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Background The UW-STI consortium seeks to develop novel antimicrobials for the treatment of syndromically similar infections caused by Neisseria gonorrhoeae (GC), Chlamydia trachomatis (CT), and Mycoplasma genitalium (MG). Methods We utilize a structure-based validation pipeline embedded with a gated series of criteria for progressing druggable enzyme targets, and for identifying and advancing compounds active against these protein targets. The pipeline includes orthologous and essential enzyme target identification, structure determination, compound library screening, antimicrobial susceptibility testing, hit optimization, and chemical-genetic target validation. Results To date, we have identified over 80 enzyme candidates that are essential, single copy genes in both GC and MG; 7 GC structures, 1 CT structure and 1 MG structure have been solved by crystallography, and soluble expression has been achieved for 19 GC, 20 CT, and 3 MG recombinant enzymes. Several structures common to two bacteria have been solved including tryptophan-tRNA synthetase, lysyl-tRNA synthetase, and ribose-5-phosphate isomerase A/B. Phenylalanyl-tRNA synthetase (PheRS) is among our highest priority targets and is presented as a proof of concept for multi-organism drug development. PheRS is a validated drug target with divergence from its human counterpart, as modeled by the group. In lieu of crystal structures, the GC PheRS alpha and beta complex was modeled using the Rosetta software suite. Multiple cloning and expression strategies have been employed including surface mutations, solubility tags, engineered truncations, and co-expression of both subunits, in hopes of producing crystals. A PheRS001 inhibitor was synthesized from published literature and proved active against GC PheRS with an IC50 of 93 nM, and in antimicrobial testing against all three bacteria: 120 µg/ml (CT MIC) and 18 µg/ml (MG and GC MIC). Conclusion PheRS is a promising example of our pipeline capabilities in our three-pronged approach to produce 5–10 therapeutic leads and aid in the global fight against antibiotic resistance in sexually-transmitted bacterial infections. Disclosure No significant relationships.

We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from T. gondii. The current work, through structure–activity relationship studies, led to the discovery of compounds (34 and 35) with improved characteristics over the starting inhibitor 1 in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds 34 and 35 were further demonstrated to be more effective than 1 in a mouse infection model and markedly reduced the amount of T. gondii in the brain, spleen, and peritoneal fluid, and 35 given at 20 mg/kg eliminated T. gondii from the peritoneal fluid.

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasiteTrypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure ofT. cruzihistidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind toT. cruziHisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors.T. cruziHisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition ofT. cruziHisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

New therapies are needed for the treatment of toxoplasmosis, which is a disease caused by the protozoan parasite Toxoplasma gondii. To this end, we previously developed a potent and selective inhibitor (compound 1) of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) that possesses anti-toxoplasmosis activity in vitro and in vivo. Unfortunately, 1 has potent human Ether-à-go-go-Related Gene (hERG) inhibitory activity, associated with long Q-T syndrome, and, consequently, presents a cardiotoxicity risk. Here, we describe the identification of an optimized TgCDPK1 inhibitor 32, which does not have a hERG liability and possesses a favorable pharmacokinetic profile in small and large animals. 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing the amount of parasite in the brain, spleen, and peritoneal fluid and reducing brain cysts by >85%. These properties make 32 a promising lead for the development of a new anti-toxoplasmosis therapy.

We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from T. gondii. The current work, through structure–activity relationship studies, led to the discovery of compounds (34 and 35) with improved characteristics over the starting inhibitor 1 in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds 34 and 35 were further demonstrated to be more effective than 1 in a mouse infection model and markedly reduced the amount of T. gondii in the brain, spleen, and peritoneal fluid, and 35 given at 20 mg/kg eliminated T. gondii from the peritoneal fluid.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known "bumped kinase inhibitor" to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.