A guide to preprinting for early-career researchers

The use of preprints, research manuscripts shared publicly before completing the traditional peer-review process, is becoming a more common practice among life science researchers. Early-career researchers (ECRs) benefit from posting preprints as they are shareable, citable, and prove productivity. However, preprinting a manuscript involves a discussion among all co-authors, and ECRs are often not the decision-makers. Therefore, ECRs may find themselves in situations where they are interested in depositing a preprint but are unsure how to approach their co-authors or advisor about preprinting. Leveraging our own experiences as ECRs, and feedback from the research community, we have constructed a guide for ECRs who are considering preprinting to enable them to take ownership over the process and to raise awareness about preprinting options. We hope that this guide helps ECRs to initiate conversations about preprinting with co-authors and encourage them to preprint their future research

Applications of Disulfide Tethering to Fragment Discovery and Protein Dynamics in Protein-Protein Interactions

The study of protein protein-interactions (PPIs) can be approached with many lenses. Interaction networks can be mapped genetically or with proteomic methods. Biophysical characterization of specific partner interactions can clarify molecular mechanism of partner interaction. Screening methods can to identify novel interaction modulators, and site-directed chemical probes can used to demonstrate the importance of protein partner interaction in disease or biology. In this work I begin with an overview of the chemical biology toolbox for targeting non-catalytic cysteines residues. I apply one such tool, disulfide tethering, to two PPIs with the goal of broadening the PPIs which are targetable with covalent chemical probes. I then describe the use of cysteine-reactive molecules in understanding protein ligandability and its connection to protein dynamics. Finally, I study a promiscuous PPI receptor (Mac-1) to understand if inhibitors could be developed for specific ligands. Taken together, these data demonstrate the applicability of cysteine reactivity generally, and disulfide tethering specifically, to the study of PPIs

Supplemental materials for preprint: A Guide to Preprinting for Early Career Researchers

Supplemental materials for: A Guide to Preprinting for Early Career Researcher

Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery.

The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an 'allele specific' manner. For the medicinal chemist, the structure-guided design of cysteine- reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteinemodifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues

A Guide to Preprinting for Early Career Researchers

The use of preprints, research manuscripts shared publicly before the traditional peer-review process, is becoming more common in the life sciences. Early career researchers (ECRs) benefit from posting preprints as they are shareable, citable, and prove productivity. However, the decision to preprint a manuscript involves a discussion among all co-authors, and ECRs are often not the decision-makers. Therefore, ECRs may find themselves in situations where they are interested in posting a preprint but are unsure how to approach their co-authors or advisor about preprinting. Leveraging our own experiences as ECRs, and feedback from the research community, we have constructed a guide for ECRs who are considering preprinting - to enable them to take ownership over the process, and to raise awareness about preprinting options. We hope that this guide helps ECRs to initiate conversations about preprinting with co-authors and encourage them to preprint their future research

Exploration of a 14-3-3 PPI Pocket by Covalent Fragments as Stabilizers.

The systematic discovery of functional fragments binding to the composite interface of protein complexes is a first critical step for the development of orthosteric stabilizers of protein-protein interactions (PPIs). We have previously shown that disulfide trapping successfully yielded covalent stabilizers for the PPI of 14-3-3 with the estrogen receptor ERα. Here we provide an assessment of the composite PPI target pocket and the molecular characteristics of various fragments binding to a specific subpocket. Evaluating structure-activity relationships highlights the basic principles for PPI stabilization by these covalent fragments that engage a relatively large and exposed binding pocket at the protein/peptide interface with a "molecular glue" mode of action

Site-directed fragment-based screening for the discovery of protein-protein interaction stabilizers

Modulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery

Exploration of a 14-33 PPI Pocket by Covalent Fragments as Stabilizers

[Image: see text] The systematic discovery of functional fragments binding to the composite interface of protein complexes is a first critical step for the development of orthosteric stabilizers of protein–protein interactions (PPIs). We have previously shown that disulfide trapping successfully yielded covalent stabilizers for the PPI of 14-3-3 with the estrogen receptor ERα. Here we provide an assessment of the composite PPI target pocket and the molecular characteristics of various fragments binding to a specific subpocket. Evaluating structure–activity relationships highlights the basic principles for PPI stabilization by these covalent fragments that engage a relatively large and exposed binding pocket at the protein/peptide interface with a “molecular glue” mode of action