Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

In Vivo Efficacy of VT-1129 against Experimental Cryptococcal Meningitis with the Use of a Loading Dose-Maintenance Dose Administration Strategy.

VT-1129 is a novel fungal enzyme-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and our desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model loading dose-maintenance dose regimens to generate different steady-state concentrations. Mice were inoculated intracranially with Cryptococcus neoformans, and oral treatment began 1 day later. Treatment consisted of placebo or one of three VT-1129 loading dose-maintenance dose regimens, i.e., loading dose of 1, 3, or 30 mg/kg on day 1, followed by once-daily maintenance doses of 0.15, 0.5, or 5 mg/kg, respectively. In the fungal burden arm, therapy continued for 14 days and brains were collected on day 15 for fungal burden assessments. In the survival arm, treatment continued for 10 days, after which mice were monitored without therapy until day 30. VT-1129 plasma and brain concentrations were also measured. All VT-1129 doses significantly improved survival and reduced fungal burdens, compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions (R2 = 0.72 and R2 = 0.67, respectively), with a plasma concentration of 1 µg/ml yielding a reduction of ∼5 log10 CFU/g. With the highest loading dose-maintenance dose regimen, fungal burdens were undetectable in one-half of the mice in the fungal burden arm and in one-fourth of the mice in the survival arm, 20 days after the final dose. These data support a loading dose-maintenance dose strategy for quickly reaching highly efficacious VT-1129 concentrations for treating cryptococcal meningitis.

The Fungal Cyp51 Inhibitor VT-1129 Is Efficacious in an Experimental Model of Cryptococcal Meningitis.

Cryptococcal meningitis is a significant cause of morbidity and mortality in immunocompromised patients. VT-1129 is a novel fungus-specific Cyp51 inhibitor with potent in vitro activity against Cryptococcus species. Our objective was to evaluate the in vivo efficacy of VT-1129 against cryptococcal meningitis. Mice were inoculated intracranially with Cryptococcus neoformans Oral treatment with VT-1129, fluconazole, or placebo began 1 day later and continued for either 7 or 14 days, and brains and plasma were collected on day 8 or 15, 1 day after therapy ended, and the fungal burden was assessed. In the survival study, treatment continued until day 10 or day 28, after which mice were monitored off therapy until day 30 or day 60, respectively, to assess survival. The fungal burden was also assessed in the survival arm. VT-1129 plasma and brain concentrations were also measured. VT-1129 reached a significant maximal survival benefit (100%) at a dose of 20 mg/kg of body weight once daily. VT-1129 at doses of ≥0.3 mg/kg/day and each dose of fluconazole significantly reduced the brain tissue fungal burden compared to that in the control after both 7 and 14 days of dosing. The fungal burden was also undetectable in most mice treated with a dose of ≥3 mg/kg/day, even ≥20 days after dosing had stopped, in the survival arm. In contrast, rebounds in fungal burden were observed with fluconazole. These results are consistent with the VT-1129 concentrations, which remained elevated long after dosing had stopped. These data demonstrate the potential utility of VT-1129 to have a marked impact in the treatment of cryptococcal meningitis.

Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT) has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids. METHODOLOGY/PRINCIPAL FINDINGS: Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8) have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors. CONCLUSIONS/SIGNIFICANCE: Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy.

Assessment of the anterior maxilla in orthodontic patients using upper anterior occlusal radiographs and dental panoramic tomography: a comparison.

OBJECTIVE: The aim of this study was to establish if dental panoramic tomographic (DPT) radiographs provide a reliable means of assessing the anterior maxilla in new orthodontic patients. STUDY DESIGN: Two hundred fifty pairs of upper anterior occlusal (UAO) and DPT radiographs were randomly selected from the records of new orthodontic patients. Ten observers each rated 50 pairs of radiographs for specific risk factors as well as for a measure of adequacy. Correlations between the 2 radiograph types and inter/intraobserver reliability were computed using Cohen kappa test. RESULTS: Intraobserver reliability was good for detection of impacted teeth/supernumeraries/periapical pathology (kappa = 0.711-1.0). Reliability was more variable for the specific root morphologies, with blunt and bent roots being reassessed as such most reliably (kappa = 0.259-0.533). There was low interobserver reliability (kappa = 0.327 for UAO and kappa = 0.223 for DPT) for "normal" or "not normal" root morphology. For every variable, however, the interexaminer agreement was higher when using UAO radiographs rather than DPTs. Observers were best able to agree on the diagnosis of eroded/resorbed roots on the UAO (kappa = 0.402) and blunt roots (kappa = 0.303) on the DPT radiographs. With UAO as the gold (reference) standard for grading root morphology, DPT had a sensitivity of 45.6% and a specificity of 71.4% for detecting abnormal root form. Risk factors were more likely to be detected on the UAO radiographs than the DPTs. DPTs had a poor ability to detect abnormal root form. DPTs were more likely to be rated as "inadequate" than UAO radiographs. On 6 occasions, supernumerary teeth that were observed on the UAO were missed on the DPT. CONCLUSION: The DPT is not an accurate means of screening the anterior maxilla prior to orthodontic treatment.