Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to Schistosoma mansoni: Structural and Mechanistic Insights into Chemotype Reactivity

Abstract

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

Keywords: azapeptide inhibitors; cysteine proteases; protein structures; schistosomiasis; structure−activity relationships.

Figure 1

Carba- and azadipeptide nitriles and their reaction with cysteine proteases. Isoelectronic CαH/N exchange in the warhead (cyan/magenta) of dipeptide nitriles (left) leads to azadipeptide nitriles (right). Two carbohydrazide nitrogens in azanitriles need to be alkylated to circumvent spontaneous heterocyclization. R¹ and R² are substituents in amino acid residues at the P1 and P2 positions (binding in the enzyme subsites S1 and S2), respectively; PG is a protecting group. The depicted azadipeptide bears an aza-alanine nitrile at the P1 position. Reactive warheads of both chemotypes form a covalent, reversible bond with the thiol of the catalytic cysteine residue (orange) of papain-family cysteine proteases (represented by SmCB1).

Figure 2

Different binding kinetics of SmCB1 inhibitors with azanitrile and carbanitrile warheads represented by 2a and 2c, respectively. Progress curves show the hydrolysis of the fluorogenic substrate Cbz-Phe-Arg-AMC by SmCB1 in the presence of increasing inhibitor concentrations. (A) The azanitrile exhibited a time-dependent inhibition characterized by nonlinear progress curves typical of slow-binding kinetics. (B) Linear progress curves obtained for the carbanitrile are characteristic of fast-binding inhibitors. In dose–response plots, the derived steady-state reaction velocities were plotted against inhibitor concentration, and the inhibition constants K_i were obtained. In the k_obs versus [I] plot, the first-order rate constants k_obs from the time-dependent progress curves were plotted against inhibitor concentrations to show a linear dependence. For details on data fitting and kinetic parameters, see the Methods.

Figure 3

Correlation of the antischistosomal activity with the inhibition of SmCB1 by azanitrile and carbanitrile analogs. (A) Pairs of analogs with azanitrile and carbanitrile warheads were phenotypically screened against S. mansoni newly transformed schistosomula (NTS) and the data arising was compared to those for the inhibition of SmCB1. Phenotypic changes in the parasite (Table S3) were observed every day for 4 days in the presence of 10 μM inhibitor. Changes were converted to a severity score on a scale from 0 (no effect) to 4 (severe; red heat map). K_i values for SmCB1 inhibition (from Table 1) are shown in the blue heat map. (B) An example of an inhibitor-induced degenerated phenotype in NTS of S. mansoni versus untreated controls (for details, see Table S3).

Figure 4

Binding mode of the azadipeptide nitrile inhibitor 3a in the SmCB1 active site. (A) Overall crystal structure of the SmCB1–3a complex in surface (enzyme) and stick (inhibitor) representation. In the SmCB1 active site (boxed), the catalytic residues Cys100 (yellow) and His270 (pink) and major subsite residues (cyan) are highlighted. (B) Chemical structure of 3a forming a covalent bond with the S atom of the catalytic Cys100. The azanitrile warhead is boxed in gray, and atom labeling is indicated (hydrogen atoms are omitted). (C) Zoomed view of (A) showing the active site residues that form nonpolar interactions (green) with 3a (in stick representation with carbon atoms in magenta); the catalytic residues are also indicated. (D) The P1 to P3 positions of the inhibitor bind the corresponding S1 to S3 subsites of the SmCB1 active site. Dashed lines indicate hydrogen bonds formed between SmCB1 residues (gray) and 3a (magenta); heteroatoms have standard color coding (O, red; N, blue; S, yellow). Coordinates are deposited under PDB code 6YI7.

Figure 5

Computational analysis of the binding reaction of azanitrile and carbanitrile inhibitors to the active site of SmCB1. (A) The “free” energy profile of the binding of azanitrile 3a and carbanitrile 3c was determined using quantum chemical calculations. Individual states along the reaction pathway (indicated by numbers) are defined by their relative “free” energies (Table S6). (B) The unbound azanitrile inhibitor has the E-configuration in solution (with minimum “free” energy) and undergoes a conformational change to the Z-configuration that was also demonstrated crystallographically in the SmCB1–3a complex. (C) Modeled states upon binding of the azanitrile inhibitor to the active site include an initial noncovalent complex (4), a transition state with proton transfer from His270 to H₂O (5), and a final covalent complex after proton transfer to the nitrile group (6). The distance (sulfur–carbon) of the catalytic Cys100 and the inhibitor’s C_AB atom is 3.2, 2.3, and 1.8 Å, respectively.

Scheme 1. Synthesis of Compound 3c

Reagents and conditions: (a) (1) ClCO₂i-Bu, NMM, THF, −25 °C and (2) H₂NCH₂CN × H₂SO₄, NaOH, H₂O, THF, −25 °C to rt, 2 h; (b) TFA, CH₂Cl₂, rt, 2 h; (c) benzyl isocyanate, Et₃N, CH₂Cl₂, 0 °C for 15 min to rt for 16 h.