Chemoproteomics - Knowledge

839: 2237: 2472: 802: 2367: 1744: 1252: 180: 680: 5323: 444: 5347: 5359: 5335: 1512: 1418: 1076:, is passed through the column and the target of interest binds to the immobilized ligand while other sample components pass through the column unretained. Under the target immobilization format, a target of interest - often a disease-relevant protein - is immobilized within a chromatography column and acts as the 675:

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

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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

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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

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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

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Affinity chromatography emerged in the 1950s as a rarely used method used to purify enzymes; it has since seen mainstream use and is the oldest among chemoproteomic approaches. Affinity chromatography is performed following one of two basic formats: ligand immobilization or target immobilization.

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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

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pharmacophore modeling. A structure-based model integrates key structural properties of the protein's binding site, such as the spatial distribution of interaction points, with features identified from ligand based pharmacophore models to generate a holistic simulation of the ligand-protein

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based modeling has improved binding predictions but has also given rise to the challenge of modeling ligand flexibility, which requires building a database of conformational models and uses large amounts of data storage space. Another approach is the so-called on-the-fly method, in which

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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: (1) a

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interaction. A major challenge in structure-based modeling is to narrow down pharmacophore features, of which many are initially identified, to a set of high priority features, as modeling too many features is a computational challenge. Another challenge is the incompatibility of

2529:. The interaction brings the E3 ubiquitin ligase close enough to the target that the target is labeled for degradation. The existence of potential covalent binding sites across the proteome suggests that many drugs can be covalently targeted using such a modality. 1604:

and used to generate methionine stability curves, which are a proxy for drug binding. There are drawbacks to the SPROX assay, namely that the only relevant peptides from SPROX samples are those with methionine residues, which account for approximately one-third of

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content. Individual proteins elute with characteristic profiles, and pre-incubated drugs should mirror the elution profiles of the targets they complex with. Correlation of drug and protein elution profiles allows for targets to be narrowed down and inferred.

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and stability curves are generated. Upon comparison to an untreated stability curve, the treated curve is expected to shift to the right, indicating that ligand-induced stabilization occurred. Historically, thermal proteome profiling has been assessed using a

2390:, and accordingly requires large libraries with corresponding experimental data to enhance predictive power. Compound structures are superimposed virtually and common elements are scored on the basis of their tendency toward bioactivity. The move away from 544:, 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 1219:

by adding a high concentration of a high-affinity binding partner to the mobile phase, bound analytes are competed off of the stationary phase; the additive can be either a protein that scavenges bound ligands or a small molecule that displaces them.

2521:. This is particularly salient in the case of proteins with no enzymatic activity to inhibit, or with mutated drug resistant proteins. In any of these cases, proteins can potentially be targeted for degradation using the novel drug modality of 2412:

models require training data sets, giving rise to another challenge—selection of the appropriate library of compounds to adequately train models. Data set size and chemical diversity significantly affect performance of the downstream product.

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modeling, without a database; this method requires significantly less storage space at the cost of high computing time. A second challenge arises from the decision of how to superimpose analog structures. A common approach is to use a

2512:, bold approaches to ligand discovery are required. The use of ABPP has coincidentally reinvigorated the search for newly ligandable sites. ABPP probes, intentionally used to label enzyme active sites, have been found to label many 767:, 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 2516:

regions on many different proteins unintentionally. Originally thought to be experimental noise, these unintended reactions have clued researchers to the presence of sites that can potentially be targeted by novel

922:. In both cases, a solid support is used as an immobilization surface bearing a bait molecule. The bait molecule can be a potential drug if the investigator is trying to identify targets, or a target, such as an 1825:. Thus, the typical range for AS-MS is 400-3,000 compounds per pool. Other considerations for screening are more practical, such as a need to balance desired compound concentration, which is usually in the 2612:

because the chemical libraries used in screening have not been optimized against any targets. However, methods like affinity chromatography and affinity selection-mass spectrometry are workhorses of the

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requires accurate modeling of both ligand and protein conformation at atomic resolution, and is therefore aided by empirical determination of protein structure, often through orthogonal methods such as

1760:. 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 1032:

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

1600:. Proteins that are stabilized by the drug will remain folded at higher concentrations of GuHCl and will experience less methionine oxidation. Oxidized methionine residues can be quantified via 2570:. If a drug produces a positive test, the next step is to determine whether it is acting on a known or novel target. Chemoproteomics is thus a follow-up to phenotypic screening. In the case of 1364:

interactions from shifts in protein thermal stability induced by ligand binding. In a typical assay setup, protein-containing samples are exposed to a ligand of choice, then those samples are

1036:-coated beads, which bind the biotin tag and allow for pull-down and identification of interaction partners. This technology can be extended to assist with preparation of samples for ABPP and 1289:

from induced fit is often limited when proteins or drugs are immobilized. Probe-based approaches also alter the three-dimensional nature of the ligand-protein interaction by introducing

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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

926:, if the investigator is screening for small molecules. The bait is exposed to a mixture of potential binding partners, which can be identified after removing non-binding components. 1521:

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

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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

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follows a similar basic assumption to TPP – that protein stability is increased by ligand binding. In DARTS, however, protein stability is assessed in response to digestion by a

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is expected to increase, so ligand-bound proteins will be more resistant to thermal denaturation. After heating, the amount of non-denatured protein remaining is analyzed using

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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

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conformation, and moves away from the unpredictable distribution of less stable conformers. Thus, ligand binding is said to stabilize proteins, making them resistant to

1574:. 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 778:

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

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Annis DA, Nickbarg E, Yang X, Ziebell MR, Whitehurst CE (October 2007). "Affinity selection-mass spectrometry screening techniques for small molecule drug discovery".

