An oral caspase inhibitor as monotherapy or with antibiotics eradicates MRSA skin infections in mice

Staphylococcus aureus is the leading cause of skin and soft tissue infections. With the emergence of antibiotic‐resistant bacteria, there is an unmet clinical need to develop immune‐based therapies to treat skin infections. Previously, we have shown pan‐caspase inhibition as a potential host‐directed immunotherapy against community‐acquired methicillin‐resistant S aureus (CA‐MRSA) and other bacterial skin infections. Here, we evaluated the role of irreversible pan‐caspase inhibitor emricasan as a monotherapy and an adjunctive with a standard‐of‐care antibiotic, doxycycline, as potential host‐directed immunotherapies against S. aureus skin infections in vivo. We used the established CA‐MRSA strain USA300 on the dorsum of WT C57BL/6J mice and monitored lesion size and bacterial burden noninvasively, and longitudinally over 14 days with in vivo bioluminescence imaging (BLI). Mice in four groups placebo (0.5% carboxymethyl cellulose [CMC] solution), placebo plus doxycycline (100 mg/kg), emricasan (40 mg/kg) plus doxycycline, and emricasan only were treated orally twice daily by oral gavage for 7 days, starting at 4 h after injection of S aureus. When compared with placebo, all three groups, placebo plus doxycycline, emricasan plus doxycycline, and emricasan treated group, exhibited biological effect, with reduction of both the lesion size (*p = .0277, ****p < .0001, ****p < .0001, respectively) and bacterial burden (***p = .003, ****p < .0001, ****p < .0001, respectively). Importantly, the efficacy of emricasan against S. aureus was not due to direct antibacterial activity. Collectively, pan‐caspase inhibitor emricasan and emricasan plus doxycycline reduced both the lesion size and bacterial burden in vivo, and emricasan is a potential host‐directed immunotherapy against MRSA skin infections in a preclinical mouse model.

Staphylococcus aureus (S. aureus) infection is currently the most common cause of skin and soft tissue infections, and increasing antibiotic-resistant strains, such as community-acquired methicillin-resistant S. aureus (CA-MRSA), are creating a serious public health threat (DMPD et al., 2020).
Currently, there is a high unmet clinical need for immunotherapy that can enhance the immune response and provide adjunctive therapy plus antibiotics or act as a monotherapy to promote clearance of S. aureus skin infections, especially those infections caused by CA-MRSA.However, the immune mechanisms that promote clearance of S. aureus skin infections are incompletely understood, and this has greatly hindered the development of effective immunotherapy or vaccine against this important human pathogen (Alphonse et al., 2021).We previously reported that a single intraperitoneal dose of the pan-caspase inhibitor, quinoline-valine-aspartic acid-difluorophenoxymethyl ketone (Q-VD-OPH), enhanced the immune response and promoted clearance against a CA-MRSA S. aureus skin infection in an established mouse model.The efficacy of Q-VD-OPH against S. aureus was found to be due to its modulation of cell death pathways, including reduced apoptosis of neutrophils and monocytes and increased necroptosis in macrophages.
This led to the increased immune cell survival and function of neutrophils and monocytes/macrophages, as well as increased tumor necrosis factor (TNF) responses that collectively promoted bacterial clearance as a single-dose monotherapy without concomitant antibiotic therapy (Alphonse et al., 2021).However, Q-VD-OPH requires parenteral administration and has mainly been used for research purposes, and clinical trials have not progressed.Furthermore, it is unclear whether a pan-caspase inhibitor might have improved efficacy if combined as adjunctive therapy when given with antibiotics or if it can work well as a monotherapy compared with conventional antibiotic therapies.
In this report, we evaluated the in vivo efficacy of emricasan, a known irreversible pan-caspase inhibitor that can be safely administered orally in humans.Specifically, emricasan has been shown to be safe in Phase 1 and 2 clinical trials in healthy humans and patients with viral infections (e.g., mild symptomatic COVID-19 patients; Histogen, 2022) and against patients with liver disorders (e.g., nonalcoholic steatohepatitis and fibrosis, islet transplantation, liver transplantation, and chronic hepatitis C infections) (Harrison et al., 2020; Safety and Tolerability of Emricasan in Symptomatic Outpatients Diagnosed with Mild-COVID-19).Therefore, this brief report evaluated emricasan as adjunctive therapy with the commonly used oral antibiotic for CA-MRSA, doxycycline, and as a monotherapy (i.e., without doxycycline therapy) in a mouse model of CA-MRSA infection in vivo.

| Mice
All mice were 6-8 weeks female C57BL/6 background, obtained from the Jackson laboratories.All animal studies were approved by the Johns Hopkins University Animal Care and Use Committee.All experiments were not blinded, and there were no exclusions of data or exclusion criteria to report in this study.

| Reagents
The pan-caspase inhibitor emricasan was provided as a dry powder and dissolved in carboxy methyl cellulose (CMC) solution as described in the protocol by Histogen.Emricasan (40 mg/kg) in 0.5% CMC at 20 mg/kg/dose was administered twice daily by oral gavage for 7 days.Doxycycline hyclate (100 mg/kg) (Millipore Sigma) was administered twice daily by oral gavage.

| Treatment groups
Mice (n = 5) in each group of four groups (placebo, placebo plus doxycycline, emricasan plus doxycycline, and emricasan) were treated orally twice daily by oral gavage with or without doxycycline, with placebo (0.5% CMC solution) or emricasan (40 mg/kg) at 20 mg/kg/ dose in 0.5% CMC solution for 7 days, starting at 4 h after injection until the duration of 14 days.Doxycycline (100 mg/kg) was administered twice daily by oral gavage following established protocols (Guo et al., 2013).

