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. 2023 Sep 5:257:115501.
doi: 10.1016/j.ejmech.2023.115501. Epub 2023 May 18.

HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues

Gordon J Lockbaum  1 , Linah N Rusere  1 , Mina Henes  1 , Klajdi Kosovrasti  1 , Desaboini Nageswara Rao  1 , Ean Spielvogel  2 , Sook-Kyung Lee  2 , Ellen A Nalivaika  1 , Ronald Swanstrom  2 , Nese Kurt Yilmaz  1 , Celia A Schiffer  3 , Akbar Ali  4
Affiliations

HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues

Gordon J Lockbaum et al. Eur J Med Chem. .

Abstract

Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants. The phosphonate moiety substantially improved potency against highly mutated and resistant HIV-1 protease variants, but only when combined with more hydrophobic moieties at the P1' and P2' positions. Phosphonate analogs with a larger hydrophobic P1' moiety maintained excellent antiviral potency against a panel of highly resistant HIV-1 variants, with significantly improved resistance profiles. The cocrystal structures indicate that the phosphonate moiety makes extensive hydrophobic interactions with the protease, especially with the flap residues. Many residues involved in these protease-inhibitor interactions are conserved, enabling the inhibitors to maintain potency against highly resistant variants. These results highlight the need to balance inhibitor physicochemical properties by simultaneous modification of chemical groups to further improve resistance profiles.

Keywords: Drug resistance; HIV-1 protease; Protease inhibitors; SAR studies; X-ray structure.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.
Structures of HIV-1 protease inhibitors. (A) Darunavir (DRV), TMC-126, and the corresponding P1 phosphonate analog GS-8374. (B) DRV and GS-8374 bound to wild-type HIV-1 protease (PDB: 6DGX and 2I4W, respectively). The protease is depicted as a gray surface and a cartoon representation, with chain A in teal and chain B in magenta. (C) DRV analogs with variations at the P1′ and P2′ positions and the corresponding P1 phosphonate analogs analyzed in this study.
Figure 2.
Figure 2.
Binding of inhibitors with the P1 phosphonate modification at the HIV-1 protease active site in cocrystal structures. (A) Superposition of all phosphonate compounds shown as sticks, with protease in surface representation. (B) Same as panel A, with protease in cartoon representation. (C) The compounds that share the main biding pose of the phosphonate moiety (10 of 13 inhibitors) are superimposed, with flap (47–53) and 80’s loop (81′–82′) residues interacting with the phosphonate moiety shown as sticks. (D) Inhibitor and residues F53, P81′, and V82′ shown with vdW spheres. (E) All phosphonate compounds from cocrystal structures superimposed and shown as sticks, with protease residues that make vdW contacts labeled. Comparing the phosphonate with the analogous parent compounds, residues with similar (black), more (~0.4–1.0 kcal/mol; orange), and substantially increased (~2–3 kcal/mol; red) vdW contacts.
Figure 3.
Figure 3.
Comparison of the binding modes of the (A) parent compounds and the (B) P1 phosphonate analogs in cocrystal structures bound to HIV-1 protease. Each inhibitor is indicated by a different color. Crystallographic B-factors of the (C) parent compounds and the (D) phosphonate analogs mapped onto the inhibitor structure. B-factor values were normalized between all inhibitors to enable a direct comparison. Warmer colors show higher B-factors, indicating relative flexibility.
Figure 4.
Figure 4.
Protease-inhibitor vdW contacts for phosphonate compounds. (A) Protease residues that have increased vdW contacts upon phosphonate addition relative to the corresponding parent compounds. (B) Mapping vdW contacts of phosphonate compounds onto the protease structure. The protease is in surface representation, with residues colored from blue to red for increasing vdW contacts (gray indicates no contact). (C) Increase in average vdW contacts compared with the parent compounds. Residues in panel A shown on the structure and are colored yellow (I47, G52, I50, and V82′) or red (G48, G49, F53, and P81′) to indicate the most increase in vdW contacts.
Scheme 1:
Scheme 1:
Synthesis of P1 Phosphonate Modified HIV-1 Protease Inhibitors. Reagents and Conditions: (a) R1-NH2, iPrOH, 80 °C, 3 h, 60–98%; (b) aq. Na2CO3, EtOAc, rt, 12 h, 51–92%; (c) NaBH4, MeOH, 0 °C, 30 min, 81%; (d) TFA, CH2Cl2, rt, 1 h, 100%; (e) DIPEA, CH3CN, 0 °C to rt, 24 h, 64–93%; (f) TBDMS-Cl, DIPEA, DMAP, CH2Cl2, 0 °C to rt, 15 h, 98%; (g) 10 wt% Pd/C, H2 gas, MeOH/EtOAc (1:1), rt, overnight, 55–98%; (h) Cs2CO3, CH3CN, 0 °C, 40 min, rt, 1 h, 62–99%; (i) TBAF, THF, 0 °C, 2 h, 98%.
Scheme 2:
Scheme 2:
Alternative Synthesis of P1 Phosphonate Modified HIV-1 Protease Inhibitors. Reagents and Conditions: (a) Benzyl chloroformate, Et3N, CH2Cl2, 0 °C to rt, 15 h, 53–64%; (b) TFA, CH2Cl2, rt, 1 h, 100%; (c) DIPEA, CH3CN, 0 °C to rt, 24 h, 50–75%; (d) 20 wt% Pd(OH)2/C, H2 gas, EtOH/EtOAc (1:1), rt, 3 h; (e) (Boc)2O, Na2CO3, dioxane/H2O (1:1), 0 °C, 30 min, rt, 2 h; 77–91% over two-steps; (f) Cs2CO3, CH3CN, 0 °C, 40 min, rt, 1 h, 82–88%; (g) TFA, CH2Cl2, rt, 1 h, 100% (h) aq. Na2CO3, EtOAc, rt, 12 h, 66–83%; (i) NaBH4, THF, −10 °C, 30 min, 88%.
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