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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

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Chemoproteomics assays can be stratified into three basic types. Solution-based approaches involve the use of drug analogs that chemically modify target proteins in

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

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to assess differences in extent of digestion, and bands are then excised and analyzed via mass spectrometry to confirm the identities of proteins that are resist

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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

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is identical to the application buffer, ligands move readily through the column but at different speeds according to their underlying affinity for the target.

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Prudent R, Annis DA, Dandliker PJ, Ortholand JY, Roche D (2020-10-21). "Exploring new targets and chemical space with affinity selection-mass spectrometry".

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ligand with a known structure is to be screened against a protein with limited structural information, modeling is done with regard to ligand structure.

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

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profiles are constructed and correlated. Target identification is supported by a strong correlation in elution profile between a drug and a protein.

1297:-free approaches aim to infer interactions by proxy, often through observations of changes to protein stability upon binding, and sometimes through 2435: 1840:, effectively capping the number of compounds screened in the thousands. After appropriate test compounds and targets are selected and incubated, 4418: 937:-based immobilization is a modular technique in that it allows the investigator to decide whether they wish to fish for protein targets from the 705:

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

4029:"Target identification by chromatographic co-elution: monitoring of drug-protein interactions without immobilization or chemical derivatization" 1389:-wide scale and stability curves can be generated for thousands of proteins at once. Thermal proteome profiling has been successfully performed 1617:

provide meaningful information, and not all differences in methionine oxidation are consistent with protein stabilization. Without enrichment,

324: 1040:. While immobilization approaches have been reproducible and successful, it is impossible to avoid the limitation of immobilization-induced 4519: 1285:

and targets are limited in their ability to move freely through space in a way that replicates the native protein-ligand interaction, and

4524: 3392:"Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification" 2483:

are heterobifunctional small molecules that contain a functional group that binds a target and another functional group that recruits an

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levels, and in living systems to ensure that labeling occurs only when exposed to light. Activation by light must also be fine-tuned, as

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simulations have emerged as relatively low-cost, high-throughput means for ranking the strength of small molecule-protein interactions.

1013:. High-affinity ligands bind to the immobilized target and resist removal by washing, so they are enriched in the sample. Conversely, a 4175:

Khedkar SA, Malde AK, Coutinho EC, Srivastava S (March 2007). "Pharmacophore modeling in drug discovery and development: an overview".

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strategies owing to the lack of functional readouts, which would otherwise require deconvolution of the source compound that generates

4361:"Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors" 2386:

modeling identifies key electronic and structural features that are associated with therapeutic activity across similarly bioactive

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rates have been on the decline over the last decade. One potential source of drug failure is the disconnect between early and late

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functional groups are common linkers to facilitate attachment. More modern approaches have benefitted from the popularization of

195:(TMTs), then labeled samples are pooled. The sample origin of each peptide can be discerned from the TMT attached to it. Labeled 135:

upon binding. Computational techniques complement the chemoproteomic toolkit as parallel lines of evidence supporting potential

4546: 2040: 2000: 2617:, and AS-MS particularly has been documented to produce a significant number of hits across many classes of difficult-to-drug 3553: 3145: 2287:

co-elution does not rely on differences in protein stability after drug treatment. Instead, it rests on the assumption that

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Because photoaffinity probes are multifunctional, they are difficult to design. Chemists incorporate the same principles of

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Deng H, Lei Q, Wu Y, He Y, Li W (April 2020). "Activity-based protein profiling: Recent advances in medicinal chemistry".

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for superimposition, but this requires user-selected anchor points and therefore introduces human bias into the process.

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is incubated with a small molecule of interest, the sample is split into aliquots, and each aliquot goes through limited

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strategies are categorized by the type of information that is already known about the ligand and protein of interest.

3255: 200: 3493:"Ligand Fishing: A Remarkable Strategy for Discovering Bioactive Compounds from Complex Mixture of Natural Products" 1733:

per experiment have been reported in the literature, and one group has reported assaying chemical libraries against

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enzyme with the reactive group and can vary depending on the downstream readout. The most widely used reporters are

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was followed with hope for a new paradigm in treating disease. Many fatal and intractable diseases were able to be

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Yang SY (June 2010). "Pharmacophore modeling and applications in drug discovery: challenges and recent advances".

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The development and application of bench-top chemoproteomics assays is often time consuming and cost-prohibitive.

5363: 2738: 2728: 1725:- and label-free approaches, AS-MS has the unique advantage of being amenable to the assessment of multiple test 1432: 791: 529: 525: 340: 2741:

as targets of purvanolol; CK1 is a Plasmodium protein, and purvanolol analogs have activity against Plasmodium.

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gradient and fractions are collected over short time intervals. Each fraction is analyzed by LC-MS/MS for both

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Under the assumption that enzymes vary in their structure, function, and associations depending on a system's

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models rely on inclusion of many potential targets, not just the therapeutic target. For example, important

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Under the ligand immobilization format, a ligand of interest - often a drug lead - is immobilized within a

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Spradlin JN, Zhang E, Nomura DK (April 2021). "Reimagining Druggability Using Chemoproteomic Platforms".

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from the column and identified using mass spectrometry. A table of elution strategies is provided below.

