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. Author manuscript; available in PMC: 2022 Aug 20.
Published in final edited form as: Biochem Biophys Res Commun. 2021 Jun 7;566:30–35. doi: 10.1016/j.bbrc.2021.05.094

Novel HIV PR inhibitors with C4-substituted bis-THF and bis-fluoro-benzyl target the two active site mutations of highly drug resistant mutant PRS17

Johnson Agniswamy a, Daniel W Kneller a,#, Arun K Ghosh b, Irene T Weber a,c,*
PMCID: PMC8286348  NIHMSID: NIHMS1712648  PMID: 34111669

Abstract

The emergence of multidrug resistant (MDR) HIV strains severely reduces the effectiveness of antiretroviral therapy. Clinical inhibitor darunavir (1) has picomolar binding affinity for HIV-1 protease (PR), however, drug resistant variants like PRS17 show poor inhibition by 1, despite the presence of only two mutated residues in the inhibitor-binding site. Antiviral inhibitors that target MDR proteases like PRS17 would be valuable as therapeutic agents. Inhibitors 2 and 3 derived from 1 through substitutions at P1, P2 and P2ʹ positions exhibit 3.4- to 500-fold better inhibition than clinical inhibitors for PRS17 with the exception of amprenavir. Crystal structures of PRS17/2 and PRS17/3 reveal how these inhibitors target the two active site mutations of PRS17. The substituted methoxy P2 group of 2 forms new interactions with G48V mutation, while the modified bis-fluoro-benzyl P1 group of 3 forms a halogen interaction with V82S mutation, contributing to improved inhibition of PRS17.

Keywords: Drug resistance, HIV protease, Protease inhibitor, X-ray crystallography

1. Introduction

Combination antiretroviral therapy (cART) has played a critical role in the suppression of human immunodeficiency virus (HIV) replication and the improved outcome for HIV-infected patients [1–3]. HIV protease (PR) inhibitors (PIs) are an integral part of cART regimens together with reverse-transcriptase (RTIs) and integrase inhibitors[4,5]. However, successful treatment is hampered by drug toxicity, side effects, and importantly, the emergence of drug resistant HIV-1 variants. PIs have a higher barrier to resistance than RTIs [6]. Currently, 3 of the 9 approved PIs, ritonavir-boosted darunavir (1), lopinavir and atazanavir are recommended in cART because of their high resistance barrier and potency [7]. Inhibitor 1, which was designed to form hydrogen bonds with the main-chain atoms of PR, is extremely potent and possesses the highest resistance barrier among PIs [8–12]. Other favorable traits of 1 include inhibition of precursor autoprocessing and inhibition of PR dimerization [13,14]. However, the emergence of drug resistant mutations to 1 and the prevalence of multidrug resistant (MDR) viral strains underscore the importance of developing more effective drugs [15,16].

The exceptional antiviral activity and picomolar enzyme inhibition of 1 has led to the design of derivatives to extend its potency, especially for poorly accessible reservoirs of virus. GRL-4410 (2) incorporates a substituted alkoxy group at the C4 position of P2 bis-THF in 1 and a methoxy group replaces the amine group in P2′ aniline of 1 [17]. Compound 2 has an excellent inhibition profile with Ki of 2.9 pM and a potent antiviral efficacy with an IC50 value of 2.4 nM as determined by MTT assay [17]. GRL-142 (3) has a 6–5-5 ring fused crown-like tetrahydropyranofuran (crn-THF) as the P2 ligand, bis-fluoro-benzene at P1 and cyclopropylamino-benzothiazole at P2′ [18,19]. Compound 3 exhibits exceptionally potent antiviral activity with an IC50 value of 0.019 nM compared to values of 3.2 to 33 nM for the nine FDA-approved PIs with tested viral variants, including drug-resistant strains [18]. Compound 3 shows around 1000-fold better inhibition of PR dimerization than 1 [19]. Furthermore, 3 shows better CNS penetration in vitro compared to 1 and studies in rats suggest it can effectively block HIV-1 replication in the brain. These traits make 3 an excellent PI for HIV/AIDS and HIV-associated neurocognitive disorder (HAND).

