Anupam Pradhan

A novel multiple-stage antimalarial agent that inhibits protein synthesis

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication. Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na+ homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Lead optimization of 3-carboxyl-4(1H)-quinolones to deliver orally bioavailable antimalarials.

Malaria is a protozoal parasitic disease that is widespread in tropical and subtropical regions of Africa, Asia, and the Americas and causes more than 800,000 deaths per year. The continuing emergence of multidrug-resistant Plasmodium falciparum drives the ongoing need for the development of new and effective antimalarial drugs. Our previous work has explored the preliminary structural optimization of 4(1H)-quinolone ester derivatives, a new series of antimalarials related to the endochins. Herein, we report the lead optimization of 4(1H)-quinolones with a focus on improving both antimalarial potency and bioavailability. These studies led to the development of orally efficacious antimalarials including quinolone analogue 20g, a promising candidate for further optimization.

Impact of intrauterine tobacco exposure on fetal telomere length

OBJECTIVE We sought to investigate whether maternal smoking during pregnancy affects telomere length of the fetus. STUDY DESIGN Pregnant women were recruited on hospital admission at delivery. A self-report questionnaire and salivary cotinine test were used to confirm tobacco exposure. Neonatal umbilical cord blood samples were collected, and genomic DNA was isolated from cord blood leukocytes and was analyzed for fetal telomere length based on quantitative polymerase chain reaction. A ratio of relative telomere length was determined by telomere repeat copy number and single copy gene copy number (T/S ratio) and used to compare the telomere length of active, passive, and nonsmokers. Bootstrap and analysis of variance statistical methods were used to evaluate the relationship between prenatal smoking status and fetal telomere length. RESULTS Of the 86 women who were included in this study, approximately 69.8% of the participants were covered by Medicaid, and 55.8% of the participants were black or Hispanic. The overall mean T/S ratio was 0.8608 ± 1.0442. We noted an inverse relationship between smoking and fetal telomere length in a dose-response pattern (T/S ratio of nonsmokers that was more than passive smokers that was more than active smokers). Telomere length was significantly different for each pairwise comparison, and the greatest difference was between active and nonsmokers. CONCLUSION Our results provide the first evidence to demonstrate a positive association between shortened fetal telomere length and smoking during pregnancy. Our findings suggest the possibility of early intrauterine programming for accelerated aging that is the result of tobacco exposure.

Chemogenomic profiling of Plasmodium falciparum as a tool to aid antimalarial drug discovery

The spread of Plasmodium falciparum multidrug resistance highlights the urgency to discover new targets and chemical scaffolds. Unfortunately, lack of experimentally validated functional information about most P. falciparum genes remains a strategic hurdle. Chemogenomic profiling is an established tool for classification of drugs with similar mechanisms of action by comparing drug fitness profiles in a collection of mutants. Inferences of drug mechanisms of action and targets can be obtained by associations between shifts in drug fitness and specific genetic changes in the mutants. In this screen, P. falciparum, piggyBac single insertion mutants were profiled for altered responses to antimalarial drugs and metabolic inhibitors to create chemogenomic profiles. Drugs targeting the same pathway shared similar response profiles and multiple pairwise correlations of the chemogenomic profiles revealed novel insights into drugs’ mechanisms of action. A mutant of the artemisinin resistance candidate gene - “K13-propeller” gene (PF3D7_1343700) exhibited increased susceptibility to artemisinin drugs and identified a cluster of 7 mutants based on similar enhanced responses to the drugs tested. Our approach of chemogenomic profiling reveals artemisinin functional activity, linked by the unexpected drug-gene relationships of these mutants, to signal transduction and cell cycle regulation pathways.

Lead Optimization of Antimalarial Propafenone Analogues

Previously reported studies identified analogues of propafenone that had potent antimalarial activity, reduced cardiac ion channel activity, and properties that suggested the potential for clinical development for malaria. Careful examination of the bioavailability, pharmacokinetics, toxicology, and efficacy of this series of compounds using rodent models revealed orally bioavailable compounds that are nontoxic and suppress parasitemia in vivo. Although these compounds possess potential for further preclinical development, they also carry some significant challenges.

Association Between Maternal-Perceived Psychological Stress and Fetal Telomere Length.