378: 155: 115:, tagging them for identification. Immobilization-based approaches seek to isolate potential targets or 5339: 4888: 4859: 4839: 4792: 2638: 2296: 128: 3441:"Recent advances in screening active components from natural products based on bioaffinity techniques" 2236: 838: 5385: 4477: 2609: 2442:

may yield high-affinity interactions with therapeutic targets, but they may also lead to undesirable

2299:. In a typical workflow, a cell lysate is incubated with a drug, then the lysate is injected onto an 2183: 1818: 1586: 664: 596:; and (3) a linker group. The site-directed covalent warhead, also called a covalent modifier, is an 561: 450: 284: 2525:. PROTACs are heterobifunctional small molecules that are designed to interact with a target and an 2170:

source under conditions gentle enough to preserve the interaction and maintain its integrity in the

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do not allow for enrichment for proteomic analysis, while biotin-based affinity tags co-purify with

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Geoghegan KF, Johnson DS (2010). "Chemical Proteomic Technologies for Drug Target Identification".

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and chemical degradation. Some examples of stability-based derivatization-free approaches follow.

1092:, and other compounds pass through the column unretained. In both cases, retained analytes can be 373:

and samples are processed using a generalizable workflow. A standard procedure begins with sample

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bind targets in solution, and are then pulled down by an enrichment handle, such as biotin. (1C)

672: 659: 541: 359:, which give insight into the functional state of a cell, is also possible. The vast majority of 328: 253: 249: 5088: 5076: 4967: 4832: 4606: 4472: 3580:

Rodriguez EL, Poddar S, Iftekhar S, Suh K, Woolfork AG, Ovbude S, et al. (November 2020).

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profiling has led to innumerable advances in the biomedical space, but the characterization of

35: 2258:. Fractions are collected every minute, then analyzed for both drug and protein content using 1052:

of both proteins and small molecules to the bead surface, which has the potential to generate

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While the approaches above have shown success, they are inherently limited by their need for

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and heated to separate individual temperature points. Upon binding to a ligand, a protein's

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Chan JN, Vuckovic D, Sleno L, Olsen JB, Pogoutse O, Havugimana P, et al. (July 2012).

3798: 2916: 2557: 1837: 1778: 1730: 1726: 1702: 1582: 1337: 1313: 1201: 1134: 982: 950: 934: 911: 859: 331:, which allows for the near complete identification and relative quantification of complex 221: 112: 47: 3785:

Lomenick B, Hao R, Jonai N, Chin RM, Aghajan M, Warburton S, et al. (December 2009).

2471: 2182:. While this was shown to be possible by Ganem and Henion in 1991, it is not suitable for 2023:

complexes are excluded from entering the beads and exit the column quickly, while unbound

1500:. Alternatively, if the target is already suspected and is being tested for validation, a 1410:, this technique is referred to as the Mass Spectrometry Cellular Thermal Shift Assay (MS- 8: 5250: 5204: 5129: 5102: 5000: 4982: 4935: 4873: 4769: 4749: 4618: 4613: 4514: 3586:

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences

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Dowden H, Munro J (July 2019). "Trends in clinical success rates and therapeutic focus".

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does not need to be tied to a functional readout, opening the possibility of identifying

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Knockaert M, Gray N, Damiens E, Chang YT, Grellier P, Grant K, et al. (June 2000).

3802: 1439:. Drug binding is expected to result in an increase in signal of the stabilized protein. 5390: 5327: 5293: 5155: 5124: 5005: 4947: 4645: 4628: 4623: 4578: 4541: 4531: 4492: 4341: 4293: 4268: 4242: 4157: 4104: 4079: 4055: 4028: 4006: 3954: 3821: 3786: 3759: 3734: 3733:

Mateus A, Kurzawa N, Becher I, Sridharan S, Helm D, Stein F, et al. (March 2020).

3710: 3685: 3663: 3606: 3581: 3519: 3492: 3465: 3440: 3416: 3391: 3362: 3337: 3304: 3278:"Advanced Activity-Based Protein Profiling Application Strategies for Drug Development" 3277: 3198: 3173: 3151: 3095: 3047: 3022: 2994: 2593: 2391: 2387: 2191: 1971: 1706: 994: 923: 736: 612: 4377: 4360: 3247: 524:

and protein expression information leave gaps in knowledge surrounding the effects of

50:, in which lead compounds are designed to interact with predetermined disease-driving 5346: 5308: 5273: 5256: 5194: 5112: 5107: 5035: 5020: 4990: 4911: 4878: 4849: 4844: 4819: 4809: 4729: 4717: 4596: 4509: 4382: 4345: 4333: 4298: 4246: 4234: 4192: 4149: 4109: 4060: 3998: 3958: 3946: 3892: 3826: 3764: 3715: 3667: 3655: 3611: 3582:"Affinity chromatography: A review of trends and developments over the past 50 years" 3559: 3549: 3524: 3470: 3421: 3367: 3309: 3251: 3203: 3155: 3141: 3099: 3087: 3052: 2998: 2986: 2896: 2582: 2395: 2355: 2342: 2338: 2167: 2156: 1975: 1851: 1810: 1798: 1678: 1626: 1590: 1542: 1526: 1436: 1407: 1369: 1045: 946: 801: 783: 760: 724: 637: 629: 545: 412: 370: 152: 144: 63: 51: 4161: 2372:

An example pharmacophore model. Each sphere represents a different scorable feature.

