Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis

Background & Aims Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC’s anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis. Methods Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC’s antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses. Results CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight. Conclusions CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC’s antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475)

Mutations in the Plasmodium falciparum

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites

Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites

Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL), a Resistance Mechanism for Two Distinct Compound Classes

MMV007564 is a novel antimalarial benzimidazolyl piperidine chemotype identified in cellular screens. To identify the genetic determinant of MMV007564 resistance, parasites were cultured in the presence of the compound to generate resistant lines. Whole genome sequencing revealed distinct mutations in the gene named Plasmodium falciparum cyclic amine resistance locus (<i>pfcarl</i>), encoding a conserved protein of unknown function. Mutations in <i>pfcarl</i> are strongly associated with resistance to a structurally unrelated class of compounds, the imidazolopiperazines, including KAF156, currently in clinical trials. Our data demonstrate that <i>pfcarl</i> mutations confer resistance to two distinct compound classes, benzimidazolyl piperidines and imidazolopiperazines. However, MMV007564 and the imidazolopiperazines, KAF156 and GNF179, have different timings of action in the asexual blood stage and different potencies against the liver and sexual blood stages. These data suggest that <i>pfcarl</i> is a multidrug-resistance gene rather than a common target for benzimidazolyl piperidines and imidazolopiperazines

SaLSSA—a low cost method for assessing the malaria transmission-blocking potential of small molecules

Effective prevention of malaria transmission is an important component of malaria control. However, most methods designed to detect whether a small molecule can block transmission are relatively low throughput and cannot be applied routinely to large chemical libraries nor can they be used to routinely calculate inhibition constants. Here we describe a ultrahigh throughput and cost effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules that have the capacity to block malaria transmission. In analysis of 13850 compounds our data show that >90% of well characterized and clinically used antimalarial compounds, including endoperoxides and 4-aminoquinolines, as well as compounds discovered by phenotypic screening of asexual blood stages lose most their killing activity as parasites develop into mature, metabolically quiescent stage V gametocytes. On the other hand, our data show that compounds with consistent low nanomolar transmission-blocking activity exist. Similar compounds can be found at a rate of 3% in blood-stage selected libraries and 0.16% in unbiased chemical libraries, including a small diversity-oriented synthesis library. We identify several scaffold families including thioureas and napthoquinones that are active in parasite sexual and asexual stages as well as a few compound classes whose activity is limited to sexual stages. The data highlight the substantial physiological differences between sexual and asexual parasites and provide starting points for altruistic transmission-blocking drugs

High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission

SummaryPreventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs

Prev Chronic Dis

IntroductionHealth-risk behaviors such as eating poorly, being physically inactive, and smoking contribute to the leading causes of morbidity and mortality in the United States and are often established during adolescence and young adulthood. The objectives of this study were to characterize the health-risk behaviors of young adults (aged 18\u201324 years) using a large population-based survey of Americans and to determine if behaviors of this group differ by weight category, as assessed by body mass index (BMI).MethodsPrevalence estimates for selected health-risk behaviors were calculated for respondents aged 18 to 24 years to the 2003 Behavioral Risk Factor Surveillance System (BRFSS). Respondents were categorized by BMI, and comparisons between sex and race and ethnicity were made within the overweight and obese categories.ResultsMore than three quarters (78.4%) of respondents consumed fewer than five fruits and vegetables per day, 43.2% reported insufficient or no physical activity, 28.9% were current smokers, 30.1% reported binge drinking, and 11.9% reported frequent mental distress. One quarter (26.1%) of respondents were overweight, and 13.6% were obese. Of obese young adults, 67.2% reported that they currently were trying to lose weight; however, only 24.3% reported having received professional advice to lose weight. More obese women (34.2%) than obese men (16.7%) reported having received professional advice to lose weight. Only 19.1% of obese non-Hispanic white respondents had received professional advice to lose weight compared with 28.0% of obese Hispanic respondents and 30.6% of obese non-Hispanic black respondents.ConclusionMany young adults engage in unhealthy behaviors, and differences exist in health-risk behaviors by BMI category and specifically by sex and race and ethnicity within BMI categories. The transition from adolescence to adulthood may be an opportune time for intervening to prevent future chronic disease.2007720

A broad analysis of resistance development in the malaria parasite

Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.Bill and Melinda Gates FoundationNational Institute of Allergy and Infectious DiseasesUniversity of California San Diego Genetics Training ProgramNational Institute of General Medical SciencesDepto. de Bioquímica y Biología MolecularFac. de FarmaciaTRUEpu