Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome

Abstract

Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K(i)=6.8 nM, whereas it inhibits the human proteasome beta5 active site following a two-step mechanism with K(i)=11.5 nM and K(i)(*)=0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.

Figure 1

Inhibition of Mtb proteasome by fellutamide B (1) A) Structure of 1. B) Reaction progress curves of cleavage of Suc-LLVY-AMC by Mtb proteasome in the absence and presence of 1 at 6.25 nM – 40 nM; the curves were fit by nonlinear regression to equation (1) to determine the apparent first-order rate constant k_obs values, which were corrected by equation (2) to yield real k_obs. C) Dependence of k_obs on inhibitor concentration. The solid line was drawn by fitting the data to equation (3), yielding k_on = (129 ± 0.8) × 10³ M⁻¹s⁻¹, and k_off = (0.96 ± 0.13) × 10⁻³ s⁻¹. D) Plot of v_s/v_o versus inhibitor concentration. The data were fitted to equation (4), yielding K_i = 6.8 ± 0.2 nM.

Figure 2

1 inhibits the human proteasome β5 via a two-step mechanism that likely involves enzyme conformational changes. (A) Reaction progress curve of hydrolysis of Suc-LLVY-AMC by hu proteasome in the presence of 1 at 0–75 nM; the curves were fit to equation (1) to determine the apparent k_obs. (B) The plot of k_obs values to [1] demonstrates a two-step inhibition of human 20S β5 by 1. Fitting to equation (6) yields K_i, k₅ and k₆. (C) Binding of 1 induces significant substrate pocket narrowing of the yeast proteasome β5. The native yeast proteasome β5 structure (gray) is superimposed with β5 bound to 1 (cyan). 1 is shown as sticks in magenta. The two red arrows indicate tightening of the substrate pocket upon binding of 1.

Figure 3

Crystal structure of fellutamide B (1) bound to the Mtb proteasome A) The 2Fo-Fc electron density map of fellutamide B shown at 1σ level is shown in blue mesh and the Fo-Fc map at 3σ in green mesh. The likely positions of the disordered alkyl tail from C24 to C32 of 1 are sketched by two dashed blue curves. B) The hydrogen-bonding network that stabilizes 1 in the substrate-binding pocket. The identities of selected proteasome residues are labeled in black, and the inhibitor residues are shown in orange. C) Scheme of the different stereochemistry of fellutamide B in the Mtb and yeast proteasomes. Upper panel, the carbonyl group takes trans-configuration, is hydrogen bonded with Gly-47 when approaching the Thr-1, and subsequently yields a (R)-hemiacetal; lower panel, the carbonyl group takes cis-configuration, is hydrogen bonded with NH₂-Thr-1, and subsequently yields a (S)-hemiacetal. D) A surface view of the substrate-binding pocket of Mtb proteasome with the resolved structure of the fellutamide B (1) (yellow). The three structures of 1 bound to yeast proteasome β1, β2, and β5 are superimposed and shown in gray, blue, and purple, respectively (PDB ID 3D29).

Scheme 1

Two types of time-dependent inhibition mechanism. (A) One-step binding mode and (B) two-step binding mode.