Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymus of mice treated with dexamethasone (dex) as well as in tumor-bearing mice treated with the apoptosis inducing monoclonal antibody Apomab. Caspase ABPs provided direct readouts of the kinetics of apoptosis in live animals, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored non-invasively and that coincided with the peak in caspase activity as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for non-invasive imaging of apoptosis in both pre-clinical and clinical settings.

MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a novel allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here, we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small molecule inhibitors of this protease domain and present the 2.35 Å structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising novel anti-toxin strategies for a number of bacterial pathogens.

SUMMARY The widespread resistance of malaria parasites to all affordable drugs has made the identification of new targets urgent. Dipeptidyl aminopeptidases (DPAPs) represent potentially valuable new targets that are involved in hemoglobin degradation (DPAP1) and parasite egress (DPAP3). Here we use activity-based probes to demonstrate that specific inhibition of DPAP1 by a small molecule results in the formation of an immature trophozoite that leads to parasite death. Using computational methods we designed stable, non-peptidic covalent inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. These compounds show signs of slowing parasite growth in a murine model of malaria, which suggests that DPAP1 might be a viable anti-malarial target. Interestingly, we found that re-synthesis and activation of DPAP1 after inhibition is rapid, suggesting that effective drugs would need to sustain DPAP1 inhibition for a period of 2–3h.

While there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma orthologue of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

Summary Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multidomain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently there are no known inhibitors of this protease. Here we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented here will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.