Novel isoforms of influenza virus PA-X and PB1-F2 indicated by automatic annotation.

The influenza A virus genome contains 8 gene segments encoding 10 commonly recognized proteins. Additional protein products have been identified, including PB1-F2 and PA-X. We report the in-silico identification of novel isoforms of PB1-F2 and PA-X in influenza virus genomes sequenced from avian samples. The isoform observed in PA-X includes a mutated stop codon that should extend the protein product by 8 amino acids. The isoform observed in PB1-F2 includes two nonsense mutations that should truncate the N-terminal region of the protein product and remove the entire mitochondrial targeting domain. Both isoforms were uncovered during automatic annotation of CEIRS sequence data. Nominally termed PA-X8 and PB1-F2-Cterm, both predicted isoforms were subsequently found in other annotated influenza genomes previously deposited in GenBank. Both isoforms were noticed due to discrepant annotations output by two annotation engines, indicating a benefit of incorporating multiple algorithms during gene annotation.

A 35-Year Review of Pre-Clinical HIV Therapeutics Research Reported by NIH ChemDB: Influences of Target Discoveries, Drug Approvals and Research Funding.

We present a retrospective analysis of trends in human immunodeficiency virus (HIV) small molecule drug development over the last thirty-five years based on data captured by ChemDB, a United States (US) National Institutes of Health (NIH) database of chemical and biological HIV testing data. These data are analyzed alongside NIH funding levels, US Food and Drug Administration (FDA) drug approvals, and new target identifications to explore the influences of these factors on anti-HIV drug discovery research. The NIH's ChemDB database collects chemical and biological testing data describing published and patented pre-clinical compounds in development as potential HIV therapeutics. These data were used as a proxy for estimating overall levels of HIV therapeutics research activities in order to assess research trends. Data extracted from ChemDB were compared with records of drug approvals from the FDA, NIH funding levels, and drug target discoveries to elucidate the influences that these factors have on levels of HIV therapeutics research activities. Despite the increasingly wide suite of HIV therapeutic options that have accumulated during decades of research, interest in HIV therapeutics research activities remains strong. While decreases in research activity levels have followed cuts in research funding, FDA-approved HIV therapeutics have continued to accumulate. The comparisons presented here indicate that HIV drug research activity levels have historically been more responsive to changes in funding levels and the identification of new drug targets, than they have been to drug approvals. Continued interest in HIV therapeutics research may reflect that fact that of the 55 drugs approved for HIV treatment as of 2018, only seven inhibitory targets are represented. Moreover, drug resistance presents substantial clinical challenges. Sustained research interest despite drug approvals and fluctuations in available funding likely reflects the clinical need for safer, more palatable and more efficacious therapeutics; robust attention to both novel therapeutics and inhibitory targets is necessary given the speed of development of drug-resistant HIV strains. Only with such continued interest will we reduce the burden of acquired immunodeficiency syndrome (AIDS) disease and control the AIDS epidemic.

Colorimetric high-throughput screen for detection of heme crystallization inhibitors.

Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.

Synthesis of spiro-1,2-dioxolanes and their activity against Plasmodium falciparum.

Artemisinin-derived compounds play an integral role in current malaria chemotherapy. Given the virtual certainty of emerging resistance, we have investigated spiro-1,2-dioxolanes as an alternative scaffold. The endoperoxide functionality was generated by the SnCl(4)-mediated annulation of a bis-silylperoxide and an alkene. The first set of eight analogs gave EC(50) values of 50-150 nM against Plasmodium falciparum 3D7 and Dd2 strains, except for the carboxylic acid analog. A second series, synthesized by coupling a spiro-1,2-dioxolane carboxylic acid to four separate amines, afforded the most potent compound (EC(50) approximately 5 nM).