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.

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.

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.

We report the refinement of a high-throughput, liquid chromatography/mass spectrometry (LC/MS)-based screening method for the identification of covalent small-molecule binders to proteins. Using a custom library of 1600 disulfide-capped fragments targeting surface cysteine residues, we optimize sample preparation, chromatography, and ionization conditions to maximize the reliability and flexibility of the approach. Data collection at a rate of 84 s per sample balances speed with reliability for sustained screening over multiple, diverse projects run over a 24-month period. The method is applicable to protein targets of various classes and a range of molecular masses. Data are processed in a custom pipeline that calculates a percent bound value for each compound and identifies false positives by calculating significance of detected masses (signal significance). An example pipeline is available through Biovia's ScienceCloud Protocol Exchange. Data collection and analysis methods for the screening of covalent adducts of intact proteins are now fast enough to screen the largest covalent compound libraries in 1 to 2 days.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ_377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.