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The Asp233-Asp246 pair is highly conserved in Class A β-lactamases, which hydrolyze β-lactam antibiotics. Here, we characterize its function using CTX-M-14 β-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in kcat /Km ranging from 20% for nitrocefin to 6-fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10-fold. The mutant structures showed movement of the 213-219 loop and the loss of the Thr216-Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX-M activity. These results demonstrate the importance of the aspartate pair to CTX-M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance.

The integrated stress response comprises multiple signaling pathways for detecting and responding to cellular stress that converge at a single event-the phosphorylation of Ser51 on the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α-P) results in attenuation of global protein synthesis via the inhibitory effects of eIF2α-P on eIF2B, the guanine exchange factor (GEF) for eIF2. Herein we describe structure-activity relationship (SAR) studies of bis-O-arylglycolamides, first-in-class integrated stress response inhibitors (ISRIB). ISRIB analogues make cells insensitive to the effects of eIF2α-P by activating the GEF activity of eIF2B and allowing global protein synthesis to proceed with residual unphosphorylated eIF2α. The SAR studies described herein support the proposed pharmacology of ISRIB analogues as binding across a symmetrical protein-protein interface formed between protein subunits of the dimeric eIF2B heteropentamer.

Antimalarial agents artemisinin and arterolane act via initial reduction of a peroxide bond in a process likely mediated by ferrous iron sources in the parasite. Here, we report the synthesis and antiplasmodial activity of arterolane-like 1,2,4-trioxolanes specifically designed to release a tethered drug species within the malaria parasite. Compared with our earlier drug delivery scaffolds, these new arterolane-inspired systems are of significantly decreased molecular weight and possess superior metabolic stability. We describe an efficient, concise and scalable synthesis of the new systems, and demonstrate the use of the aminonucleoside antibiotic puromycin as a chemo/biomarker to validate successful drug release in live Plasmodium falciparum parasites. Together, the improved drug-like properties, more efficient synthesis, and proof of concept using puromycin, suggests these new molecules as improved vehicles for targeted drug delivery to the malaria parasite.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.

Diels−Alder cycloadditions of oximinosulfonate 8 with a variety of 1,3-dienes proceed with regiochemistry opposite to that observed with conventional imino dienophiles, providing expeditious synthetic routes to substituted pyridines, tetrahydropyridines, and pyrrolines. The oximinosulfonate 8 is prepared in one convenient synthetic operation from Meldrum's acid and reacts with conjugated dienes at −78 °C in the presence of 2 equiv of dimethylaluminum chloride to afford [4 + 2] cycloadducts in good to excellent yield. Exposure of these cycloadducts to the action of NaOMe and N-chlorosuccinimide in methanol−THF at room temperature then produces substituted pyridines. The utility of this new two-step annulation protocol is demonstrated in total syntheses of the pyridine alkaloids fusaric acid and (S)-(+)-fusarinolic acid. Heating the [4 + 2] cycloadducts derived from 8 in a mixture of acetonitrile and pH 7 phosphate buffer induces an unusual Stieglitz-type rearrangement leading to the formation of interestin...