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and has the advantage of more efficient removal of unbound compounds as compared to

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modeling against therapeutic targets is only one component of the compound's total

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binding confers protection to proteins from manners of degradation, this time from

1537:. Drug binding is expected to protect methionine from oxidation by stabilizing the 1290: 1278: 1138: 1081: 1041: 1006: 942: 787: 517: 480: 385: 105: 75: 4284: 3597: 1805:

across a range of affinities can be identified; inversely, assays can contain low

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Stability of Proteins from Rates of Oxidation also rests upon the assumption that

62:, chemoproteomics provides valuable follow-up strategies to narrow down potential 5209: 5165: 5160: 5054: 5030: 4864: 4827: 4680: 4670: 4553: 4329: 4230: 4145: 2879: 2827: 2447: 2036: 1992: 1953: 1921: 1913: 1893: 1877: 1861: 1753:

A pool of test compounds is added to a protein sample, which is passed through a

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Hybrid solution- and immobilization-based strategies have been applied, in which

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is used to move the sample through the column. SpeedScreen is not coupled to an

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must travel through the beads and are retained by the column for a longer time.

1609:, and for which there are currently no viable enrichment techniques. Only those 997:

properties, which make them very easy to remove from solution using an external

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A drug scaffold acts as the first interaction site between probe and protein. A

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Proceedings of the National Academy of Sciences of the United States of America

3787:"Target identification using drug affinity responsive target stability (DARTS)" 3456: 3083: 2906: 2855:

protein, which has a large binding site and strong competing endogenous ligand

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that can be screened in a single assay. Researchers at the iHuman Institute at

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An example target identification by chromatographic co-elution (TICC) workflow.

2152: 2144: 2128: 2092: 1988: 1933: 1917: 1901: 1857: 1830: 1782: 1754: 1722: 1718: 1710: 1666: 1654: 1538: 1294: 1274: 1152: 970: 891: 887: 867: 740: 732: 690: 576: 509: 396: 356: 280: 268: 264: 233: 163: 159: 132: 120: 94: 82: 79: 31: 3509: 3407: 3174:"Activity-based protein profiling for biochemical pathway discovery in cancer" 2099:

system and requires further processing prior to final analysis. In this case,

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after an investigation into off-target effects of repurposed COVID-19 drugs.

2549: 2509: 2451: 2439: 2427: 2409: 2400: 2383: 2366: 2304: 2278: 2251: 2226: 2080: 1802: 1694: 1686: 1630: 1251: 1156: 728: 702: 640: 621: 580: 564:) is a technique that was developed to monitor the availability of enzymatic 484: 460: 456: 408: 348: 288: 276: 260: 237: 208: 151:

have been identified using chemoproteomics, and the continued improvement of

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used AS-MS to successfully screen for ligands against the difficult to drug

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employed of scheme in which 20,000 compounds per pool were screened against

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that elute from the column early on are therefore inferred to be bound to a

1589:, proteins will unfold to varying degrees. Each sample is then reacted with 1175:

Elution conditions may not be strong enough to elute unoptimized compounds.

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Haas P, Muralidharan M, Krogan NJ, Kaake RM, Hüttenhain R (February 2021).

4238: 4196: 4153: 4113: 4080:"Molecular docking: a powerful approach for structure-based drug discovery" 4064: 4002: 3950: 3896: 3830: 3768: 3719: 3659: 3615: 3528: 3474: 3425: 3371: 3313: 3207: 3091: 3056: 2990: 2567: 2497: 2259: 2199: 2003:(HPLC) based and spin column formats. In either case, a mixture of unbound 1963: 1945: 1501: 1378: 1142: 1133:

composition shifts the local chemical environment of the stationary phase;

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Phenylazide, a photoreactive group commonly used in photoaffinity labeling.

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technology indicates that chemoproteomics will play a large role in future

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to drug, with a 0.12% hit rate, leading to several high affinity ligands.

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An example stability of proteins from rates of oxidation (SPROX) workflow.

1320:, with many of them different from one another despite being identical in 1141:

is decreased, causing bound ligands to preferentially distribute into the

427:. For more accurate quantification, different samples can be reacted with 179: 4573: 4269:"Proteomic Approaches to Study SARS-CoV-2 Biology and COVID-19 Pathology" 3975:

Zehender H, Le Goff F, Lehmann N, Filipuzzi I, Mayr LM (September 2004).

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from up to 96 samples. Samples are also passed through porous beads, but

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Protein extracts from different samples are extracted and digested using

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of the target by the E3 ubiquitin ligase, leading to target degradation.

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Immobilization-based chemoproteomic techniques encompass variations on

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concentrations to allow for distinction between high and low affinity

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noise can drown out relevant signal. Therefore, SPROX samples require

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An example drug affinity responsive target stability (DARTS) workflow.

459:

warhead and reporter tag are connected by a linker group. The warhead

5261: 4563: 4428: 3977:"SpeedScreen: The "missing link" between genomics and lead discovery" 3543: 3390:

Chen X, Wang Y, Ma N, Tian J, Shao Y, Zhu B, et al. (May 2020).

1614: 1567: 986: 978: 827: 593: 537: 228:, providing a starting point to better understand the roles of their 74:. Chemoproteomics also attempts to address the inherent challenge of 43: 3189: 2504:

of druggable proteins, these define only about 15% of the annotated

1740:

pools. The basic steps of AS-MS are described in more detail below.

1451: 1001:. In a simplified workflow, magnetic beads are used to immobilize a 303:. Chemoproteomic tools offer a solution to bridge the gap between a 275:

validation of a target, which is a strong predictor of success, but

89:-wide scale. A major goal of chemoproteomics is to characterize the 5283: 3735:"Thermal proteome profiling for interrogating protein interactions" 3338:"Photoaffinity labeling in target- and binding-site identification" 3129: 2618: 2571: 2562: 2542: 2505: 2421:

Ideally, the structure of a drug target is known, which allows for

2292: 2072: 1618: 1594: 1493: 1473: 1461: 1391: 1386: 1382: 1018: 973:

to attach a bait of choice. In the case of using proteins as bait,

938: 898: 795: 719:

Unlike ABPP, which results in protein labeling upon probe binding,

605: 548:, chemical tools are required to detect drug-protein interactions. 424: 367: 332: 304: 272: 101: 86: 67: 3634:"Recent advances in identifying protein targets in drug discovery" 2035:. The automated ligand identification system (ALIS), developed by 1852:

Separation of unbound small molecules and ligand-protein complexes

1625:

is challenging, as the contribution of other sample components to

1431:

and the extent of protein digestion can either be visualized on a

1236:

Compounds not previously tested may have higher affinity than the

735:

makes this possible. These probes are composed of three connected

315:

by identifying the many proteins involved in therapeutic success.