Recently, MDR variant PRS17 was chosen by mean-shift clustering on genotype-phenotype data using a unified encoding of sequence and 3D structure [20,21]. PRS17 has 17 mutations relative to wild-type PR and exhibits 1.5 to 5 orders of magnitude poorer inhibition relative to wild-type PR for 8 clinical inhibitors [22,23]. PRS17 also shows enhanced binding to substrate analogs[24]. NMR spectroscopy and X-ray crystallography studies show that the dynamic equilibrium conformation of PRS17, unlike that of wild-type PR, is shifted toward the open flap conformation in the absence of inhibitor [25]. Other studied MDR variants, PR20 and MDR769, also exhibit wide open flap conformations and poor binding affinity for inhibitors [26,27]. However, unlike PR20 and MDR769, PRS17 has only two mutations in the inhibitor-binding cavity (G48V and V82S). Hence, PRS17 is an excellent prototype to evaluate inhibitors targeting MDR PR variants with minimal alterations in the binding site.

We have determined the inhibitory activity and crystal structures of PRS17 in complex with 2 and 3. The structures are compared to corresponding wild-type PR complexes and PRS17/1 complex. Insights from this analysis will benefit the design of better drugs for MDR variants like PRS17.

2. Materials and methods

2.1. Expression and Purification of PRS17

The synthetic gene derived from genotype data for PRS17 was expressed in E. coli and purified as described in [24]

2.2. Kinetic inhibition measurements

Compounds 2 and 3 (>95% purity by HPLC) were dissolved in 100% DMSO. Inhibition values (Ki) for PRS17 were measured in a spectroscopic assay with FRET-substrate (BACHEM H-2992) at 37°C and pH 5.6 as described in [24].

2.3. Crystallization

PRS17 was mixed with inhibitor at 1:6 molar ratios and incubated on ice for 30 minutes. PRS17 complex at 5 mg/mL was used in hanging drop vapor diffusion crystallization trials at room temperature. PRS17/2 crystallized in 1.15 M sodium chloride, 0.1 M sodium acetate at pH 5.5. PRS17/3 crystallized from 1.2 M sodium chloride, 0.1 M sodium acetate at pH 5.5. The crystals were cryo-cooled in the respective mother liquor and 30% glycerol.

2.4. X-ray data collection and structure determination

X-ray diffraction data were collected at 100 K on beamline 22-ID (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory. The data were integrated and scaled with HKL2000 [28]. Structures were solved using molecular replacement with PHASER [29,30] with PRS17/1 (5T2Z)[26] as the starting model. Structures were refined using REFMAC5.2 [31] and refitted with COOT [32]. Solvent molecules were inserted at stereochemically reasonable positions using 2Fo-Fc and Fo-Fc maps at 1 and 3 sigma levels, respectively. Hydrogen bonds (2.4–3.5 Å) and hydrophobic contacts (3.6–4.2 Å) were inferred from interatomic distances and chemistry. Molecular figures were prepared with PyMOL (http://www.pymol.org). Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 7MYP for PRS17/2 and 7MYY for PRS17/3.

3. Results

3.1. Compounds 2 and 3 are excellent inhibitors of PRS17

The Ki values of compounds 2 and 3 were 15.8 ± 4.8 and 17 ± 1.3 nM, respectively, for PRS17. These values are 3- to 500-fold better than those of clinical inhibitors (Ki values of 50 – 8400 nM), except for amprenavir (Ki value of 11 nM) [23]. Interestingly, non-hydrolyzable substrate analogs CA-p2 (Ki = 22 nM) and p2-NC (Ki = 514 nM) also show better inhibition than most clinical inhibitors for PRS17 [24]. Compounds 2 and 3 had similar inhibitory activity to CA-p2 and better inhibition than p2-NC analog for PRS17.

