Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

Identification of Abundant and Evolutionarily Conserved Opioid Receptor Circular RNAs in the Nervous System Modulated by Morphine.

Circular RNAs (circRNAs) are a distinct category of single-stranded, covalently closed RNAs formed by backsplicing. The functions of circRNAs are incompletely known and are under active investigation. Here, we report that in addition to traditional linear mRNAs (linRNA), mouse, rat, and human opioid receptor genes generate exonic circRNA isoforms. Using standard molecular biologic methods, Oprm1 circRNAs (circOprm1) were detected in RNAs of rodent and human brains and spinal cords, as well as human neuroblastoma cells, suggesting evolutionary conservation. Sequencing confirmed backsplicing using canonical splice sites. Oprm1 circRNAs were sense-stranded circRNAs resistant to RNase R digestion. The relative abundance of Oprm1 circRNA to linRNA determined by quantitative reverse transcription polymerase chain reaction varied among mouse brain regions, with circRNA isoforms predominating in rostral structures and less abundant in brain stem. Chronic morphine exposure in mice increased brain circOprm1e2.3 and circOprm1.e2.e3.e4(302) levels by 1.5- to 1.6-fold relative to linRNA. Sequence analysis predicted numerous microRNA binding sites within Oprm1 circRNA sequences, suggesting a potential role in microRNA sequestration through sponging. In addition, we observed that other opioid receptor genes including δ, κ, and nociceptin receptor genes produced similar circRNAs. In conclusion, all members of the opioid receptor gene family express circRNAs, with Oprm1 circRNA levels exceeding those of linear forms in some regions. SIGNIFICANCE STATEMENT: The modulation of Oprm1 circular RNA (circRNA) expression by morphine, coupled with the high abundance and existence of potential miRNA binding sites with circRNA sequences suggests the potential role of Oprm1 circRNAs in chronic opioid effects such as tolerance.