Thioamide-based fluorescent protease sensors.

Thioamide quenchers can be paired with compact fluorophores to design "turn-on" fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually any protease. We demonstrate the value of this method in three model applications: (1) characterization of papain enzyme kinetics using rapid-mixing experiments, (2) selective monitoring of cleavage at a single site in a peptide with multiple proteolytic sites, and (3) analysis of the specificity of an inhibitor of calpain in cell lysates.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

Parallel publication of articles and congress presentations for industry-sponsored research: strategies for success.

Recent increases in the practice of parallel publication, during which a peer-reviewed manuscript is published concurrently with the first dissemination of the same key data at a medical congress as a late-breaking abstract, have highlighted substantial value for this method of publication. Parallel publication can increase access to new clinical information for healthcare providers and patients, as well as promote engagement and reach of the publication and presentation. As the practice becomes more common, there is a need for strategies to address the multiple challenges involved in the development process, such as shortened timelines, journal and congress policies, and stakeholder alignment. We surveyed journals, congresses, and publication professionals on the challenges of parallel publication and recommendations for success. Recommendations from journal editors and congress officials included the importance of adhering to timelines and early communication. Insights from a community of publication professionals showed that timelines and the author review process were among the key challenges of parallel publication development and stressed the importance of clear roles and expectations for authors. To provide real-world insights, we present 3 case studies of successful parallel publication development, highlighting the crucial role of journal selection, planning around data availability, and adapting to unpredictable circumstances. The recommendations described here may provide publication professionals with strategies to successfully plan, execute, and carry out parallel publication.

Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase.

The malarial PfA-M1 metallo-aminopeptidase is considered a putative drug target. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1. Herein we present a new, efficient, and high-yielding protocol for the synthesis of bestatin derivatives from natural and unnatural N-Boc-d-amino acids. A diverse library of bestatin derivatives was synthesized with variants at the side chain of either the α-hydroxy-ß-amino acid (P1) or the adjacent natural α-amino acid (P1'). Surprisingly, we found that extended aromatic side chains at the P1 position resulted in potent inhibition against PfA-M1. To understand these data, we determined the X-ray cocrystal structures of PfA-M1 with two derivatives having either a Tyr(OMe) 15 or Tyr(OBzl) 16 at the P1 position and observed substantial inhibitor-induced rearrangement of the primary loop within the PfA-M1 pocket that interacts with the P1 side chain. Our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme that may eventually lead to new therapies for malaria.