| Bacterial strains
The bioluminescent S. aureus USA300 LAC::lux strain was previously generated from the community-acquired methicillinresistant S. aureus (MRSA) USA300 LAC isolate obtained from a skin infection outbreak in the Los Angeles County Jail (Los Angeles, California, USA) and was kindly provided by Tammy Kielian (University of Nebraska).

| Bacterial preparation
As previously described, S. aureus USA300 LAC::lux bacteria were streaked onto a tryptic soy agar (TSA) plate (tryptic soy broth [TSB] plus 1.5% bacto agar; BD Biosciences) and grown overnight at 37°C in a bacterial incubator.Single colonies were cultured in TSB at 37°C in a shaking incubator (240 rpm) overnight (~18 h), followed by a 1:50 subculture at 37°C for 2 h to obtain midlogarithmic growth phase bacteria (Alphonse et al., 2021).

| Measurement of total lesion size
Total lesion size (cm 2 ) was measured from digital photographs of the back skin of anesthetized mice (2% isoflurane) using ImageJ software (https://imagej.nih.gov/ij/,NIH) and a millimeter ruler as a reference.In vivo bioluminescence imaging (BLI) to provide an approximation of the in vivo bacterial burden (when used in conjunction with the USA300 LAC::lux bioluminescent bacterial strain) was performed on anesthetized mice (2% isoflurane) using Lumina III in vivo imaging system (IVIS) (Perkin Elmer).For in vivo BLI, data are presented on a color scale overlaid on a grayscale photograph of the mice and quantified within a 1 × 10 3 pixel circular region of interest (ROI) as total flux (photons/s).

| Bacterial growth kinetics
Bacterial broth culture of S. aureus USA300 LAC::lux was prepared as described above.After overnight culture, the cultures were diluted 1:100 in their respective growth media.The bacterial cultures were either incubated with vehicle (Veh) or various logarithmic concentrations of emricasan (1 μg/mL, 10 μg/mL, 100 μg/mL, and 1000 μg/mL) in a total volume of 200 μL.The bacterial growth (OD600) was measured in triplicate for 10 h cultures at 37°C, and measurements recorded at 20-min intervals in a Gen5 plate reader (BioTek).

| Statistical analysis
Data for longitudinal comparisons across multiple groups (≥3 groups) were compared using a two-way analysis of variance multiple comparisons test.p Values from multiple comparisons were adjusted by the step-up Bonferroni method to control the overall family-wise error rate.Data are presented as means ± standard error of the mean (SEM).All statistical analyses were calculated using Prism software (GraphPad) for macOS (v11).p < .05 was considered statistically significant.

| RESULTS
The immune mechanisms that protect against S. aureus infections remains elusive and have hindered the development of an effective immunotherapy or vaccine against this human pathogen that is becoming increasingly resistant to antibiotics (Alphonse et al., 2021).
Previously we have shown that pan-caspase inhibition could be a potential host-directed immunotherapy against MRSA.Furthermore, we showed that the potential inhibition of cell death pathways (pyroptosis, apoptosis, and necroptosis) by the systemically administered pan-caspase inhibitor Q-VD-OPH promoted host defense against a mouse model of CA-MRSA skin infection (Alphonse et al., 2021).Here we sought to evaluate the in vivo efficacy of emricasan, a known irreversible pan-caspase inhibitor that can be safely administered orally in humans.Wild-type C57BL/6 mice were separated into four groups (placebo, placebo + doxycycline, emricasan + doxycycline, and emricasan alone), with five mice/experimental group.The treatments were initiated 4 h after S. aureus intradermal skin injection (using the bioluminescent CA-MRSA strain USA300 LAC::lux) through 14 days, as was previously described in an established approach comparing the efficacy of different antibiotics in an S. aureus skin infection mouse model (Guo et al., 2013).Compared with placebo alone, placebo + doxycycline, emricasan + doxycycline, and emricasan alone, all had statistically significant (p < .05)reductions in lesion size (*p < .05,****p < .0001,and ****p < .0001,respectively) (Figure 1a) and in vivo BLI (***p < .01,****p < .0001,and ****p < .0001,respectively) (Figure 1b).Remarkably, and perhaps unexpectedly, there was no significant difference between the lesion sizes (p = .998)and in vivo BLI signals (p = .2577)between the emricasan + doxycycline and emricasan alone experimental groups, suggesting that emricasan alone as monotherapy as effective when emricasan was combined with doxycycline in promoting immune clearance of the CA-MRSA skin infection.The lack of synergism of ermricasan and doxycycline could have been due to a number of factors.In particular, emricasan resulted in rapid bacterial clearance (i.e., in the first 1-3 days) that could have obscured the antibacterial effects of doxycycline, which we previously showed to take >5 days to reach the same degree of bacterial clearance in a similar mouse model (Guo et al., 2013).The mean lesion size (mm) ± SEM and mean in vivo BLI signals (photons/ second) ± SEM (log scale) measurements are shown in Figure 1c,d.
The BLI highly correlates with the burden of live bacterial CFU in the skin during the infection (R 2 = .9996)(Guo et al., 2013).Visible skin lesions form soon after bacteria skin inoculation as a result of the activity of secreted cytolytic toxins from S. aureus (especially α-toxin), F I G U R E 2 Emricasan does not have direct in vitro antibacterial activity against Staphylococcus aureus.Bacterial broth cultures were incubated with vehicle (Veh) or various concentrations of emricasan (1 μg/mL, 10 μg/mL, 100 μg/mL, and 1000 μg/mL).The bacterial growth (OD 600 ) was measured (n = 3) in triplicate for 10 h, with measurements recorded at 20-min intervals.