3.2. Overall structure

Crystal structures of PRS17 with investigational inhibitors 2 and 3 derived from compound 1 (Figure 1) were determined at 1.65 and 1.50 Å resolution, respectively, and R-factors of 20% (Table 1). The structures were solved in space group P61 with one PRS17 dimer per asymmetric unit. Residues in the two subunits are numbered 1–99 and 1′−99′ (Figure 1D). The inhibitors were observed in two mutually exclusive orientations related by 180° rotation with relative occupancies of 0.55 and 0.45 for PRS17/2 complex and 0.5 each for PRS17/3 complex. Both inhibitors and all mutations were unambiguously modelled in the structures. The two subunits in PRS17/2 and PRS17/3 dimers are essentially identical with low root mean square deviation (RMSD) values of 0.07 and 0.05 Å for 99 Cα atoms, respectively.

Figure 1. Compounds 1, 2, 3 and sites of mutation in PRS17 dimer.

Figure 1.

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A. Chemical structure of darunavir (1). B. Chemical structure and Fo-Fc omit map of 2 contour d at 3σ. C. Chemical structure and Fo-Fc omit map of 3 contour d at 3σ. D. PRS17 dimer in cartoon representation showing the sites of 17 mutations. The two active site mutations are shown as red spheres and the other mutations are blue spheres. Compound 3 bound at the active site is shown as pink sticks

Table 1.

Crystallographic data and refinement statistics

PRS17 Complexes PRS17/2 PRS17/3
Space group P61 P61
Cell Dimensions
a (Å) 62.94 62.89
b (Å) 62.94 62.89
c (Å) 82.72 83.11
Resolution range (Å) 50.0 – 1.65 50.0 – 1.5
Unique reflections 21295 28781
Redundancy 4.4 (3.8) 4.9 (3.9)
Completeness 95.2 (71.1)a 96.4 (81.5)
<I/σ(I)> 21.0 (3.1) 34.0 (2.7)
Rsym (%) 5.9 (44.0) 3.9 (42.6)
Refinement resolution range (Å) 50 – 1.65 50.0 – 1.50
R(%) 20.0 20.2
Rfree (%) 24.8 24.8
Number of water molecules 97 124
Average B-factor (Å2)
 Main-chain 28.2 25.7
 Side-chain 33.0 30.3
 Inhibitor 22.4 21.5
 Water 37.4 35.1
RMS deviations from ideality
Bond length (Å) 0.01 0.01
Angles(°) 1.6 1.7
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a

Values in parentheses are for the highest resolution shell

3.3. New interaction of 2 with G48V of PRS17 contributes to its improved inhibition over 1

The P2 alkoxy group at the C4-position of bis-THF of 2 was designed to form additional interactions with the flexible flaps of PR [17]. The dimers of PRS17/2 and wild-type PR/2 [17] superposed with a RMSD of 0.8 Å for 198 equivalent Cα atoms, however, PRS17/2 is more similar to PRS17/1 [25] with a low RMSD of 0.17 Å. The protein residues in the active site cavity share similar conformations in the three structures except at the 80′s loop and flaps, where V82S and G48V mutations are located in PRS17. Mutation V82′S substitutes the polar serine for β-branched hydrophobic valine. The main-chain atoms of Thr80′ to Ser82′ in the S1 pocket of PRS17/2 complex shift by about 0.7–1.0 Å towards P1 of the inhibitor compared to the position in the wild-type PR/2 complex (Figure 2A). This shift maintains the van der Waals contacts of the smaller Ser82′ mutation and Pro81′ of PRS17 with P1 Phe of compound 2. A similar shift in the other subunit acts to maintain the hydrophobic contact between P1′ Leu of 2 and Ser82 mutation of PRS17. PRS17/1 complex shows a similar conformational change, which confirms the importance of V82S mutation.

Figure 2. Interactions of P2 group of compound 2 with G48V mutation of PRS17.

Figure 2.

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A. The main-chain of residues 80–82 in PRS17/2 complex shifts by ~1 Å due to V82S mutation to maintain van der Waals contacts with P1 group of 2 observed in wild-type PR/2 complex. PR/2 complex is shown as grey sticks colored by element in panels A and B. PRS17/2 amino acids are in green sticks and inhibitor 2 is in pink. Green and black (― - ―) lines represent van der Waals contacts in PRS17/2 and PR/2 complexes, respectively. B. Comparison of P2 methoxy group interaction in the S2 pocket of PRS17/2 and PR/2 complexes. The new interactions of P2 group of 2 are shown in red lines in panels B and C. Green and black (- - -) lines represent hydrogen bonds in mutant and wild-type PR complexes. C. Comparison of interactions at the S2 site of PRS17 by substituted P2 methoxy group of 2 in PRS17/2 complex and bis-THF of 1 in PRS17/1 complex. PRS17/1 is shown as sticks with yellow carbons.

