Short-course combination treatment for experimental chronic Chagas disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of people in the Americas and across the world, leading to considerable morbidity and mortality. Current treatment options, benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead to adverse side effects because of long treatment durations. Better treatment options are therefore urgently required. Here, we describe a pyrrolopyrimidine series, identified through phenotypic screening, that offers an opportunity to improve on current treatments. In vitro cell-based washout assays demonstrate that compounds in the series are incapable of killing all parasites; however, combining these pyrrolopyrimidines with a subefficacious dose of BNZ can clear all parasites in vitro after 5 days. These findings were replicated in a clinically predictive in vivo model of chronic Chagas disease, where 5 days of treatment with the combination was sufficient to prevent parasite relapse. Comprehensive mechanism of action studies, supported by ligand-structure modeling, show that compounds from this pyrrolopyrimidine series inhibit the Qi active site of T. cruzi cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Knowledge of the molecular target enabled a cascade of assays to be assembled to evaluate selectivity over the human cytochrome b homolog. As a result, a highly selective and efficacious lead compound was identified. The combination of our lead compound with BNZ rapidly clears T. cruzi parasites, both in vitro and in vivo, and shows great potential to overcome key issues associated with currently available treatments.

DNDI-6174 is a preclinical candidate for visceral leishmaniasis that targets the cytochrome bc1.

New drugs for visceral leishmaniasis that are safe, low cost, and adapted to the field are urgently required. Despite concerted efforts over the last several years, the number of new chemical entities that are suitable for clinical development for the treatment of Leishmania remains low. Here, we describe the discovery and preclinical development of DNDI-6174, an inhibitor of Leishmania cytochrome bc1 complex activity that originated from a phenotypically identified pyrrolopyrimidine series. This compound fulfills all target candidate profile criteria required for progression into preclinical development. In addition to good metabolic stability and pharmacokinetic properties, DNDI-6174 demonstrates potent in vitro activity against a variety of Leishmania species and can reduce parasite burden in animal models of infection, with the potential to approach sterile cure. No major flags were identified in preliminary safety studies, including an exploratory 14-day toxicology study in the rat. DNDI-6174 is a cytochrome bc1 complex inhibitor with acceptable development properties to enter preclinical development for visceral leishmaniasis.

mTORC1 amplifies the ATF4-dependent de novo serine-glycine pathway to supply glycine during TGF-ß1-induced collagen biosynthesis.

The differentiation of fibroblasts into a transient population of highly activated, extracellular matrix (ECM)-producing myofibroblasts at sites of tissue injury is critical for normal tissue repair. Excessive myofibroblast accumulation and persistence, often as a result of a failure to undergo apoptosis when tissue repair is complete, lead to pathological fibrosis and are also features of the stromal response in cancer. Myofibroblast differentiation is accompanied by changes in cellular metabolism, including increased glycolysis, to meet the biosynthetic demands of enhanced ECM production. Here, we showed that transforming growth factor-ß1 (TGF-ß1), the key pro-fibrotic cytokine implicated in multiple fibrotic conditions, increased the production of activating transcription factor 4 (ATF4), the transcriptional master regulator of amino acid metabolism, to supply glucose-derived glycine to meet the amino acid requirements associated with enhanced collagen production in response to myofibroblast differentiation. We further delineated the signaling pathways involved and showed that TGF-ß1-induced ATF4 production depended on cooperation between canonical TGF-ß1 signaling through Smad3 and activation of mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). ATF4, in turn, promoted the transcription of genes encoding enzymes of the de novo serine-glycine biosynthetic pathway and glucose transporter 1 (GLUT1). Our findings suggest that targeting the TGF-ß1-mTORC1-ATF4 axis may represent a novel therapeutic strategy for interfering with myofibroblast function in fibrosis and potentially in other conditions, including cancer.

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Effects of mid-respiratory chain inhibition on mitochondrial function in vitro and in vivo.

Relating the in vitro mitochondrial effects of drug candidates to likely in vivo outcomes remains challenging. Better understanding of this relationship, alongside improved methods to assess mitochondrial dysfunction in vivo, would both guide safer drug candidate selection and better support discovery programmes targeting mitochondria for pharmacological intervention. The aim of this study was to profile the in vivo effects of a compound with suspected complex III electron transport chain (ETC) inhibitory activity (GSK932121A) at doses associated with clinical signs, and relate findings back to in vitro data with the same compound. Control liver mitochondria or HepG2 cells were treated in vitro with GSK932121A to assess mitochondrial effects on both calcium retention capacity (CRC) and oxygen consumption rate (OCR) respectively. The same assessments were then performed on liver mitochondria isolated from Crl:CD(SD) rats, 5 hours following intraperitoneal (IP) administration of GSK932121A. Lactate/pyruvate assessment, hepatic microscopy, blood gas analysis, glutathione profiling and transcriptomics were used to characterise the acute toxicity. In vivo, GSK932121A caused hypothermia, increased levels of hepatocellular oxidative stress and a metabolic shift in energy production, resulting in an increased lactate/pyruvate ratio, liver steatosis and glycogen depletion, together with gene expression changes indicative of a fasted state. As would be expected of an ETC inhibitor, GSK932121A reduced the CRC of liver mitochondria isolated from naive control animals and the OCR of HepG2 cells when treated directly in vitro. In contrast, mitochondria isolated from animals treated with GSK932121A in vivo unexpectedly showed an increase in CRC and basal OCR. Whilst seemingly contradictory, these differences likely reflect an adapted state in vivo resulting from the initial insult in combination with compensatory changes made by the tissue to maintain energy production. Only the initial, unconfounded, response is observable in vitro. These findings improve current understanding of the toxicological and molecular consequences of ETC inhibition. Furthermore, this work highlights key differences in the way that mitochondrial perturbation is manifest in vivo versus in vitro in terms of functional endpoints and helps guide endpoint selection for future studies with potential mitochondrial toxicants or drugs designed to modulate mitochondrial function for therapeutic benefit.