Structure-Driven Discovery of α,γ-Diketoacid Inhibitors Against UL89 Herpesvirus Terminase

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing a variety of severe viral infections, including irreversible congenital disabilities. Nowadays, HCMV infection is treated by inhibiting the viral DNA polymerase. However, DNA polymerase inhibitors have several drawbacks. An alternative strategy is to use compounds against the packaging machinery or terminase complex, which is essential for viral replication. Our discovery that raltegravir (<b>1</b>), a human immunodeficiency virus drug, inhibits the nuclease function of UL89, one of the protein subunits of the complex, prompted us to further develop terminase inhibitors. On the basis of the structure of <b>1</b>, a library of diketoacid (α,γ-DKA and β,δ-DKA) derivatives were synthesized and tested for UL89-C nuclease activity. The mode of action of α,γ-DKA derivatives on the UL89 active site was elucidated by using X-ray crystallography, molecular docking, and in vitro experiments. Our studies identified α,γ-DKA derivative <b>14</b> able to inhibit UL89 in vitro in the low micromolar range, making <b>14</b> an optimal candidate for further development and virus-infected cell assay

Crystal structure of the CsdE dimer.

<p>(A) Top (left) and side (right) views of the disulfide-bridged CsdE dimer, shown in cartoon representation with the two chains in cyan and slate blue colors. Cys61 side chain and the disulfide bridge between them is represented in sticks, with the sulfur atoms in yellow and the carbon atoms in chain colors. (B) Zoom-in into the disulfide bridge holding together the CsdE dimer. The electron density map is a σ_A-weighted 2<i>DF</i>_O−<i>mF</i>_C map contoured at 1.2 σ in grey. (C) Angle between the vectors joining the center-of-mass of the disulfide bridge and those of monomers of the CsdE dimer during the simulation. The red line indicates the value determined from the X-ray structure.</p

Trajectory followed by the Cys61 side chain of CsdE as it approaches CsdA for reaction.

<p>Three structures of CsdE are superimposed and represented in cartoons, with helices shown as cylinders. NMR CsdE is colored white, X-ray CsdE is in cyan, and the fully rearranged CsdE observed in the persulfurated (CsdA-CsdE)₂ complex is in green. Helices α6, α7, and, for persulfurated CsdE, α8’ and α8, are labeled in order to facilitate comparisons. The side chains of Cys61 and Glu62 are shown in sticks with carbon colors according to the corresponding CsdE structure and sulfur/oxygen atoms in CPK colors.</p

Cys61 as an interface hub on CsdE exposed surface.

<p>(A) Opaque molecular surfaces of CsdE (white) with the position of Cys61 mapped out in yellow. From the top right corner and following a clockwise rotation, the following interaction surfaces are shown: TcdA (in pink), CsdA (persulfurated complex, in green), the two non-symmetric CsdE interaction surfaces (in cyan and in blue slate). (B) Close-up on the molecular surface of CsdE around Cys61 (yellow; labeled C61). The outlines of the interaction surfaces shown in (A) are drawn in thick line with the same color code; each area is labeled with the protein that occupies the respective surface area.</p

Structural comparison of CsdE along the proposed conformational change.

<p>(A) Superposition in cartoon with helices shown as cylinders of the CsdE free monomer from X-Ray (overlaid structures colored in green, white and red to highlight the movement, with the Cys61 Cα atom represented as a sphere), the CsdE monomer of dimer of the present study (pale blue) and CsdE monomer from the X-Ray structure of the (CsdA-CsdE)₂ complex (cyan). The CsdE free monomer is depicted over the ensemble-weighted maximally correlated mode contributing to the change in the selected distance (d[Cys61(Cα)–Val88(Cα)]). (B) Insight of the CsdE free monomer’s movement of the loop is shown with Cys61 side chain represented as balls and sticks.</p

Functional consequences of CsdE inactivation by disulfide bridge formation.

<p>A functional CsdA-CsdE sulfur mobilization system is depicted inside a cyan outline. CsdA, CsdE, and TcdA are represented as molecular surfaces. The tRNA molecules in the TcdA-tRNA complex are bead models derived from SAXS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186286#pone.0186286.ref041" target="_blank">41</a>]. CsdE is color coded in light cyan (CsdA unbound form) or bright cyan (bound to CsdA). CsdA subunits are colored in green and violet. TcdA subunits are shown in pale green and wheat. The tRNA bead models are in yellow. The inactivation of CsdE during oxidative stress conditions would likely lead to the impairment of the CsdA-CsdE downstream effector functions, the best known of which is the effect on ribosomal translation efficiency and fidelity through the TcdA-tRNA^ANN complex.</p

Distance between the Cα atoms of Cys61 and Val88 during the MD simulation of the CsdE monomer in solution with an anionic Cys61.

<p>Representative structures over the MD simulation are depicted, where the protein is colored in green for the buried conformation of Cys61 and in red for the solvent exposed conformation, Cys61 residue side chain is represented with balls.</p

Interaction network at the CsdE dimer interface.

<p>(A) The two CsdE monomers are shown in ribbon representation. Cys61 side-chain atoms are shown in yellow to mark its position with respect to the monomer-monomer interfaces. The structural elements of each monomer contacted by the opposite monomer are shown mapped in slate blue (top) or cyan (below). (B) Close-up around the disulfide bridge depicting the details of the interaction network that consolidates the dimer. Helices α7 are labeled H7 and β-strands β1, β2, and β3 are labeled as B1, B2, and B3, respectively, only in the top monomer (cyan). Cys61 is harbored in the small loop connecting β2 and β3. Interacting amino acid residues are represented in sticks (main and side chains) and the interactions are depicted by black dashed lines.</p

Scheme of the net reaction catalyzed by alanine transaminase (glutamic acid-pyruvic acid transaminase, GPT).

<p>In the first half-reaction (1) L-alanine is converted to pyruvate with the concomitant conversion of the Lys-PLP Schiff-base linked cofactor to free Lys and PMP (in AlaA, the catalytic lysine residue is Lys240). In the second and last half-reaction (2), a molecule of 2-oxoglutarate is converted to L-glutamate and PMP is recycled back to the enzyme’s resting state cofactor (Lys240-PLP). In the net reaction scheme, a lonely electron pair is shown beside the reactive amine groups of L-alanine and L-glutamate. In the case of valine-pyruvate transaminase (AvtA), the incoming oxo acid is 3-methyl-2-oxobutanoate yielding L-valine as the corresponding α-amino acid instead of L-glutamate.</p

Dual substrate recognition in alanine aminotransferases: Catalytic pocket for dicarboxylic acid substrates.

<p>Schematic representation of the active site of AlaA in complex with acetate (<b>A</b>) and of <i>T. termophilus</i> α-aminoadipate transaminase LysN (PDB 2zyj) (<b>B</b>, orange) and <i>A. thaliana</i> LL-aminopimelate aminotransferase (PDB 3ei5) (<b>B</b>, blue) crystallized in complex with the glutamate external aldimine of PLP (PGU). AlaA residues Tyr15, Arg18 and Tyr129 (shadowed) are equivalent to residues known to stabilize the γ-carboxylate moiety of PGU, including Arg23 in α-aminoadipate transaminase and Tyr37 and Tyr152 in LL-aminopimelate aminotransferase.</p