5303: 2522: 2500:

targets. While historically successful drugs target well-defined

2351: 2316: 2308: 2263: 2218: 2195: 2148: 2132: 2056: 2032: 2020: 2008: 1909: 1869: 1845: 1806: 1790: 1737: 1674: 1662: 1650: 1634: 1622: 1606: 1530: 1505: 1485: 1402: 1395: 1361: 1329: 1237: 1230: 1216: 1212: 1184: 1165: 1101: 1093: 1002: 990: 966: 962: 958: 894: 847: 782:

handles are suitable for enrichment of labeled proteins prior to

513: 472: 420: 296: 241: 229: 196: 188: 27: 2496:

Chemoproteomic strategies have been used to expand the scope of

2446:, and they may also be substrates of metabolic enzymes, such as 1952:

were used to stabilize the protein and prevent its aggregation.

1912:

on the basis of size. Multiple washing steps are required after

1817:. The choice of a chemical library is less stringent than other 844:

Three strategies for immobilization-based target identification.

3974: 3023:"How chemoproteomics can enable drug discovery and development" 2766: 2760: 2622: 2597: 2586: 2581:, which was among the first repurposed drugs to be used in the 2247: 2175: 1774:

A generalized AS-MS workflow begins with the pre-incubation of

1022: 998: 609: 601: 589: 533: 488: 476: 468: 4174: 3928: 2702:

Lekostaj et al identified the chloroquine binding site on the

1669:

are removed from the mixture, and the components of remaining

763:

of a drug whose mechanism is being studied, and, importantly,

3686:"Identification of direct protein targets of small molecules" 3276:

Wang S, Tian Y, Wang M, Wang M, Sun GB, Sun XB (2018-04-09).

2911: 2872: 2794: 2788: 2749: 2270: 1761: 1549: 1457: 1443: 1411: 1304:

The stability-based methods below are thought to work due to

1261: 1088:, ligands are retained through binding interactions with the 974: 902: 706: 492: 471:

and the reporter tag is used to enrich or detect the labeled

374: 212: 55: 3732: 2585:. Remdesivir was tested via thermal proteome profiling in a 2560:

is usually employed to identify drugs with a desired effect

1480:

is critical, since complete proteolysis would render even a

119:

by anchoring their binding partners to an immobile support.

16:

Techniques used to study protein–small molecule interactions

5278: 4266: 2431: 2320: 2288: 1698: 1541:

form of a protein. Extent of oxidation can be monitored by

1325: 1316:

may be represented by individual molecules in a variety of

148: 136: 124: 3579: 3439:

Hou X, Sun M, Bao T, Xie X, Wei F, Wang S (October 2020).

2295:, and that those complexes are robust enough to survive a 2279:

Target identification by chromatographic co-elution (TICC)

2103:

must be freed from their targets and analyzed separately.

1072:. A complex sample consisting of many proteins, such as a 431:(TMTs), a form of chemical barcode that allows for sample 5288: 4358: 2682:

inhibitor ABX1431 using a competitive inhibition system.

2179: 2163:

approach requires direct infusion of the complex into an

1856:

Affinity selection is followed by the removal of unbound

1308:-induced shifts in equilibrium concentrations of protein 1273:, which jeopardizes the affinity of the interaction that 244:

absence, particularly in the development of disease, and

3874: 2139:

detection of intact complexes - or indirectly - through

1693:. Since AS-MS measures binding in an unbiased manner, a 1511: 1417: 2797:

as a target of the longevity-associated small molecule

2399:

conformational models are tested during the process of

1644: 1381:

against a known target of interest. With the advent of

1148: 823: 4026: 2882:

4513-0042 and followed up with validation experiments

2574:, Friman et al investigated off-target effects of the 1789:. Assays can be designed to contain sufficiently high 1137:

is changed, causing release of ligands; alternatively

667:

has benefitted from ABPP, particularly in the area of

516:

expression is limited in its ability to inform on the

479:

is an example of an activity-based probe that targets

85:

by analyzing protein-small molecule interactions on a

2709:

resistance transporter using photoaffinity labeling.

2645:

Real World Applications of Chemoproteomic Techniques

2119:

from their targets, and subsequent identification of

1558:

Stability of proteins from rates of oxidation (SPROX)

1260:

its stability curve (2). This image was adapted from

1084:

are then passed through the column in an application

3784: 2862:

Target identification by chromatographic co-elution

1876:

available for downstream analysis. Several types of

1229:

Allows for concentration of analytes with immediate

1017:

of interest can be immobilized and screened against

632:. There are drawbacks to each strategy, namely that 4216: 2621:. This is due in large part to the sheer volume of 2432:

quantitative structure-activity relationship (QSAR)

