Kirschning, A. | Search bioRxiv

bioRxiv2024

Cystobactamid off-target profiling reveals favorable safety, superoxide reduction, and SCARB1 inhibition in eukaryotes

Antimicrobial resistance (AMR) poses a fundamental global threat, necessitating new strategies for effective therapies. Cystobactamids (CYS), a class of antibacterial agents targeting bacterial gyrase and topoisomerase IV, represent a non-traditional chemical scaffold with broad-spectrum activity. For toxicological de-risking, we performed a comprehensive profiling on eukaryotic cells, focusing on cytotoxicity, genotoxicity, and mitochondrial toxicity, demonstrating cellular safety and superoxide scavenging properties. Studies in zebrafish embryos assessed developmental, cardiovascular, and hepatic toxicity, indicating a favorable in vivo safety profile. Metabolism studies revealed glucuronidation and amide bond hydrolysis as key pathways, whereby CYS metabolic stability substantially improved by cobicistat co-treatment. Affinity-based protein profiling identified the cholesterol- and HCV-receptor scavenger receptor class B member 1 (SCARB1) as a primary eukaryotic off-target protein, with cystobactamids shown to inhibit SCARB1s function, preventing hepatitis C virus pseudoparticle entry into cells. These findings suggest a high therapeutic potential for cystobactamids and highlight SCARB1 as a primary eukaryotic target.

pharmacology and toxicology↗

bioRxiv2021

Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer iPSC-based cellular therapies

Drug-inducible suicide systems may help to minimize risks of cellular therapies due to the tumor forming potential of human induced pluripotent stem cells (hiPSCs). Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed that showed elimination or silencing of the transgene. We have introduced a drug-inducible Caspase9 suicide system (iCASP9) into the AAVS1 safe harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. In mice, drug treatment generally led to rapid elimination of teratomas, but individual animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ~ 3x10-8. Transgene elimination, presumably via Loss of Heterozygosity (LoH), or methylation of the CAG promoter but not methylation of the O_SCPLOWPPPC_SCPLOWO_SCPCAP1C_SCPCAPO_SCPLOWRC_SCPLOWO_SCPCAP12C_SCPCAPO_SCPLOWCC_SCPLOW locus were identified as underlying mechanisms. In contrast, we never observed any escapees from biallelic iCASP9 hiPSCs, even after treatment of up to 0.8 billion hiPSCs. In conclusion, biallelic integration of an iCASP9 system in the AAVS1 locus may substantially contribute to the safety level of iPSC-based therapies, which should be calculated by relating clonal escapee frequencies to the cell number in tumors of a size that is readily detectable during routine screening procedures.

molecular biology↗