Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

Dissecting EXP2 sequence requirements for protein export in malaria parasites.

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In Plasmodium parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in Toxoplasma gondii. EXP2, however, has an additional function in Plasmodium parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function. This revealed that EXP2 is a unique pore-forming protein, and its protein export role in P. falciparum cannot be complemented by T. gondii GRA17. This was despite the addition of the EXP2 assembly strand and part of the linker helix to GRA17, which are regions necessary for the interaction of EXP2 with the other core PTEX components. This indicates that the body region of EXP2 plays a critical role in PTEX assembly and/or that the absence of other T. gondii GRA proteins in P. falciparum leads to its reduced efficiency of insertion into the PVM and complementation potential. Altering the timing and abundance of EXP2 expression did not affect protein export but affected parasite viability, indicating that the unique transcriptional profile of EXP2 when compared to other PTEX components enables it to serve an additional role in nutrient exchange.

Epidemiology of Invasive Pneumococcal Disease in Bangladeshi Children Before Introduction of Pneumococcal Conjugate Vaccine.

BACKGROUND: Because Bangladesh intended to introduce pneumococcal conjugate vaccine (PCV)-10 in 2015, we examined the baseline burden of invasive pneumococcal disease (IPD) to measure impact of PCV. METHODS: During 2007-2013, we performed blood and cerebrospinal fluid cultures in children <5 years old with suspected IPD identified through active surveillance at 4 hospitals. Isolates were serotyped by quellung and tested for antibiotic susceptibility by disc diffusion and E-test. Serotyping of culture-negative cases, detected by Binax or polymerase chain reaction, was done by sequential multiplex polymerase chain reaction. Trends in IPD case numbers were analyzed by serotype and clinical syndrome. RESULTS: The study identified 752 IPD cases; 78% occurred in children <12 months old. Serotype information was available for 78% (442/568), including 197 of 323 culture-negative cases available for serotyping. We identified 50 serotypes; the most common serotypes were 2 (16%), 1 (10 %), 6B (7%), 14 (7%) and 5 (7%). PCV-10 and PCV-13 serotypes accounted for 46% (range 29%-57% by year) and 50% (range 37%-64% by year) of cases, respectively. Potential serotype coverage for meningitis and nonmeningitis cases was 45% and 49% for PCV-10, and 48% and 57% for PCV-13, respectively. Eighty-two percent of strains were susceptible to all antibiotics except cotrimoxazole. CONCLUSION: The distribution of serotypes causing IPD in Bangladeshi children is diverse, limiting the proportion of IPD cases PCV can prevent. However, PCV introduction is expected to have major benefits as the country has a high burden of IPD-related mortality, morbidity and disability.

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods.

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of $87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.