Chemoproteomics

A major goal of chemoproteomics is to characterize the interactome of drug candidates to gain insight into mechanisms of off-target toxicity and polypharmacology.

Computational techniques complement the chemoproteomic toolkit as parallel lines of evidence supporting potential drug-target pairs, and are used to generate structural models that inform lead optimization.

Many fatal and intractable diseases were able to be mapped to specific genes, providing a starting point to better understand the roles of their protein products in illness.

[3] Early drug discovery focuses on genetic validation of a target, which is a strong predictor of success, but knock-out and overexpression systems are simplistic.

[5] In addition to proteomic analysis, the detection of post-translational modifications, like phosphorylation, glycosylation, acetylation, and recently ubiquitination, which give insight into the functional state of a cell, is also possible.

The most widely used reporters are fluorescent moieties that enable imaging and affinity tags, such as biotin, that allow for pull-down of labeled enzymes and analysis via mass spectrometry.

There are drawbacks to each strategy, namely that fluorescent reporters do not allow for enrichment for proteomic analysis, while biotin-based affinity tags co-purify with endogenously biotinylated proteins.

[7] These probes are composed of three connected moieties: (1) a drug scaffold; (2) a photoreactive group, such as an phenylazide, phenyldiazirine, or benzophenone; and (3) an identification tag, such as biotin, a fluorescent dye, or a click chemistry handle.

The probe must remain stable in storage, across buffers, at various pH levels, and in living systems to ensure that labeling occurs only when exposed to light.

Microbead-based immobilization is a modular technique in that it allows the investigator to decide whether they wish to fish for protein targets from the proteome or drug-like compounds from chemical libraries.

The macroscopic properties of microbeads make them amenable to relatively low labor enrichment applications, since they are easily to visualize and their bulk mass is readily removable protein solutions.

[12] Hybrid solution- and immobilization-based strategies have been applied, in which ligands functionalized with an enrichment tag, such as biotin, are allowed to float freely in solution and find their target proteins.

After an incubation period, ligand-protein complexes can be reacted with streptavidin-coated beads, which bind the biotin tag and allow for pull-down and identification of interaction partners.

While immobilization approaches have been reproducible and successful, it is impossible to avoid the limitation of immobilization-induced steric hindrance, which interferes with induced fit.

A single protein type in solution may be represented by individual molecules in a variety of conformations, with many of them different from one another despite being identical in amino acid sequence.

With the advent of high resolution Orbitrap mass spectrometers, this type of experiment can be executed on a proteome-wide scale and stability curves can be generated for thousands of proteins at once.

In SPROX, a lysate is split and treated with drug or a DMSO control, then each group is further aliquoted into separate samples with increasing concentrations of the chaotrope and denaturant guanidinium hydrochloride (GuHCl).

[16] While adoption of affinity selection-mass spectrometry (AS-MS) has led to an expansion of assay formats,[19] the general technique follows a simple scheme.

Since AS-MS measures binding in an unbiased manner, a hit does not need to be tied to a functional readout, opening the possibility of identifying drugs that act beyond active sites, such as allosteric modulators and chemical chaperones, all in a single assay.

Assays can be designed to contain sufficiently high protein concentrations to prevent competition for binding sites between structural analogs, ensuring that hits across a range of affinities can be identified; inversely, assays can contain low protein concentrations to allow for distinction between high and low affinity analogs and to inform structure-activity relationships.

[20] The choice of a chemical library is less stringent than other high-throughput screening strategies owing to the lack of functional readouts, which would otherwise require deconvolution of the source compound that generates biological activity.

[19] Affinity selection is followed by the removal of unbound small molecules via ultrafiltration or size-exclusion chromatography, making only protein-bound ligands available for downstream analysis.

[19] Size-exclusion chromatography (SEC) is more widely used in industrial drug discovery and has the advantage of more efficient removal of unbound compounds as compared to ultrafiltration.

[19] Novartis' SpeedScreen uses SEC in 96-well spin column format, also known as gel filtration chromatography, which allows for simultaneous removal of unbound ligands from up to 96 samples.

[19] The top-down approach requires direct infusion of the complex into an electrospray ionization mass spectrometry source under conditions gentle enough to preserve the interaction and maintain its integrity in the transition from liquid to gas.

[19] Interestingly, electron capture dissociation, which is typically used in structure elucidation of peptides, has been used to identify ligand binding sites during top-down analysis.

A common approach is to use a least-squares regression for superimposition, but this requires user-selected anchor points and therefore introduces human bias into the process.

