Matylda Anna Izert-Nowakowska

Kinases have proven to be one of the most fertile target classes for new drug approvals, however, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we established a systematic workflow to assess the cellular impact of minimal electrophilic modifications to ATP-site binding scaffolds. Amongst multiple potential starting points for monovalent kinase degradation that were revealed, we identified that one of our scout molecules was able to control the abundance of the master regulator of autophagy, Unc-51-like autophagy activating kinase 1 (ULK1) and that a scaffold-hop could be performed to generate a targeted monovalent ULK1 degrader chemical tool, we named ALP-1. Remarkably, further investigation of how ALP-1 engages ULK1 unveiled an atypical conjugation event between the chloroacetamide warhead of ALP1 and an aspartate residue on the lip of the ULK1 ATP-binding site. These discoveries reveal new understanding of the potential for non-cysteine reactivity of the commonly employed chloroacetamide warhead, as well as new opportunities for the targeting and covalent engagement of kinases.

Fluorescent reporters provide a useful tool for studying degron motifs. Fusing a degron of interest to a fluorescent protein allows to accurately track protein levels overtime to characterise the degradation kinetics of studied degrons. Here we describe a rapid and simple method to study degron peptides in Escherichia coli using plasmid-encoded eGFP-degron fusion constructs. The described methods provide an accessible workflow to evaluate degrons. We provide protocols for generation of pBAD plasmids encoding the studied constructs and two different methods for evaluating degrons – an end-point fluorescence measurement on agar plates and a kinetic measurement in liquid cultures in a 96-well format for high-throughput degron studies.

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We describe a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrate the mode of action of the degraders on a challenging essential target, GroEL. The studies in bacteria are complemented by in vitro binding and structural studies. Expression of degrader peptides results in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels is accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial-targeted protein degradation.

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We have created a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrated the mode-of-action of the degraders on a challenging essential target GroEL. The studies in bacteria were complemented by in vitro binding and structural studies. Expression of degrader peptides resulted in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels was accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial targeted protein degradation.

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo . Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches. SUMMARY: This work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control.