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The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects., A protease-activated chemosensor for in vivo molecular imaging enables the detection of activated immune cells by specifically harnessing granzyme B catalysis.

Antiviral therapeutics to treat SARS-CoV-2 are much desired for the on-going pandemic. A well-precedented viral enzyme is the main protease (MPro), which is now targeted by an approved drug and by several investigational drugs. With the inevitable liabilities of these new drugs, and facing viral resistance, there remains a call for new chemical scaffolds against MPro. We virtually docked 1.2 billion non-covalent and a new library of 6.5 million electrophilic molecules against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 μM and 20 μM, respectively. Several series were optimized, resulting in inhibitors active in the low micromolar range. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. Together, these compounds reveal new chemotypes to aid in further discovery of MPro inhibitors for SARS-CoV-2 and other future coronaviruses.

To fight the SARS-CoV-2 pandemic, much effort has been directed toward drug repurposing, testing investigational and approved drugs against several viral or human proteins in vitro. Here we investigate the impact of colloidal aggregation, a common artifact in early drug discovery, in these repurposing screens. We selected 56 drugs reported to be active in biochemical assays and tested them for aggregation by both dynamic light scattering and by enzyme counter screening with and without detergent; seventeen of these drugs formed colloids at concentrations similar to their literature reported IC50s. To investigate the occurrence of colloidal aggregators more generally in repurposing libraries, we further selected 15 drugs that had physical properties resembling known aggregators from a common repurposing library, and found that 6 of these aggregated at micromolar concentrations. An attraction of repurposing is that drugs active on one target are considered de-risked on another. This study suggests not only that many of the drugs repurposed for SARS-CoV-2 in biochemical assays are artifacts, but that, more generally, when screened at relevant concentrations, drugs can act artifactually via colloidal aggregation. Understanding the role of aggregation, and detecting its effects rapidly, will allow the community to focus on those drugs and leads that genuinely have potential for treating COVID-19.Abstract FigureTable of Contents Graphic