For example, important pharmacophores may yield high-affinity interactions with therapeutic targets, but they may also lead to undesirable off-target activity, and they may also be substrates of metabolic enzymes, such as Cytochrome P450s.

Originally thought to be experimental noise, these unintended reactions have clued researchers to the presence of sites that can potentially be targeted by novel covalent drugs.

Researchers at the iHuman Institute at ShanghaiTech University employed of scheme in which 20,000 compounds per pool were screened against A2AR, a difficult G-protein coupled receptor to drug, with a 0.12% hit rate, leading to several high affinity ligands.

An example quantitative proteomics workflow. Protein extracts from different samples are extracted and digested using trypsin . Separate samples are labeled using individual isobaric tandem mass tags (TMTs), then labeled samples are pooled. The sample origin of each peptide can be discerned from the TMT attached to it. Labeled peptides are then detected and fragmented by LC-MS/MS , and quantified by comparing relative amounts of TMT fragments in each mass spectrum . This image was adapted from BioRender.com .
A prototypical activity-based protein profiling probe . A covalent warhead and reporter tag are connected by a linker group. The warhead covalently bonds with the active site of an enzyme and the reporter tag is used to enrich or detect the labeled protein . Fluorophosphonate- biotin is an example of an activity-based probe that targets serine hydrolases . It is connected to a biotinylated enrichment handle by an alkane chain. This image was made using BioRender.com .
A prototypical photoaffinity probe. A drug scaffold acts as the first interaction site between probe and protein. A photoreactive group , here an arylazide , can be activated by light to form a reactive intermediate that bonds with a non-specific site on the protein. A tag can then be used to enrich and identify or image and detect the target. This image was made using BioRender.com .
Phenylazide, a photoreactive group commonly used in photoaffinity labeling.
Three strategies for immobilization-based target identification. In all cases, protein mixtures are incubated with the ligand and bound targets are detected downstream. (1A) Ligands are attached to a solid support, such as a microbead or chromatography column , via derivatization . (1B) Ligands bind targets in solution, and are then pulled down by an enrichment handle, such as biotin. (1C) Ligands contain a cross-linking group, which is activated, often with light, labeling the target. An enrichment handle is used to pull down the labeled target. (2) After attachment, the target can be eluted from the ligand or trypsinized directly on-bead. Trypsinized peptides are analyzed via LC-MS/MS . This image was adapted from BioRender.com .
An example thermal proteome profiling workflow. Binding of a drug to a protein often leads to ligand-induced stabilization of the protein (1), which can be measured by comparing the amount of non-denatured protein remaining in a drug-treated sample to an untreated control. The change in protein stability can be visualized as a rightward shift in its stability curve (2). This image was adapted from BioRender.com .
An example drug affinity responsive target stability (DARTS) workflow. Binding of a drug to a protein often leads to ligand-induced stabilization of the protein. In DARTS, drug and control treated proteins are subjected to limited proteolysis and the extent of protein digestion can either be visualized on a gel or measured by mass spectrometry . Drug binding is expected to result in an increase in signal of the stabilized protein. This image was made using BioRender.com .
An example stability of proteins from rates of oxidation (SPROX) workflow. Binding of a drug to a protein often leads to ligand-induced stabilization of the protein. In SPROX, drug and control treated proteins samples are exposed to increasing amounts of a denaturant . Hydrogen peroxide is added to oxidize exposed methionine residues . Drug binding is expected to protect methionine from oxidation by stabilizing the folded form of a protein. Extent of oxidation can be monitored by mass spectrometry and used to generate stability curves. This image was made using BioRender.com .
Affinity selection via gel filtration. A pool of test compounds is added to a protein sample, which is passed through a gel filtration column . Protein-bound compounds move around the beads and exit the column quickly. Unbound compounds are small enough to travel through beads and take a longer path before elution. This image was made using BioRender.com .
An example target identification by chromatographic co-elution (TICC) workflow. Drug-spiked lysate is fractionated using ion exchange chromatography . Fractions are collected every minute, then analyzed for both drug and protein content using LC-MS/MS. Drug and protein elution profiles are constructed and correlated. Target identification is supported by a strong correlation in elution profile between a drug and a protein. This image was made using BioRender.com .
An example pharmacophore model. Each sphere represents a different scorable feature.
PROTAC: Proteolysis targeting chimera . PROTACs are heterobifunctional small molecules that contain a functional group that binds a target and another functional group that recruits an E3 ubiquitin ligase. Binding to both proteins induces proximity-based ubiquitination of the target by the E3 ubiquitin ligase, leading to target degradation.