2225:into the precipitation solution, which can then be 2087:, which allows for simultaneous removal of unbound 1347: 751:; and (3) an identification tag, such as biotin, a 551: 3683: 1848:complexes can be separated by a variety of means. 1721:is needed to confirm the validity of a hit. Among 1293:to the ligand, which can alter compound activity. 833: 48:target-based drug discovery (reverse pharmacology) 4077: 3020: 2487:Binding to both proteins induces proximity-based 2206:analysis. A simpler method for analysis of bound 1452:Drug affinity responsive target stability (DARTS) 1246: 929: 536:activity, and that enzyme activity varies across 283:systems are simplistic. Spatially and temporally 5377: 3275: 3233: 3172:Nomura DK, Dix MM, Cravatt BF (September 2010). 3171: 2332: 1689:and can be used to provide a starting point for 854:and bound targets are detected downstream. (1A) 256:compounds against disease-causing proteins. Yet 191:. Separate samples are labeled using individual 4078:Meng XY, Zhang HX, Mezei M, Cui M (June 2011). 2608:Approved drugs are never identified as hits in 2011:, ligand-protein complexes is passed through a 1385:, this type of experiment can be executed on a 1257:An example thermal proteome profiling workflow. 914:-based affinity pull-down, which is similar to 3684:Lomenick B, Olsen RW, Huang J (January 2011). 3631: 3491:Zhuo R, Liu H, Liu N, Wang Y (November 2016). 3490: 3389: 2962: 2808:Stability of proteins from rates of oxidation 2541:. This is particularly relevant in the era of 2071:system for on-line analysis of bound ligands. 1456:The Drug Affinity Responsive Target Stability 1197:Allows for comparison of compound affinities. 588:(reactive group); (2) a reporter tag, such as 325:liquid chromatography-tandem mass spectrometry 4412: 2965:"Chemoproteomic-enabled phenotypic screening" 1277:compounds are said to emulate and introduces 1129:Bound ligands are released after a change in 993:from treated samples. Magnetic beads exhibit 291:systems have improved the level of nuance in 127:-target interactions by observing changes in 3632:Ha J, Park H, Park J, Park SB (March 2021). 3438: 3335: 2603: 2545:, which saw a dire need to rapidly identify 2106: 1356:) is recently popularized strategy to infer 438: 185:An example quantitative proteomics workflow. 58:do not provide confirmation of a compound's 4315: 3069: 2327: 1880:have been reported with varying degrees of 1585:(GuHCl). Depending on the concentration of 1383:high resolution Orbitrap mass spectrometers 1200:Can be time consuming; requires continuous 858:are attached to a solid support, such as a 4419: 4405: 3021:Moellering RE, Cravatt BF (January 2012). 2780:Drug affinity responsive target stability 2416: 1058: 759:handle. The drug scaffold is typically an 323:The chemoproteomic toolkit is anchored by 34:interactions. Chemoproteomics complements 4426: 4376: 4292: 4103: 4054: 4044: 3992: 3820: 3810: 3758: 3709: 3649: 3605: 3518: 3508: 3464: 3415: 3361: 3303: 3293: 3197: 3046: 2980: 2963:Conway LP, Li W, Parker CG (March 2021). 2600:as a potential off-target of Remdesivir. 1944:published a screening strategy involving 798:, to validate interaction with a target. 714: 377:, in which proteins are extracted into a 3396:Signal Transduction and Targeted Therapy 2523:proteolysis-targeting-chimeras (PROTACs) 2470: 2365: 2291:form stable complexes with their target 2235: 2151:complexes followed by identification of 1742: 1510: 1416: 1250: 1005:target, then the beads are mixed with a 837: 815:structure-activity relationship modeling 800: 678: 442: 178: 139:-target pairs, and are used to generate 4318:European Journal of Medicinal Chemistry 2537:Chemoproteomics is at the forefront of 2361: 2135:interactions either directly - through 2125:liquid chromatography-mass spectrometry 2049:liquid chromatography-mass spectrometry 1999:approaches have been described in both 731:to a protein occurs. The presence of a 5378: 4262: 4260: 4258: 4256: 4212: 4210: 4208: 4206: 4022: 4020: 3870: 3868: 3866: 3864: 3862: 3860: 3780: 3778: 3679: 3677: 3627: 3625: 3541: 3486: 3484: 3385: 3383: 3381: 3016: 3014: 3012: 3010: 3008: 2001:high-performance liquid chromatography 1916:to ensure complete removal of unbound 1749:Affinity selection via gel filtration. 1701:that act beyond active sites, such as 1545:and used to generate stability curves. 697:, can be activated by light to form a 518:functional characteristics of proteins 335:in biological samples. In addition to 42:on the basis of alleviating a disease 4400: 4127: 4125: 4123: 3970: 3968: 3924: 3922: 3920: 3918: 3916: 3914: 3912: 3910: 3908: 3906: 3858: 3856: 3854: 3852: 3850: 3848: 3846: 3844: 3842: 3840: 3575: 3573: 3236:Annual Reports in Medicinal Chemistry 3117: 3115: 3113: 3111: 3109: 2958: 2956: 2837:Affinity selection-mass spectrometry 2477:PROTAC: Proteolysis targeting chimera 2127:. AS-MS offers means for identifying 1769: 1657:to allow for the formation of stable 5334: 4131: 3336:Smith E, Collins I (February 2015). 3331: 3329: 3327: 3325: 3323: 3271: 3269: 3267: 3229: 3227: 3225: 3223: 3221: 3219: 3217: 3167: 3165: 2940: 2592:, along with the controversial drug 1713:can be directly identified by their 1645:Affinity selection-mass spectrometry 676:at only a small number of proteins. 