Chemoproteomics
Chemoproteomics entails a broad array of techniques used to identify and interrogate protein-small molecule interactions. Chemoproteomics complements phenotypic drug discovery, a paradigm that aims to discover lead compounds on the basis of alleviating a disease phenotype, as opposed to target-based drug discovery, in which lead compounds are designed to interact with predetermined disease-driving biological targets. As phenotypic drug discovery assays do not provide confirmation of a compound's mechanism of action, chemoproteomics provides valuable follow-up strategies to narrow down potential targets and eventually validate a molecule's mechanism of action. Chemoproteomics also attempts to address the inherent challenge of drug promiscuity in small molecule drug discovery by analyzing protein-small molecule interactions on a proteome-wide scale. A major goal of chemoproteomics is to characterize the interactome of drug candidates to gain insight into mechanisms of off-target toxicity and polypharmacology.
Chemoproteomics assays can be stratified into three basic types. Solution-based approaches involve the use of drug analogs that chemically modify target proteins in solution, tagging them for identification. Immobilization-based approaches seek to isolate potential targets or ligands by anchoring their binding partners to an immobile support. Derivatization-free approaches aim to infer drug-target interactions by observing changes in protein stability or drug chromatography upon binding. 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. Several targets of high profile drugs have been identified using chemoproteomics, and the continued improvement of mass spectrometer sensitivity and chemical probe technology indicates that chemoproteomics will play a large role in future drug discovery.
Background
Context
The conclusion of the Human Genome Project was followed with hope for a new paradigm in treating disease. 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. Drug discovery has made use of animal knock-out models that highlight the impact of a protein's absence, particularly in the development of disease, and medicinal chemists have leveraged computational chemistry to generate high affinity compounds against disease-causing proteins. Yet FDA drug approval rates have been on the decline over the last decade. One potential source of drug failure is the disconnect between early and late drug discovery. 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. Spatially and temporally conditional knock-out/knock-in systems have improved the level of nuance in in vivo analysis of protein function, but still fail to completely parallel the systemic breadth of pharmacological action. For example, drugs often act through multiple mechanisms, and often work best by engaging targets partially. Chemoproteomic tools offer a solution to bridge the gap between a genetic understanding of disease and a pharmacological understanding of drug action by identifying the many proteins involved in therapeutic success.Basic tools
The chemoproteomic toolkit is anchored by liquid chromatography-tandem mass spectrometry based quantitative proteomics, which allows for the near complete identification and relative quantification of complex proteomes in biological samples. 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 vast majority of proteomic studies are analyzed using high-resolution orbitrap mass spectrometers and samples are processed using a generalizable workflow. A standard procedure begins with sample lysis, in which proteins are extracted into a denaturing buffer containing salts, an agent that reduces disulfide bonds, such as dithiothreitol, and an alkylating agent that caps thiol groups, such as iodoacetamide. Denatured proteins are proteolysed, often with trypsin, and then separated from other mixture components prior to analysis via LC-MS/MS. For more accurate quantification, different samples can be reacted with isobaric tandem mass tags, a form of chemical barcode that allows for sample multiplexing, and then pooled.Solution-based approaches
Broad proteomic and transcriptomic profiling has led to innumerable advances in the biomedical space, but the characterization of RNA and protein expression is limited in its ability to inform on the functional characteristics of proteins. Given that transcript and protein expression information leave gaps in knowledge surrounding the effects of post-translational modifications and protein-protein interactions on enzyme activity, and that enzyme activity varies across cell types, disease states, and physiological conditions, specialized tools are required to profile enzyme activity across contexts. Additionally, many identified enzymes have not been sufficiently characterized to yield actionable mechanisms on which to base functional assays. Without a basis for a functional biochemical readout, chemical tools are required to detect drug-protein interactions.Activity-based protein profiling
Activity-based protein profiling is a technique that was developed to monitor the availability of enzymatic active sites to their endogenous ligands. ABPP uses specially designed probes that enter and form a covalent bond with an enzyme's active site, which confirms that the enzyme is an active state. The probe is typically an analog of the drug whose mechanism is being studied, so covalent labeling of an enzyme is indicative of drug binding. ABPP probes are designed with three key functional units: a site-directed covalent warhead ; a reporter tag, such as biotin or rhodamine; and a linker group. The site-directed covalent warhead, also called a covalent modifier, is an electrophile that covalently modifies a serine, cysteine, or lysine residue in the enzyme's active site and prevents future interactions with other ligands. ABPP probes are generally designed against enzymatic classes, and thus can provide systems-level information about the impact of cell state on enzymatic networks. The reporter tag is used to confirm labeling of the enzyme with the reactive group and can vary depending on the downstream readout. 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. A linker group is used to connect the reactive group to the reporter, ideally in a manner that does not alter the activity of probe. The most common linker groups are long alkyl chains, derivatized PEGs, and modified polypeptides.Under the assumption that enzymes vary in their structure, function, and associations depending on a system's physiological or developmental state, it can be inferred that the accessibility of an enzyme's active site will also vary. Therefore, the ability of an ABPP probe to label an enzyme will also vary across conditions. Thus, the binding of a probe can reveal information around an enzyme's functional characteristics in different contexts. High-throughput screening has benefitted from ABPP, particularly in the area of competitive inhibition assays, in which biological samples are pre-incubated with drug candidates, then made to compete with ABPP probes for binding to target enzymes. Compounds with high affinity to their targets will prevent binding of the probe, and the degree of probe binding can be used as an indication of compound affinity. Because ABPP probes label classes of enzymes, this approach can also be used to profile drug selectivity, as highly selective compounds will ideally outcompete probes at only a small number of proteins.
File:Photoaffinity_labeling.png|thumb|380x380px|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.
Photoaffinity labeling
Unlike ABPP, which results in protein labeling upon probe binding, photoaffinity labeling probes require activation by photolysis before covalent bonding to a protein occurs. The presence of a photoreactive group makes this possible. These probes are composed of three connected moieties: a drug scaffold; a photoreactive group, such as an phenylazide, phenyldiazirine, or benzophenone; and an identification tag, such as biotin, a fluorescent dye, or a click chemistry handle. The drug scaffold is typically an analog of a drug whose mechanism is being studied, and, importantly, binds to the target reversibly, which better mimics the interaction between most drugs and their targets. There are several varieties of photoreactive groups, but they are fundamentally different from ABPP probes: while ABPP specifically labels nucleophilic amino acids in a target's active site, photoaffinity labeling is non-specific, and thus is applicable to labeling a wider range of targets. The identification tag will vary depending on the type of analysis being done: biotin and click chemistry handles are suitable for enrichment of labeled proteins prior to mass spectrometry based identification, while fluorescent dyes are used when using a gel-based imaging method, such as SDS-PAGE, to validate interaction with a target.Because photoaffinity probes are multifunctional, they are difficult to design. Chemists incorporate the same principles of structure-activity relationship modeling into photoaffinity probes that apply to drugs, but must do so without compromising the drug scaffold's activity or the photoreactive group's ability to bond. Since photoreactive groups bond indiscriminately, improper design can cause the probe to label itself or non-target proteins. 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. Activation by light must also be fine-tuned, as radiation can damage cells.