ALS-4 is a small molecule which inhibits dehydrosqualene desaturase of S. aureus (incl. MRSA), an enzyme that is critically involved in the biosynthesis of staphyloxanthin, a commonly visible “golden pigment” covering the bacteria. Staphyloxanthin is believed to be primarily responsible for the bacteria’s defense mechanism against the attack from reactive oxygen species (ROS) deployed by phagocytic cells and neutrophils.1
Through inhibiting the production of staphyloxanthin, we believe that ALS-4 renders S. aureus highly susceptible to the host’s immune defense (see below for in vivo data and experimental outline). This novel mechanism is significantly different from the bactericidal approach found in currently marketed antibiotics used to treat S. aureus, which are experiencing increasing drug resistance issues2. Specifically, MRSA infections in humans typically exhibit high rates of morbidity and mortality and can cause metastatic or complicated infections such as infective endocarditis or sepsis, with relapse and hospital readmission after S. aureus bacteremia common and costly3.
Based on our testing in a rat bacteremia survival model, a lethal (109 CFU) dose of MRSA (USA300-LAC) was introduced through the tail vein. ALS-4 was administered orally at 10mg/kg per animal 30 minutes after the infection for twice a day thereafter (N=9). A control untreated group was given a sterile vehicle solution (N=9). Survival was monitored for 7 days. 0 out of 9 animals (0%) in the control untreated group survived past day 4, in contrast, 5 out of 9 animals (56%) treated with ALS-4 survived past day 7, which is determined to be statistically significant compared with the control group (p=0.013).
In addition we conducted a study in a non-lethal rat bacteremia infection model. The animals were challenged with a non-lethal (107 CFU) dose of MRSA (USA300-LAC) through the tail vein. In order to simulate a more realistic clinical scenario, treatment was introduced 14-days after the model induction, where ALS-4 was administered orally twice a day at 10mg/kg per animal (N=8). A control untreated group was given a sterile vehicle solution (N=8). After 7 days of ALS-4 treatment, the kidneys were collected and the bacterial titers were measured. Remarkably, ALS-4 reduced the organ bacterial load by 99.5%, from 63,096±18 CFU/g in the control group to 316±49 CFU/g in the ALS-4 treated group, which is determined to be statistically significant (p=0.01).
Last but not least, ALS-4 has successfully inhibited staphyloxanthin production in 11 strains of S. aureus. These include 5 strains of Methicillin-sensitive S. aureus (MSSA): SH1000, HG003, USA300-JE2, Newman, and ATCC29213 with an IC50 of 70.5±6nM, 54.4±4nM, 37.7±4nM, 23.7±1nM, and 30.02±5nM respectively; 5 strains of Methicillin-resistant S. aureus (MRSA): USA300, USA300-3, USA300-LAC, ST239III, and COL, with an IC50 of 30.8±5nM, 42.8±6nM, 43.6±5nM, 16.3±8nM, and 0.9±1nM respectively; and 1 strain of vancomycin-intermediate S. aureus (VISA), Mu3 with an IC50 of 2.6±1nM.
Based on our testing, we believe ALS-4 increases the susceptibility of S. aureus including MRSA to oxidative damage by inhibiting production of staphyloxanthin,. In a hydrogen peroxide killing assay, after the addition of 1.5% H2O2, ALS-4 demonstrated an additional reduction of bacterial CFU by 93.5%, from 61,600±6437 CFU/ml in the untreated group to 4,000±230 CFU/ml in the ALS-4 treated group, which is determined to be statistically significant (p=0.003).
With respect to the study carried out to investigate the capability of ALS-4 to induce antibiotic resistance in S. aureus after prolonged exposure, USA300-LAC was cultured in 3 different conditions for 10 days. For the treatment group 1 µM of ALS-4 was added; for the positive control group 0.12 µg/mL of clindamycin and 16 µg/mL of erythromycin was added from day 1 to day 4, after which clindamycin was withdrawn. For the negative control group, dimethyl sulfoxide (DMSO) was added. On day 11, the bacteria were harvested and then cultured for 16 hr for the determination of the MIC of clindamycin. The prolonged exposure to ALS-4 or DMSO does not affect the MIC value of clindamycin (0.12 µg/mL); while the prolonged exposure to clindamycin + erythromycin triggers antibiotics resistance rapidly with the MIC increased from 0.12 µg/mL to greater than 5 µg/mL.
Based on our study we believe ALS-4 is unlikely to be prone to drug resistance since it is non-bactericidal. Growth inhibition studies were performed on different strains of S. aureus and other bacteria, including 3 strains of MSSA (ATCC29212, SH1000 and HG003), 1 strain of MRSA (USA300), 1 strain of VISA (ATCC700698 Mu3), as well as 6 different bacteria (E. coli, A. baumannii, S. cerevisiae, B. subtilis, E. faecalis, and K. pneumoniae). In all of the tested strains of bacteria, no growth inhibition effect was observed at the highest tested concentration of ALS-4 (250uM). Therefore ALS-4 does not appear to have any direct bacteriostatic or bactericidal activity against many species of bacteria, thus greatly reducing the selection pressure for drug resistance to emerge.
We also assessed the potential impact on the efficacy of vancomycin, the mainstay of treatment for infections caused by MRSA, when used in conjunction with ALS-4. 8 different strains of S. aureus (USA300 FPR3757, USA300-3, USA300-LAC, USA300-JE2, Mu3, HG003, ATCC29213 and clinical isolate ST239III) were used in this study. Our data showed that no effect on the MIC of vancomycin was observed when the concentration of ALS-4 was below 25 μM. Therefore, we believe that ALS-4 does not interfere with the action of vancomycin.
In addition, compared with the current mainstay of treatment for S. aureus infections such as vancomycin or daptomycin which is typically administered in an IV injectable form (with the exception of an oral form vancomycin specifically for treatment of Clostridium difficile diarrhea and staphylococcal enterocolitis only), an oral active agent enables wider market penetration targeting both outpatient as well as potential prophylactic markets.
GLP Toxicity Data
ALS-4 is currently undergoing IND-enabling studies and has so far shown positive safety profiles. As elucidated in our previous press release dated September 9, 2019, ALS-4 did not show any mutagenicity in the in vitro Ames tests. Our currently generated in vitro micronucleus test results also showed that ALS-4 is not genotoxic, indicating the non-mutagenic nature of the drug. Furthermore, the results of the in vitro hERG assay study predicts a low risk of ALS-4 causing cardiac QT prolongation.
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About Aptorum Group Limited
Aptorum Group Limited (Nasdaq: APM) is a pharmaceutical company dedicated to developing and commercializing novel therapeutics to tackle unmet medical needs. Aptorum Group is pursuing therapeutic projects in orphan diseases, infectious diseases, metabolic diseases and other disease areas.
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1 mBio 2017 8(5): e01224-17
2 Microbiol Spectr. 2019 Mar;7(2)
3 Clin Infect Dis. 2019 Nov 27;69(12):2112-2118
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