp120 and cell surface receptor CD4, followed by a second interaction between gp120 and a cell surface chemokine receptor. CD4 residue Phe43 makes a significant contribution to the high-affinity interaction between CD4 and env. We and others have used scorpion toxin scaffolds to display and examine CD4 epitopes used for gp120 recognition. These peptides, which have a beta-turn Phe that acts as a Phe43 surrogate, compete with CD4 for gp120 binding and enhance the binding of gp120 to 17b, an antibody that binds near the co-receptor-binding site. In the current study, a scyllatoxin-scaffolded peptide, identified via phage epitope randomization and lacking a beta-turn Phe (indeed, containing no aromatic residues), was shown to behave in a distinctly CD4-like manner. This peptide, denoted [20EGLV23]ST, not only competed with CD4 for gp120 binding, but also enhanced the binding of gp120 to 17b. Quantitatively, an [20EGLV23]ST-gp120 complex exhibited the same 17b binding on-rate as a complex of gp120 with [20AGSF23]ST, a scyllatoxin-based CD4 mimetic peptide containing a beta-turn Phe. In view of this result, we examined the role of Phe43 in CD4 itself by comparing F43V D1D2 sCD4 versus D1D2 sCD4. Like the peptides, a close similarity was observed for both Phe43 and Phe43-less D1D2 sCD4s in enhancing gp120 binding to 17b. Further, when examined for their ability to enhance binding of gp120 to CCR5+ cells, [20EGLV23]ST and [20AGSF23]ST were found to have the same efficacy, after correcting for the difference in their gp120 affinities. These results show that, although Phe43 is important in maintaining high affinity in gp120 ligands, the aromatic residue is not necessary for triggering the conformational isomerization in gp120 that results in formation or exposure of the binding sites for the 17b antibody and the CCR5 receptor.

Binding Sites↗
PubMed2002

Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target.

Plasmepsin II is a key enzyme in the life cycle of the Plasmodium parasites responsible for malaria, a disease that afflicts more than 300 million individuals annually. Since plasmepsin II inhibition leads to starvation of the parasite, it has been acknowledged as an important target for the development of new antimalarials. In this paper, we identify and characterize high-affinity inhibitors of plasmepsin II based upon the allophenylnorstatine scaffold. The best compound, KNI-727, inhibits plasmepsin II with a K(i) of 70 nM and a 22-fold selectivity with respect to the highly homologous human enzyme cathepsin D. KNI-727 binds to plasmepsin II in a process favored both enthalpically and entropically. At 25 degrees C, the binding enthalpy (DeltaH) is -4.4 kcal/mol and the entropic contribution (-TDeltaS) to the Gibbs energy is -5.56 kcal/mol. Structural stability measurements of plasmepsin II were also utilized to characterize inhibitor binding. High-sensitivity differential scanning calorimetry experiments performed in the absence of inhibitors indicate that, at pH 4.0, plasmepsin II undergoes thermal denaturation at 63.3 degrees C. The structural stability of the enzyme increases with inhibitor concentration in a manner for which the binding energetics of the inhibitor can quantitatively account. The effectiveness of the best compounds in killing the malaria parasite was validated by performing cytotoxicity assays in red blood cells infected with Plasmodium falciparum. EC50s ranging between 6 and 10 microM (3-6 microg/mL) were obtained. These experiments demonstrate the viability of the allophenylnorstatine scaffold in the design of powerful and selective plasmepsin inhibitors.

Amides↗
PubMed2002

Overcoming drug resistance in HIV-1 chemotherapy: the binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease.

Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 x 10(9) M(-1) or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 x 10(11) M(-1) or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.

Binding Sites↗
PubMed2001

The linkage between protein folding and functional cooperativity: two sides of the same coin?

During the course of their biological function, proteins undergo different types of structural rearrangements ranging from local to large-scale conformational changes. These changes are usually triggered by their interactions with small-molecular-weight ligands or other macromolecules. Because binding interactions occur at specific sites and involve only a small number of residues, a chain of cooperative interactions is necessary for the propagation of binding signals to distal locations within the protein structure. This process requires an uneven structural distribution of protein stability and cooperativity as revealed by NMR-detected hydrogen/deuterium exchange experiments under native conditions. The distribution of stabilizing interactions does not only provide the architectural foundation to the three-dimensional structure of a protein, but it also provides the required framework for functional cooperativity. In this review, the statistical thermodynamic linkage between protein stability, functional cooperativity, and ligand binding is discussed.

Algorithms↗
PubMedDate not supplied

Protease inhibition in African subtypes of HIV-1.

Of the 42 million people infected with HIV-1 worldwide, 30 million are in Africa. However, the HIV-1 subtypes prevalent in Africa are not the same that are prevalent in North America and Western Europe. In these developed regions, subtype B is responsible for the vast majority of HIV infections, whereas in sub-Saharan Africa subtypes A and C, and to a lesser extent subtype G, account for most of the infections. These subtypes exhibit genomic differences as large as 30% with respect to subtype B. These differences involve current drug targets, including the HIV-1 protease. Since protease inhibitors have been developed and tested against the HIV-1 B subtype, and proteases from other subtypes carry up to ten amino acid polymorphisms, it is important to assess the influence of these naturally occurring polymorphisms on the potency of existing inhibitors, as well as their synergistic interactions with mutations known to cause drug resistance. This review will examine the effects of naturally occurring polymorphisms on the efficacy of current protease inhibitors and the effects of well characterized drug-resistant mutations within the framework of non-B subtypes. At the biochemical level, non-B-subtype polymorphisms lower the binding affinities of existing clinical inhibitors, but not to the point of causing drug resistance. However, these polymorphisms amplify the effects of mutations causing drug resistance and may play a role in the long-term viability of these inhibitors.

Drug Resistance, Viral↗