Chemical Evolution of Antivirals Against Enterovirus D68 through Protein-Templated Knoevenagel Reactions

Abstract

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C-C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development.

Keywords: aldol condensations; antivirals; chemical evolution; protease inhibition; protein-templated reactions.

Figure 1

Discovery of protein‐catalyzed C−C couplings. a. Postulated Zimmerman–Traxler transition state of the aldol reaction of aldehyde 1 with the E‐enol tautomer of fragment F bound to EV D68 3C protease. b. Binding hypothesis of 3‐formyl‐benzamide 1 to EV D68 3C protease (3ZVG). c. Enolizable fragments F1–F13. d. Fragment ligation assay of EV D68 3C protease with aldehyde 1 (19 μM) and fragments F1–F13 (19 μM) for 2 h. Activity of protease was determined using a fluorogenic FRET substrate (see text). e. Binding simulation of the presumed anti‐aldol addition product of 1 and F1. f. Covalent binding mode of the Knoevenagel condensation product 2. Color code: white balls—protein carbon atoms, grey sticks—ligand carbon atoms, red balls and sticks—oxygen atoms, blue balls and sticks—nitrogen atoms, yellow balls—protein sulfur atom, red and green arrows—hydrogen bond acceptors and donors, respectively, yellow spheres—lipophilic contacts.

Figure 2

Kinetic analysis of protein‐catalyzed C−C couplings. Time‐dependent formation of 5 at r.t. for 60–150 min with different concentrations of F4, 1 and EV D68 3C. Quantification of 5 was conducted by HPLC‐QToF‐MS using a freshly recorded calibration curve. Time‐dependent concentration of 5 was fitted to the exponential saturation function c(t)=c ₀+(c _max−c ₀)(1−e^(−Kt)). a. F4: 100 μM, 1: 100 μM, c _max=12.6 μM and t _1/2=17 min. b. 1: 100 μM, F4: 10 μM, c _max=12.2 μM and t _1/2=115 min. c. 1: 100 μM, F4: 50 μM, c _max=9.2 μM and t _1/2=25 min. d. 1: 5 μM, F4: 100 μM, c _max=4.8 μM and t _1/2=100 min.

Figure 3

Reversibility of antiviral protease inhibitors. a. Linear conversion of the FRET substrate over time suggested reversibility of inhibitors 2, 5 and 12. b. Dilution of assays of 2, 5 and 12 with protease from 20× IC₅₀ to 0.2× IC₅₀ indicated reversible inhibition. c. Native protein MS of EV D68 3C protease (7.5 μM) with inhibitors 2 and 5 (100 μM) in 50 mM ammonium bicarbonate buffer with 100 μM DTT, pH 8 showing protein‐inhibitor complex. d. Denaturing protein MS of EV D68 3C protease (8.2 μM) with inhibitor 12 (6.5 μM) in 100 mM HEPES buffer with 1 mM EDTA and 10 μM DTT, pH 8, using a water‐acetonitrile gradient with 0.1 % formic acid. e. Saturation of protein‐ligand complex with inhibitor 2 determined by native MS. Apparent K _D: 16 +/− 2.4 μM. For experimental details see Supporting Information.

Figure 4

Thermodynamics and kinetics of protease‐interactions for covalent inhibitors 2, 5, and 12. a. Isothermal titration calorimetry of a solution of 2 (800 μM) added dropwise to EV D68 3C protease (60 μM) in buffer with 10 mM DTT. Binding of 2 was driven by binding enthalpy; ΔG=−28.5 kJ mol⁻¹, N=1, K _D=6.5 μM. b. ITC of 5 showed a strong entropic contribution, which was attributed to the release of DTT from the inhibitor‐DTT adduct; ΔG=−27.2 kJ mol⁻¹, N=0.93, K _D=12.5 μM. c. Biolayer interferometry (BLI) with EV D68 3C protease immobilized on an NTA‐agarose chips indicated the complete reversibility of the binding for 2 with k _on‐rates in the range of 100–200 M⁻¹ s⁻¹ and k _off‐rates around 0.015 s⁻¹. d. Binding for 12 was characterized by significantly smaller off‐rates and only partial dissociation resulting in a stronger deviation of the fit using a reversible 1‐to‐1 binding model. e. Symbolic representation of the biolayer interferometry experiment: EV D68 3C protease (grey ribbon) is attached to the sensor (grey balls and plane indicate nickel—nitrilotriacetic acid agarose) via its His‐tag (indicated as imidazole rings). The association and dissociation of inhibitor 2 (indicated as ball and stick model) association—dissociation is recorded as a shift in the interference pattern of visible light as a result of an alteration of the biolayer by small molecule binding. For further details see Supporting Information.

Figure 5

Chemical evolution of antivirals in a dynamic reaction system. Formation and reactivity of antivirals through protein‐catalyzed (A,B) or solution‐based (A′,B′) reversible aldol reactions of aldehyde 1 and enolizable carbonyl fragments F1–F12. Aldol additions were followed by Knoevenagel condensations (C,C′). Either aldol addition products or the aldol condensation products were released from the protein (reactions D,E). Condensation products subsequently reacted either with protein‐based thiols (reaction F) or thiol nucleophiles in solution (reaction F′). Michael additions in solution (F′) can reduce the antiviral activity of Knoevenagel products (E,F) but also can disfavor the hydration reaction (reaction C′) leading to decomposition of inhibitors via the retro‐aldol reaction (B′). EWG=electron‐withdrawing group.