Eberhard, C. D. | bioRxiv Science

bioRxiv2023

Tenofovir Activation is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection, and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can form tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, in vitro, and identify nine missense mutations (C74S, R96P, S128R, R132H, R172P, R236Q, C283S, R292Q, and H296R) that diminish this activity. Additional characterization of these mutations reveal that five (R96P, R132H, R236Q, C283S, and R292Q) have ATP dephosphorylation catalytic efficiencies less than 20% of wild-type (WT), and seven (C74S, R96P, R132H, R172P, R236Q, C283S, and H296P) induce thermal instabilities. To determine the extent CKB contributes to TFV activation in vivo, we generated a CKB knockout mouse strain, Ckbtm1Nnb. Using an in vitro assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5% and 77.4% less TFV-DP than wild-type brain lysates of the same sex, respectively. Additionally, we observe that Ckbtm1Nnb male mice treated with tenofovir disoproxil fumarate for 14 days exhibit a 22.8% reduction in TFV activation in liver compared to wild-type male mice. Lastly, we utilize mass spectrometry-based proteomics to elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how loss of CKB may be impacting tenofovir activation in these tissues. Together, our data suggest that disruptions in CKB may lower levels of active drug in brain and liver. ABSTRACT GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/559370v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1f8d26aorg.highwire.dtl.DTLVardef@be3d8corg.highwire.dtl.DTLVardef@1bab6d0org.highwire.dtl.DTLVardef@1381293_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

EstG is a novel esterase required for cell envelope integrity

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the -proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

microbiology↗