Biology subjects

Teixeira, J. E.

Publications and source records attributed to Teixeira, J. E..

4 recordsLinked to original sources

bioRxiv2025

A Propidium Iodide-based In Vitro Screen of the "Bug Box" Against Babesia duncani Reveals Potent Inhibitors

Incidence and endemic range of human babesiosis are expanding. Standard therapy for human babesiosis consists of antimicrobials developed for other indications. While these treatments are adequate in immunocompetent hosts, infections in the immunocompromised can be severe, relapsing, and drug-resistant despite use of multi-drug regimens. Existing drugs are ineffective because they cannot safely achieve and maintain adequate serum concentrations to inhibit Babesia. Discovery of improved agents against Babesia spp. is of growing importance and efficient techniques for high throughput compound screening can assist in this effort. We developed a high throughput in vitro drug screening assay for Babesia duncani that is conducted in 384 well plates and makes use of the fluorescent DNA stain propidium iodide (PI) with relative fluorescence measured by a microplate reader. A Z factor of >0.81 was calculated which suggests an excellent ability to detect inhibitory compounds. A screen of the 41-compound library Structural Genomics Consortium Bug Box was conducted yielding five hits: trimethoprim, atovaquone, SDDC M7, diphenyleneiodonium chloride, and panobinostat. Panobinostat, a histone deacetylase complex (HDAC) inhibitor, was selected for further evaluation given that its target had not been previously explored in B. duncani. Follow up dose-response testing of structurally related compounds revealed multiple potential leads including nanatinostat and quisinostat, both of which were potent at the nanomolar level and showed favorable selectivity index in cytotoxicity studies. High throughput screening using PI and 384 well plates is an advance in drug discovery for babesiosis and HDAC inhibitors show promise as lead compounds worthy of further investigation.

microbiology↗

bioRxiv2023

Cryptosporidium Life Cycle Small Molecule Probing Implicates Translational Repression And An Apetala 2 Transcription Factor In Sexual Differentiation

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate sexual differentiation and maturation. mRNA-seq studies confirmed selective modulation of sexual differentiation for 10 hit compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in sexual differentiation. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for gamont formation.

microbiology↗

bioRxiv2023

Identification of potent and orally efficacious phosphodiesterase inhibitors in Cryptosporidium parvum-infected immunocompromised mice

Cryptosporidium species, mostly C. parvum and C. hominis in humans, are intestinal apicomplexan parasites that cause life-threatening diarrhea in young children and people with cell-mediated immune defects, such as due to AIDS. There is only one approved treatment for cryptosporidiosis, but it is ineffective for immunocompromised people and only modestly effective for children. In this study, screening 278 compounds from the Merck KGaA, Darmstadt, Germany collection and accelerated follow-up work enabled by prior investigation of the compounds resulted in identification of a series of pyrazolopyrimidine human phosphodiesterase (PDE)-V inhibitors with potent anticryptosporidial activity and efficacy following oral administration in C. parvum-infected immunocompromised mice. The novel PDE inhibitor leads (compounds PDEi2 and PDEi5) affect parasite egress from infected host cells. They have comparable activity against C. parvum and C. hominis, rapidly eliminate C. parvum in tissue culture, and have minimal off-target effects in a panel of safety screening assays. In comparison, the potent human PDE-V inhibitors sildenafil and the 4-aminoquinoline compound 7a have no useful activity against C. parvum. Based on homology modeling and in silico compound docking, PDEi5 interacts directly with an active-site metal ion and docks well to two C. parvum PDEs. In contrast, larger amino acid side groups (Val900/Tyr11128 and His884/Asn1112) in both C. parvum PDEs replace alanine in human PDE-V and block sildenafil binding, explaining its lack of efficacy. These results identify a promising new drug target and lead series for anticryptosporidial drug development and validates a route to target-based optimization.

microbiology↗

bioRxiv2021

Spontaneous selection of Cryptosporidium drug resistance in a calf model of infection

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last [~]10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50S of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence. ImportanceCryptosporidium is a leading protozoan cause of diarrhea in young children with no reliable treatment. We report results of a dairy calf drug efficacy trial and the spontaneous emergence of drug resistance. Cryptosporidium parvum infected calves initially improved with drug treatment, but infection relapsed in two animals. Parasites shed by each recrudescent calf had mutations in the gene encoding the drug target that altered its amino acid sequence. Recapitulation of the drug target mutations by CRISPR/Cas9 genome editing resulted in highly drug-resistant parasites, and recombinant mutant enzymes were resistant to inhibition. This is the first report of naturally emerging Cryptosporidium drug resistance. There is a currently a great opportunity to impact public health with new drugs to treat cryptosporidiosis, and this report highlights the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence in order to realize their full potential. One-sentence summaryDrug-target point mutations mediating anticryptosporidial resistance spontaneously arose in the dairy calf C. parvum infection model.

microbiology↗