Biology subjects

Groborz, K.

Publications and source records attributed to Groborz, K..

4 recordsLinked to original sources

bioRxiv2020

Design of optical imaging probes by screening of diverse substrate libraries directly in disease tissue extracts

Fluorescently-quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection and surgical guidance. These probes are often designed to target specific enzymes associated with disease by direct optimization using single purified targets. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of mouse mammary tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino acid residues that could be used to generate highly efficient tumor-specific fluorescently quenched substrate probes. The most effective probe is significantly brighter than any of our previously reported tumor imaging probes designed for specific proteases and robustly discriminates tumor tissue from adjacent healthy tissue in a mouse model of cancer. Importantly, although the probes were developed by screening mouse mammary tumor tissues, they are able to effectively distinguish human ductal carcinomas from normal breast tissue with similar reactivity profiles to those observed in mouse tissues. This new strategy simplifies and enhances the process of probe optimization by direct screening in a tissue of interest without any a priori knowledge of enzyme targets. It has the potential to be applied to advance the development of probes for diverse disease states for which clinical or animal model tissues are available.

bioengineering↗

bioRxiv2020

Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.

biochemistry↗

bioRxiv2019

The Activome: multiplexed probing of activity of proteolytic enzymes using mass cytometry-compatible activity-based probes (TOF-probes)

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.

biochemistry↗

bioRxiv2018

Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies different amino acid preferences between peptide-based substrates and inhibitors.

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of non-optimal peptide substrates. Importantly, analysis of previously published data about the specificity of other proteases also unveiled significant discrepancies in the amino acid preference between substrates and inhibitors. In this article, we also discuss important structural and theoretical reasons that might account for these discrepancies. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.

biochemistry↗