In Silico Analysis of Mechanisms of Maribavir-Induced Inhibition and Drug Resistance Mutations in pUL97 Kinase Structural Prediction with AlphaFold2

Abstract

Infections with cytomegalovirus (CMV) can result in increased morbidity and mortality in immunocompromised patients. The pUL97 kinase is a critical enzyme in the regulation of CMV replication. Although it does not phosphorylate deoxynucleosides, this enzyme is involved in the first phosphorylation step of ganciclovir (GCV), a viral DNA polymerase inhibitor. In contrast, maribavir (MBV) is a specific inhibitor of pUL97 kinase activity. In this paper, we analyzed the already-reported amino acid changes, conferring resistance to MBV and cross-resistance to GCV, in the pUL97 protein structure, predicted with AlphaFold2. Docking experiments suggest that MBV is a dual-site inhibitor, targeting ATP binding and substrate phosphorylation. Substitutions that confer resistance to MBV only may directly or indirectly alter the shape of the cavity in the vicinity of the invariant K355 in the putative ATP binding site, without affecting the viral growth. The most frequently encountered T409M substitution may correspond to a gatekeeper mutation. Substitutions that induce cross-resistance to MBV and GCV may directly or indirectly affect the environment of D456 and N461 residues in the catalytic loop, with reduced viral replicative capacity. These results have implications for the clinical use of MBV as well as for the design of novel pUL97 kinase inhibitors.

Keywords: AlphaFold2; cyclopropavir; docking; drug resistance; ganciclovir; gatekeeper residue; human cytomegalovirus; maribavir; pUL97 kinase; predicted protein structure.

Figure 4

Distribution of amino acids that are substituted to confer resistance (yellow sticks) or hypersusceptibility (wheat-colored sticks) to maribavir (A) or cross-resistance to maribavir and ganciclovir (magenta sticks; (B)) in predicted pUL97 protein structure (pale cyan). Invariant K355 and catalytic D456 residues are represented with red sticks. Residues represented by green sticks may be indirectly involved in drug resistance and their implication is discussed with each specific amino acid substitution in text. Projected positions of maribavir (blue sticks) and ganciclovir (orange sticks) are also shown. Dots show atomic radius.

Figure 1

Predicted pUL97 protein structure and quality assessment. Structural representation of pUL97 protein structure predicted with AlphaFold2 colored according to different ranges of predicted local distance difference test (pLDDT) scores (A). Color code for ranges of pLDDT scores is shown in table (B). Predicted aligned error (PAE) plot for pUL97 protein structure (C). PAE plot shows scores of residue x with respect to aligned residue y for all pairs of residues in the protein. Dark green color indicates low PAE score, which corresponds to high reliability of relative position of residue, whereas light green color corresponds to lower confidence.

Figure 2

Conserved structural sub-domains and functional domains of pUL97 kinase. Amino acid sequence of pUL97 kinase of human cytomegalovirus strain AD169 annotated with conserved structural sub-domains (SD) among cellular and viral protein kinases and functional domains [56,57,58] that include nuclear localization signal 1 (in yellow), nuclear localization signal 2 (in magenta), putative ATP binding site (in green), P-loop (in red), and putative catalytic site (in blue) (A). α-helices (α) and β-strands (β) in predicted pUL97 protein are shown with gray and green boxes, respectively. These secondary structures are labeled according to convention for cAMP-dependent protein kinase. Phosphosites (as reviewed in [25]) are also indicated in bold red. Structural representation of functional domains on predicted pUL97 kinase structure (B).

Figure 3

Chemical structures of adenosine triphosphate (ATP), maribavir (1-H-β-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole), ganciclovir (9-[1,3-dihydroxy-2-propoxymethyl] guanine), and cyclopropavir ((Z)-9-((2,2-bis-(hydroxymethyl)cyclopropyldiene)methyl)guanine) (A). Selected views of most probable poses of ATP with ganciclovir (B), ATP with cyclopropavir (C), maribavir with ATP (D), and maribavir with cyclopropavir (E), docked separately and then superimposed onto predicted pUL97 protein structure (in pale cyan) to show absence (B,C) and presence (D,E) of clash between the different pairs of ligands. ATP, maribavir, ganciclovir, and cyclopropavir are shown using yellow, blue, orange, and red sticks, respectively. Catalytic D456 and invariant K355 residues are also shown using green sticks.

Figure 5

The structural representation of T409M and H411L/N/Y amino acid substitutions. The predicted wild-type (T409) pUL97 protein structure (in pale cyan; (A)) and a model of the M409 mutant (in wheat color; (B)). Wild-type T409 and mutated M409 residues are shown using green sticks and using yellow sticks, respectively. Residues K355 and T365 are also shown using green sticks. The predicted wild-type (H411) pUL97 protein structure (in pale cyan; (C)) and a model of L411 (D), N411 (E) and Y411 (F) mutants (in wheat color). The wild-type H411 residue is shown using green sticks, whereas mutated L411, N411, and Y411 residues are shown using yellow sticks. Residues L397, T409, and K355 are also shown using green sticks. Dots show the atomic radius.

Figure 6

Structural representation of C480F and C480R amino acid substitutions. Wild-type (C480) predicted pUL97 structure (in pale cyan; (A)) and model of F480 (B) and R480 (C) mutants (wheat color). Wild-type C480 residue is shown using green sticks whereas mutated F480 and R480 residues are shown using yellow sticks. Residue K355 and D481 are also shown using green sticks. Dots show the atomic radius.

Figure 7

Structural representation of F342Y and F342S amino acid substitutions. Wild-type (F342) predicted pUL97 structure (in pale cyan; (A)) and models of Y342 (B) and S342 (C) mutants (wheat color). Wild-type F342 residue is shown using green sticks, whereas mutated Y342 and S342 residues are shown using magenta sticks. Residues L484, D456, and N461 are also shown using green sticks. Dots show atomic radius.