Anti-infective Activity of 2-Cyano-3-Acrylamide Inhibitors with Improved Drug-Like Properties against Two Intracellular Pathogens

Abstract

Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection.

FIG 1

Structures of DUB inhibitors showing progression of small-molecule design from the lead compounds. (A) Lead compound WP1130 and analog compound G9 have anti-infective effects against Listeria monocytogenes and murine norovirus. (B) Structures of compounds C6 and E3, which have anti-infective activity toward the two intracellular pathogens in macrophages. Arrows point out the different halogens used during the progression of small-molecule design, and squares show side-chain variation from the lead molecule WP1130 to compound G9 (double lines in panel A) and then from compound G9 to compounds C6 and E3 (dashed lines in panel B). (C) Strategy for initial testing of test compound effects on RAW 264.7 cell viability by WST-1 reagent (see Tables S1 to S3 in the supplemental material).

FIG 2

Two of the 39 small-molecule DUB inhibitors reduced intracellular growth of MNV-1 and Listeria monocytogenes in RAW 264.7 cells similar to parental compound G9. (A) Overall scheme for in vitro infections testing the anti-infective effects of 39 small-molecule compounds on MNV-1 (MNV) (left) and Listeria monocytogenes (right). (B and C) Results of cell culture infections of RAW cells with MNV-1 (B) and L. monocytogenes (C) using compounds C6 (left) and E3 (right) at 5 μM. Also included are results using previously characterized compound G9. Treatment with compounds C6 and E3 resulted in a reduction of greater than 2 orders of magnitude in viral replication and greater than 50% reduction in bacterial growth compared to levels for vehicle (DMSO)-treated cells (results for vehicle-treated cells do not significantly differ from those for untreated cells; not shown). One-way analysis of variance with Dunnett's posttest comparing vehicle-treated and compound-treated cells was used. ****, P < 0.0001. Data show results from four (MNV-1) or three (Listeria) independent experiments with three replicates per experiment.

FIG 3

Compound C6 inhibits DUB activity in human cells and increases global ubiquitination in murine cells. (A) HA blot showing DUB inhibition by WP1130 and compound C6 at 5 μM and 10 μM. Arrows show regions of DUB activity inhibition. (B) Western blot using anti-ubiquitin antibody (poly- and monoubiquitin) in whole-cell lysates of murine RAW 264.7 cells with a 30-min 5 μM pretreatment (left) or 30 min at 2.5 μM and then at 1.0 μM (right) for the times indicated. Blots are representative of three independent experiments with similar results.

FIG 4

Growth of L. monocytogenes in the presence of compound C6. Compound C6 exerts a dose-dependent effect on the in vitro growth of L. monocytogenes 10403S in BHI broth culture. The graph plots the averages from triplicate samples for each of two independent experiments on a linear scale with error bars representing standard deviations.

FIG 5

Effect of compound C6 on RAW 264.7 cell viability under a variety of treatment conditions. Concentration and length of exposure of compound C6 on RAW 264.7 cells results in different levels of cell viability. (A) Three different strategies for testing the effects of C6 on RAW cell viability. Strategy 1, constant exposure; strategy 2, pretreatment only; strategy 3, varied exposures (higher pretreatment and lower concentration for duration, mimicking infection protocol). Dashed lines represent exposure of RAW cells to C6. (B to E) Resazurin cell viability assay of RAW 264.7 cells. The upper panels show cell viability at 8 h (B) and 24 h (C) of incubation according to strategy 1. The lower panels show cell viability according to strategies 2 and 3 for 8 h (D) and 24 h (E). Results are shown as the percentage of absorbance at 570 nm of untreated cells (percent viability). Indicated concentrations are in micromolars. VC, vehicle control. Vehicle control experiments used a DMSO volume/volume match with C6.

FIG 6

Intracellular growth of MNV-1 and Listeria monocytogenes in RAW 264.7 cells using a minimal pretreatment and subsequent return of compound C6 during incubation. RAW cells were pretreated with 2.5 μM compound C6 for 30 min before infection, and then medium containing 1.0 μM was added back to the culture after infection. (A) In vitro infection strategy for MNV. (B) MNV-1 viral loads were determined by plaque assay at the indicated time points. (C) In vitro infection strategy for L. monocytogenes. (D and E) Results of 8- and 24-h infections with L. monocytogenes using a high (1.0) and low (0.1) MOI, respectively. P < 0.05 (*) and P < 0.0001 (****) by Student's unpaired t test; unmarked time points showed no significant difference.

FIG 7

Compound C6 does not significantly affect uptake of MNV-1 into RAW 264.7 cells but may slow internalization of Listeria monocytogenes. (A) Difference in number of PFU in cells infected with light-sensitive MNV-1 between RAW cells treated with 2.5 μM compound C6 and vehicle-treated cells. Total, total input virus kept in the dark; replicated, viral particles that entered and were uncoated within 1 h. The graph represents a compilation of three independent experiments with two replicates per experiment. No PFU were detected for samples done entirely in the light. ns, not significant by Student's two-tailed t test. (B) Inside-out immunofluorescence microscopy of L. monocytogenes in RAW 264.7 cells 60 min postinfection (see Materials and Methods). Single, single-stained bacteria; double, double-stained bacteria. C6 cells were treated with 2.5 μM for 30 min, washed, infected with bacteria, washed again, and further treated with 1.0 μM C6 for 30 min after infection. Two independent experiments with two coverslips each were done, and at least 80 bacterium-positive cells were counted per coverslip. Ratios of single-stained to double-stained cells (with standard deviations [SD]) and total number of cells assessed (n) are the following: vehicle, 0.89 (0.29), n = 337; C6, 0.35 (0.01), n = 351.

FIG 8

Metabolic mouse liver microsome stability and short pharmacokinetic studies of compound C6 in C57BL/6 mice. (A) LC-MS/MS analysis of compound C6 in mouse liver microsomes. The metabolic half-life is 20.12 min. (B) Short pharmacokinetic analysis of compound C6 administered orally and intravenously (2 mice per condition). Concentrations in blood were measured at 0.5, 2, 4, and 7 h postadministration. Oral administration was 30 mg/kg, and i.v. injection was 10 mg/kg.