DNA-Compatible Conditions for the Formation of N-Methyl Peptide Bonds.

DNA-encoded libraries (DELs) are a powerful platform in drug discovery. Peptides have unique properties that make them attractive pharmaceutical candidates. N-methylation of the peptide backbone can confer beneficial properties such as increased proteolytic stability and membrane permeability. Herein, we evaluate different DEL reaction systems and report a DNA-compatible protocol for forming N-methylated amide bonds. The DNA-compatible, bis(trichloromethyl)carbonate-mediated amide coupling is efficient for the formation of N-methyl peptide bonds, which promises to increase the opportunity to identify passively cell-permeable macrocyclic peptide hits by DNA-encoded technology.

StARD9 is a novel lysosomal kinesin required for membrane tubulation, cholesterol transport and Purkinje cell survival.

The pathological accumulation of cholesterol is a signature feature of Niemann-Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease.

Reevaluation of a Bicyclic Pyrazoline as a Selective 15-Lipoxygenase V-Type Activator Possessing Fatty Acid Specificity.

Regulation of lipoxygenase (LOX) activity is of great interest due to the involvement of the various LOX isoforms in the inflammatory process and hence many diseases. The bulk of investigations have centered around the discovery and design of inhibitors. However, the emerging understanding of the role of h15-LOX-1 in the resolution of inflammation provides a rationale for the development of activators as well. Bicyclic pyrazolines are known bioactive molecules that have been shown to display antibiotic and anti-inflammatory activities. In the current work, we reevaluated a previously discovered bicyclic pyrazoline h15-LOX-1 activator, PKUMDL_MH_1001 (written as 1 for this publication), and determined that it is inactive against other human LOX isozymes, h5-LOX, h12-LOX, and h15-LOX-2. Analytical characterization of 1 obtained in the final synthesis step identified it as a mixture of cis- and trans-diastereomers: cis-1 (12%) and trans-1 (88%); and kinetic analysis indicated similar potency between the two. Using compound 1 as the cis-trans mixture, h15-LOX-1 catalysis with arachidonic acid (AA) (AC50 = 7.8 +/- 1 µM, A max = 240%) and linoleic acid (AC50 = 5.3 +/- 0.7 µM, A max = 98%) was activated, but not with docosahexaenoic acid (DHA) or mono-oxylipins. Steady-state kinetics demonstrate V-type activation for 1, with a ß value of 2.2 +/- 0.4 and an K x of 16 +/- 1 µM. Finally, it is demonstrated that the mechanism of activation for 1 is likely not due to decreasing substrate inhibition, as was postulated previously. 1 also did not affect the activity of the h15-LOX-1 selective inhibitor, ML351, nor did 1 affect the activity of allosteric effectors, such as 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) and 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA). These data confirm that 1 binds to a distinct activation binding site, as previously postulated. Future work should be aimed at the development of selective activators that are capable of activating h15-LOX-1 catalysis with DHA, thus enhancing the production of DHA-derived pro-resolution biomolecules.

Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial Pyrazolo[3,4-b]pyridines.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.