628:of labeled enzymes and analysis via 5358: 4253: 4203: 4033:Molecular & Cellular Proteomics 4017: 3877:Current Opinion in Chemical Biology 3775: 3674: 3622: 3481: 3378: 3121: 3005: 2532: 1829:range, with the fact that compound 1685:identified are then categorized as 1048:. Another drawback is non-specific 685:A prototypical photoaffinity probe. 97:to gain insight into mechanisms of 38:, a paradigm that aims to discover 13: 4120: 4084:Current Computer-Aided Drug Design 3965: 3903: 3837: 3570: 3106: 2953: 2083:spin column format, also known as 2067:and diverts the complex toward an 1974:was detected and quantified using 1352:Thermal proteome profiling (also, 556:Activity-based protein profiling ( 147:. Several targets of high profile 14: 5407: 3981:Journal of Biomolecular Screening 3320: 3264: 3214: 3162: 2875:protein ERG6P as a target of the 2667:Activity based protein profiling 1729:per experiment—as many as 20,000 1709:, all in a single assay. Because 1504:protocol can be used to identify 818:remain stable in storage, across 624:, such as biotin, that allow for 491:chain. This image was made using 5357: 5345: 5333: 5322: 5321: 2212:protein precipitation extraction 1896:. Under both pressure-based and 1815:structure-activity relationships 1492:. Samples are then analyzed via 1348:Thermal proteome profiling (TPP) 957:were historically made of inert 850:mixtures are incubated with the 552:Activity-based protein profiling 526:post-translational modifications 451:activity-based protein profiling 341:post-translational modifications 4352: 4309: 4168: 4071: 3726: 3535: 3432: 3125:Advances in Chemical Proteomics 2596:, and investigators discovered 2466: 2461: 2456:structure-activity relationship 1328:, the majority of ligand-bound 989:separation of bead-immobilized 834:Immobilization-based approaches 575:. ABPP uses specially designed 307:understanding of disease and a 240:that highlight the impact of a 54:. As phenotypic drug discovery 3651:10.1016/j.chembiol.2020.12.001 3072:Nature Reviews. Drug Discovery 3063: 3039:10.1016/j.chembiol.2012.01.001 2982:10.1016/j.chembiol.2021.01.012 2934: 2819:Geer Wallace et al discovered 2051:(LC-MS) system that separates 1247:Derivatization-free approaches 1021:proteins by incubation with a 930:Microbead-based immobilization 901:. This image was adapted from 765:binds to the target reversibly 318: 211:. This image was adapted from 123:-free approaches aim to infer 1: 4686:Interface and colloid science 4440:Glossary of chemical formulae 4378:10.1016/s1074-5521(00)00124-1 4285:10.1021/acs.jproteome.0c00764 4219:Accounts of Chemical Research 3598:10.1016/j.jchromb.2020.122332 3248:10.1016/s0065-7743(10)45021-6 2927: 2556:activity. In this context, a 2352:cryogenic electron microscopy 2333:Molecular docking simulations 2190:, which is typically used in 2188:electron capture dissociation 2085:gel filtration chromatography 1984:(SEC) is more widely used in 1982:Size-exclusion chromatography 1866:size-exclusion chromatography 1795:competition for binding sites 1785:) with chemical libraries in 1677:complexes are analyzed using 739:: (1) a drug scaffold; (2) a 723:probes require activation by 169: 4330:10.1016/j.ejmech.2020.112151 4273:Journal of Proteome Research 4231:10.1021/acs.accounts.1c00065 4146:10.1016/j.drudis.2010.03.013 2748:Thermal proteome profiling ( 2590:cellular thermal shift assay 2198:, has been used to identify 1884:, including pressure-based, 1354:Cellular Thermal Shift Assay 1190:Transient interactions with 941:or drug-like compounds from 786:based identification, while 530:protein-protein interactions 7: 4963:Bioorganometallic chemistry 4450:List of inorganic compounds 3445:Acta Pharmaceutica Sinica B 2890: 2301:ion-exchange chromatography 2269:This image was made using 2256:ion exchange chromatography 1833:are typically stored as 10 1653:targets are incubated with 1548:This image was made using 1442:This image was made using 1312:. A single protein type in 774:in a target's active site, 600:that covalently modifies a 363:studies are analyzed using 339:analysis, the detection of 10: 5412: 4889:Dynamic covalent chemistry 4860:Enantioselective synthesis 4840:Physical organic chemistry 4793:Organolanthanide chemistry 4189:10.2174/157340607780059521 4096:10.2174/157340911795677602 3943:10.1038/s41570-020-00229-2 3889:10.1016/j.cbpa.2007.07.011 3457:10.1016/j.apsb.2020.04.016 3342:Future Medicinal Chemistry 3084:10.1038/d41573-019-00074-z 2871:Chan et al identified the 2823:as a target of the potent 2793:Lomenick et al discovered 2733:Knockaert et al confirmed 2639:G-protein coupled receptor 2508:. To continue growing our 2297:chromatographic separation 1793:concentrations to prevent 1299:chromatographic co-elution 327:(LC-MS/MS or LC-MS) based 301:engaging targets partially 174: 66:and eventually validate a 5317: 5220: 4981: 4897: 4818: 4768: 4644: 4587: 4478:Electroanalytical methods 4463: 4435: 3739:Molecular Systems Biology 3510:10.3390/molecules21111516 3408:10.1038/s41392-020-0186-y 3282:Frontiers in Pharmacology 3122:Yao, Xudong, ed. (2021). 