Objective Our study aimed to investigate the association between maternal-perceived psychological stress and fetal telomere length. Methods We recruited women in labor upon hospital delivery admission. Based on responses to the Perceived Stress Scale, we categorized participants as having “high,” “normal,” or “low” perceived stress. We collected umbilical cord blood samples (N = 71) and isolated genomic DNA from cord blood leukocytes using quantitative polymerase chain reaction. We used a ratio of relative telomere length derived by telomere-to-single-copy gene ratio (T/S ratio). We applied analysis of variance and bootstrapping statistical procedures. Results Sixteen (22.5%) women were classified as having low perceived stress, 42 (59.2%) were classified as having normal perceived stress, and 13 (18.3%) were classified as having high perceived stress. Fetal telomere length differed significantly across the three stress groups in a dose–response pattern (T/S ratio of those with low perceived stress was greater than those with normal perceived stress, which was greater than those with high perceived stress [P < 0.05]). Conclusions Our findings support our hypothesis that maternal-perceived psychological stress during pregnancy is associated with shorter fetal telomere length and suggest maternal stress as a possible marker for early intrauterine programming for accelerated chromosomal aging.

A positive association between umbilical cord RBC folate and fetal TL at birth supports a potential for fetal reprogramming

Telomere length (TL) has been studied extensively in adults; however, limited information exists regarding maternal influences on TL in utero. The objective of this study was to investigate the relationship between fetal red blood cell (RBC) folate levels, a surrogate measure for maternal folate levels, and TL. We hypothesized that umbilical cord RBC folate concentrations would positively correlate with fetal TL. Data for this analysis were collected as part of a prospective cohort study that recruited pregnant women upon admission into labor and delivery. Cord blood was collected for 96 maternal-fetal dyads, and DNA analysis was performed using quantitative polymerase chain reaction. The telomere to single copy gene ratio method was used to determine TL, and RBC folate levels were measured. Statistical analysis was conducted by incorporating a bootstrapping approach into generalized linear modeling-based analyses. Consistent significant positive correlations were observed between RBC folate and TL (telomere to single copy gene ratio) with 9880 of the 10000 (98.8%) iterations performed having a P value less than .05. Our study shows a positive association between umbilical cord RBC folate and fetal TL at birth. These findings may provide a pathway of understanding and preventing adult-onset disease and mortality through intrauterine reprogramming.

Homocysteine Levels Are not Related to Telomere Length in Cord Blood Leukocytes of Newborns.

Objective Elevated homocysteine (HC) levels and/or shortened telomere length (TL) are associated with adverse medical conditions. Our objective is to investigate the relationship between HC and TL in cord blood leukocytes of newborns. Study Design This is a nested study from a prospective cohort from 2011 to 2012 in pregnant women admitted for delivery at a university-affiliated hospital. Cord blood was collected at delivery and genomic DNA was analyzed using quantitative PCR. The telomere-to-single copy gene ratio method was employed to quantify TL. Newborn HC levels were measured. generalized linear regression modeling (GLM) and bootstrap statistical analyses were performed. Results Seventy-seven maternal-fetal dyads with a mean gestational age of 39 weeks were included. The distribution of the coefficient of homocysteine showed most values greater than zero demonstrating that homocysteine had a positive relationship with TL. In 915 of 10,000 (9.15%) iterations, the p-value was < 0.05 demonstrating a positive effect. Conclusion Increasing newborn concentrations of HC are not associated with decreasing TL. Larger, prospective studies are needed to confirm these findings and long-term implications.

Evidence of altered brain regulatory gene expression in tobacco-exposed fetuses

Abstract Aim: We sought to determine the association between prenatal smoking status and expression of fetal brain regulatory genes. Methods: At delivery, we collected information from parturient women on prenatal smoking habits and analyzed salivary cotinine levels. We obtained neonatal umbilical cord blood and extracted total RNA. We then employed the quantitative polymerase chain reaction (QPCR) analyses and the comparative CT method to calculate the relative gene expression of selected fetal brain regulatory genes responsible for (1) brain growth (brain-derived neutrotrophic factor, BDNF), (2) myelination (proteolipidic protein 1, PLP1 and myelin basic protein, MBP), and (3) neuronal migration and cell-cell interactions during fetal brain development or RLN. The χ2-test, analysis of variance (ANOVA), and the Grubb test were used to evaluate the relationship between prenatal smoking status and relative gene expression levels. Further analysis using bootstrapping was performed to assess the precision of our estimates. Results: Of the 39 maternal-infant dyads included in this study, 25.6% were non-smokers, 43.6% were passive smokers and 30.8% were active smokers. The results showed down-regulation of the selected fetal brain regulatory genes among active smokers. Conclusions: These findings represent preliminary evidence in humans that intrauterine tobacco exposure impacts fetal brain programming. Future studies are warranted to examine whether our findings represent potential mechanisms through which adverse childhood/adult-onset cognitive and behavioral outcomes that have been previously linked to intrauterine exposure occur.