All hydrogen bond interactions between 2 and the main-chain atoms of PR are retained in PRS17/2 complex. The carbonyl group of G48V in PRS17/2 is in a single conformation in contrast to the two conformations in the wild-type PR complex. The substituted methoxy group of P2 bis-THF of 2 forms similar van der Waals contact with the carbonyl oxygen of G48V in PRS17/2 and PR/2 (Figure 2B). The water-mediated hydrogen bond observed between the oxygen of the P2 methoxy group and the amide of Gly48 in PR/2 is conserved in the new PRS17/2 complex. However, the P2 methoxy group forms additional hydrophobic contacts with the side-chain of G48V mutation cannot occur in the wild-type complex. In addition, the P2 group forms water-mediated interactions with Asp30 in PRS17/2 unlike in PR/2. Comparison with PRS17/1 reveals that P2 bis-THF of 1 lacks the water-mediated hydrogen bonds with G48V and Asp30 and has no hydrophobic contacts with G48V (Figure 2C). Thus, the P2 alkoxy group of 2 retains interactions with the main-chain of 48 in wild-type PR/2 and in PRS17/2 complexes. The absence of these interactions in PRS17/1 explains the improved inhibition of compound 2 relative to 1 for PRS17.

3.4. Halogen bond between 3 and V82S confers enhanced inhibition constant for PRS17 over 1

Compound 3 has larger groups compared to 1 with crn-THF as P2-ligand, aminobenzothiazole (Cp-Abt) as P2′-ligand, and bis-fluoro-benzene as P1-ligand. The dimer of PRS17/3 superimposes on wild-type PR/3 with RMSD of 0.79 Å for 198 equivalent Cα atoms. PRS17/3 complex is more similar to PRS17/1 with RMSD of 0.23 Å. PRS17/3 retains all hydrogen bonds observed between 3 and main-chain atoms of protein in previously reported structures of PR/3 and PRS17/1. The crn-THF P2 group of 3 forms similar van der Waals contacts with Ile 47 in the wild-type PR/3 and PRS17/3 structures, while the bis-THF P2 group in PRS17/1 complex has no contacts with Ile47 (Figure 3A). Like in the PR/3 structure, the Cp-Abt at P2′ of PRS17/3 forms two hydrogen bonds with the side-chain of Asp30′. The P2′ cyclopropyl group of 3 in PR/3 and PRS17/3 complexes forms van der Waals interactions with the side-chain of Asp29′. In contrast, the P2′ aminobenzene in PRS17/1 forms a hydrogen bond (3.5 Å) with the side-chain of Asp30′ (Figure 3B). Thus, unlike 1, the large P2ʹ group of 3 makes extensive interactions with Asp29ʹ and Asp30ʹ of PRS17.

Figure 3. Interactions of P1, P2 and P2ʹ groups of 3 with PRS17 in comparison to 1.

Figure 3.

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PRS17/1 complex is shown in sticks with yellow carbons. PRS17/3 is shown with cyan carbons for protein and blue carbons for inhibitor. Lines (― - ―) and (- - -) represent van der Waals and hydrogen bond interactions. Black and cyan lines represent interactions observed in PRS17/1 and PRS17/3, respectively. New interactions observed in PRS17/3 are shown as red dashed lines. A. The substituted P2 crown-THF of 3 forms new van der Waals contacts with Ile47 at the S2 pocket of PRS17/3 in comparison to PRS17/1. B. The P2ʹ Cp-Abt of 3 forms 2 new hydrogen bonds with Asp30ʹ and several van der Waals contacts with Asp29ʹ in PRS17/3 compared to PRS17/1. C. one of the fluorine in the P1 bis-fluro-benzyl group of 3 forms two polar interactions with the flap residues Gly49 and Ile50 in PRS17/3 compared to PRS17/1. D. The second fluorine in the P1-ligand of 3 forms polar interactions with Arg8ʹ and the critical active site mutation V82ʹS in PRS17/3 in comparison to PRS17/1.

