Design, synthesis, and evaluation of in vivo potency and selectivity of epoxysuccinyl-based inhibitors of papain-family cysteine proteases.

The papain-family cathepsins are cysteine proteases that are emerging as promising therapeutic targets for a number of human disease conditions ranging from osteoporosis to cancer. Relatively few selective inhibitors for this family exist, and the in vivo selectivity of most existing compounds is unclear. We present here the synthesis of focused libraries of epoxysuccinyl-based inhibitors and their screening in crude tissue extracts. We identified a number of potent inhibitors that display selectivity for endogenous cathepsin targets both in vitro and in vivo. Importantly, the selectivity patterns observed in crude extracts were generally retained in vivo, as assessed by active-site labeling of tissues from treated animals. Overall, this study identifies several important compound classes and highlights the use of activity-based probes to assess pharmacodynamic properties of small-molecule inhibitors in vivo.

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels.

Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca(2+) stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca(2+)-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2ß, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2ß does not by itself strongly bind Orai1, it is recruited to Orai1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2ß mutants and Orai1-STIM2ß chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with Orai1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca(2+) signals.

Identification of Orai1 channel inhibitors by using minimal functional domains to screen small molecule microarrays.

Store-operated calcium (SOC) channels are vital for activation of the immune cells, and mutations in the channel result in severe combined immunodeficiency in human patients. In lymphocytes, SOC entry is mediated by the Orai1 channel, which is activated by direct binding of STIM1. Here we describe an alternative approach for identifying inhibitors of SOC entry using minimal functional domains of STIM1 and Orai1 to screen a small-molecule microarray. This screen identified AnCoA4, which inhibits SOC entry at submicromolar concentrations and blocks T cell activation in vitro and in vivo. Biophysical studies revealed that AnCoA4 binds to the C terminus of Orai1, directly inhibiting calcium influx through the channel and also reducing binding of STIM1. AnCoA4, unlike other reported SOC inhibitors, is a molecule with a known binding site and mechanism of action. These studies also provide proof of principle for an approach to ion channel drug discovery.

Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes.

Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.