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Biology subjects

Tholen, M.

Publications and source records attributed to Tholen, M..

4 recordsLinked to original sources

Tyrosine kinase inhibitors trigger lysosomal damage-associated cell lysis to activate the NLRP3 inflammasome

Inflammasomes are intracellular protein complexes that control proteolytic maturation and secretion of inflammatory interleukin-1 (IL-1) family cytokines and are thus important in host defense. While some inflammasomes are activated simply by binding to pathogen-derived molecules, others, including those nucleated by NLRP3 and NLRP1, have more complex activation mechanisms that are not fully understood. We screened a library of small molecules to identify new inflammasome activators that might shed light on activation mechanisms. In addition to validating dipeptidyl peptidase (DPP) inhibitors as NLRP1 activators, we find that clinical tyrosine kinase inhibitors (TKIs) including imatinib and masitinib activate the NLRP3 inflammasome. Mechanistically, these TKIs cause lysosomal swelling and damage, leading to cathepsin-mediated destabilization of myeloid cell membranes and cell lysis. This is accompanied by potassium (K+) efflux, which activates NLRP3. Both lytic cell death and NLRP3 activation but not lysosomal damage induced by TKIs are prevented by the cytoprotectant high molecular weight polyethylene glycol (PEG). Our study establishes a screening method that can be expanded for inflammasome research and immunostimulatory drug development, and provides new insight into immunological off-targets that may contribute to efficacy or adverse effects of TKIs. One Sentence SummaryA functional small molecule screen identifies imatinib, masitinib and other tyrosine kinase inhibitors that destabilize myeloid cell lysosomes, leading to cell lysis and K+ efflux-dependent NLRP3 inflammasome activation.

immunology↗

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↗

The clinical drug candidate ebselen attenuates inflammation and promotes microbiome recovery after antibiotic treatment for Clostridium difficile infection

Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We recently reported that the safe in human clinical drug candidate ebselen (NCT03013400, NCT01452607, NCT00762671, NCT02603081) has biochemical, cell-based and in vivo efficacy against the bacterial toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster relapse model of CDI. Furthermore, ebselen treatment does not alter microbiome diversity but promotes its recovery back to that of healthy controls after antibiotic-induced dysbiosis in both healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.

microbiology↗

Multivariate ‘AND-gate’ substrate probes as enhanced contrast agents for fluorescence-guided surgery

The greatest challenges for surgical management of cancer are precisely locating lesions and clearly defining the margins between tumors and normal tissues. This is confounded by the characteristics of the tissue where the tumor is located as well as its propensity to form irregular boundaries with healthy tissues. To address these issues, molecularly targeted optical contrast agents have been developed to define margins in real-time during surgery1,2. However, selectivity of a contrast agent is often limited by expression of a target enzyme or receptor in both tumor and healthy tissues. Here we introduce a concept of multivariate AND-gate optical imaging probes that require sequential processing by multiple tumor-specific enzymes to produce a fluorescent signal. This results in dramatically improved specificity as well as overall enhanced sensitivity. This general approach has the potential to be broadly applied to selectively target complex patterns of enzyme activities in diverse disease tissues for detection, treatment and therapy response monitoring.

bioengineering↗