The fluorine atoms in the P1 bis-fluoro-benzene of 3 play an important role in its binding to PR. One of the fluorine atoms forms a polar interaction (C-F···H-N) to the main-chain amide group of Ile50 in both PR/3 and PRS17/3 complexes. The fluorine also forms an orthogonal multipolar interaction (C-F···C-O) interaction with the main-chain carbonyl of Gly49 in both complexes. Inhibitor 1 lacks these halogen interactions and instead forms weaker van der Waals contacts with the flap residues in PRS17/1 complex (Figure 3C). In the wild-type PR/3, the second fluorine atom forms polar interactions with the guanidinium group of Arg8′. In PRS17/3, the second fluorine retains the polar interaction with Arg8′ in one conformation of 3 while the second conformation forms a water-mediated interaction with Arg8′. The second fluorine also forms a new polar interaction with side-chain of V82′S mutation in PRS17/3 complex (Figure 3D). This interaction is not possible in PR/3 complex with Val82′ nor in PRS17/1 where P1 lacks fluorine atoms. Thus, the new halogen interactions formed by P1 group of 3 with V82ʹS, Arg8ʹ and flap residues Gly49 and Ile50 of PRS17, together with added interactions of substituted P2 and P2′, contribute to its improved inhibition relative to 1 for PRS17.

4. Discussion

Among the 17 mutations, PRS17 has only two mutations, G48V and V82S, in the active site cavity. Drug resistant mutations of Val82 are among the first to emerge in patients undergoing antiviral therapy [33] and are associated with resistance to all clinical drugs except for 1 [34]. Flap mutation G48V is selected by PIs saquinavir, atazanavir, indinavir, lopinavir and nelfinavir [35–37]. Mutations of Gly48 are common in MDR variants [38] like PRS17. Hence, inhibitors that target Gly48 mutations are likely to perform well against MDR PRs. In addition, G48V and V82S mutations were shown to play a vital role in the enhanced binding of substrate analogs CA-p2 and p2-NC to PRS17 thereby contributing to viral fitness [24]. The role of V82 mutations is confirmed by studies of PR with single mutation V82A, which also displays enhanced binding to substrate analogs CA-p2 and p2-NC [24,39]. Amprenavir with the smaller THF at P2 exhibits better inhibition constant for PRS17 compared to other PIs as well as 2 and 3. However, inhibitors 2 and 3 with bigger P2 groups perform better against MDR mutants like PR20 with expanded S2 pockets, whereas amprenavir is a poorer inhibitor of variants with an expanded S2 pocket or active site mutations like V32I or V82I. It is likely that a smaller P2 group may result in improved inhibition profile against MDR PRs with minimal active site mutations such as PRS17. The current study reveals that specific modifications to compound 1 result in better inhibition of MDR PRS17. The substituted P2 moiety of inhibitor 2 targets flap mutation G48V and these interactions contribute to its improved inhibition of PRS17. The modified P1 group of 3 targets V82S mutation through halogen interactions to improve its inhibition of PRS17. These insights will be valuable for the design of improved inhibitors of MDR PRs. A new inhibitor in the scaffold of 1 that combines the P1 and P2 substitutions of 2 and 3 may be more effective for mutants like PRS17.

Clinical inhibitors of HIV-1 protease are ineffective against drug-resistant mutant PRS17

Two new antiviral compounds derived from darunavir show better inhibition of PRS17

Structural analysis reveals new interactions of inhibitors with mutated amino acids in PRS17

Acknowledgments

This research was supported by the National Institute of Health grants AI150461 (ITW) and AI150466 (AKG). We thank the staff at the Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory, for assistance during X-ray data collection. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

Funding

This research was supported by the National Institute of Health grants AI150461 (ITW) and AI150466 (AKG) and a Georgia State University Molecular Basis of Disease fellowship.

Footnotes

Conflicts of interest

None declared.

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