2604:High-throughput screening 2283:Target identification by 2107:Analysis of bound ligands 1972:ligand-stabilized protein 1819:high-throughput screening 1583:guanidinium hydrochloride 1324:sequence. Upon binding a 665:High-throughput screening 562:activity-based proteomics 439:Solution-based approaches 429:isobaric tandem mass tags 193:isobaric tandem mass tags 36:phenotypic drug discovery 5233:Nobel Prize in Chemistry 5149:Supramolecular chemistry 4788:Organometallic chemistry 3994:10.1177/1087057104267605 3931:Nature Reviews Chemistry 3295:10.3389/fphar.2018.00353 2941:Wang, Lei (2020-08-21). 2765:Friman et al identified 2576:broad-spectrum antiviral 2566:, such as inhibition of 2406:least-squares regression 2328:Computational approaches 2111:The final step requires 1100:Affinity Chromatography 1044:, which interferes with 1009:to screen for potential 620:that enable imaging and 542:physiological conditions 487:enrichment handle by an 226:mapped to specific genes 222:the Human Genome Project 5171:Combinatorial chemistry 5082:Food physical chemistry 5045:Environmental chemistry 4929:Bioorthogonal chemistry 4855:Retrosynthetic analysis 4676:Chemical thermodynamics 4659:Spectroelectrochemistry 4602:Computational chemistry 4365:Chemistry & Biology 4046:10.1074/mcp.M111.016642 3812:10.1073/pnas.0910040106 3545:Affinity Chromatography 3138:10.1016/C2019-0-03583-2 3027:Chemistry & Biology 2717:Affinity chromatography 2627:ShanghaiTech University 2615:pharmaceutical industry 2610:high-throughput screens 2417:Structure-based methods 2165:electrospray ionization 2059:complexes from unbound 1930:non-specific adsorption 1464:. Briefly, a sample of 1374:quantitative proteomics 1334:energetically favorable 1059:Affinity chromatography 920:affinity chromatography 663:in different contexts. 483:. It is connected to a 329:quantitative proteomics 250:computational chemistry 236:has made use of animal 5243:of element discoveries 5089:Agricultural chemistry 5077:Carbohydrate chemistry 4968:Bioinorganic chemistry 4833:Alkane stereochemistry 4778:Coordination chemistry 4607:Mathematical chemistry 4473:Instrumental chemistry 3178:Nature Reviews. Cancer 2947:Doctoral Dissertations 2690:Photoaffinity labeling 2678:Cesar et al optimized 2568:viral plaque formation 2493: 2394:based modeling toward 2375: 2307:. Lysate proteins are 2275: 1942:University of Illinois 1906:semipermeable membrane 1766: 1554: 1448: 1266: 1038:photoaffinity labeling 907: 810: 790:are used when using a 776:photoaffinity labeling 747:, phenyldiazirine, or 721:photoaffinity labeling 715:Photoaffinity labeling 711: 669:competitive inhibition 579:that enter and form a 540:, disease states, and 497: 217: 199:are then detected and 5238:Timeline of chemistry 5135:Post-mortem chemistry 5120:Clandestine chemistry 5050:Atmospheric chemistry 4973:Biophysical chemistry 4805:Solid-state chemistry 4755:Equilibrium chemistry 4664:Photoelectrochemistry 3751:10.15252/msb.20199232 3638:Cell Chemical Biology 3242:. Elsevier: 345–360. 2969:Cell Chemical Biology 2474: 2369: 2348:x-ray crystallography 2239: 2229:and identified on an 1904:are forced through a 1746: 1703:allosteric modulators 1637:of interest prior to 1514: 1420: 1310:conformational states 1287:conformational change 1254: 1171:proteins or ligands. 1126:Non-specific elution 1066:chromatography column 981:, a type of magnetic 864:chromatography column 841: 804: 699:reactive intermediate 682: 634:fluorescent reporters 446: 285:conditional knock-out 232:products in illness. 182: 5228:History of chemistry 5183:Chemical engineering 4958:Bioorganic chemistry 4708:Structural chemistry 4445:List of biomolecules 4134:Drug Discovery Today 3690:ACS Chemical Biology 2917:Phenotypic screening 2840:Screening compounds 2769:as an off-target of 2485:E3 ubiquitin ligase. 2434:profiling. Accurate 2362:Ligand-based methods 2200:ligand binding sites 2115:separation of bound 1613:that are exposed to 1406:. When coupled with 1209:Biospecific elution 1135:protein conformation 618:fluorescent moieties 475:. Fluorophosphonate- 5396:Branches of biology 5251:The central science 5205:Ceramic engineering 5130:Forensic toxicology 5103:Chemistry education 5001:Radiation chemistry 4983:Interdisciplinarity 4936:Medicinal chemistry 4874:Fullerene chemistry 4750:Microwave chemistry 4619:Molecular mechanics 4614:Molecular modelling 4177:Medicinal Chemistry 3803:2009PNAS..10621984L 3797:(51): 21984–21989. 2646: 2527:E3 ubiquitin ligase 2444:off-target activity 2075:' SpeedScreen uses 1966:and remove unbound 1823:biological activity 1707:chemical chaperones 1665:complexes, unbound 1598:methionine residues 1572:methionine residues 1535:methionine residues 1224:Competitive binding 1202:fraction collection 1105: 916:immunoprecipitation 741:photoreactive group 733:photoreactive group 691:photoreactive group 546:biochemical readout 435:, and then pooled. 72:mechanism of action 60:mechanism of action 24:chemical proteomics 5294:Chemical substance 5156:Chemical synthesis 5125:Forensic chemistry 5006:Actinide chemistry 4948:Clinical chemistry 4629:Molecular geometry 4624:Molecular dynamics 4579:Elemental analysis 4532:Separation process 4140:(11–12): 444–450. 4039:(7): M111.016642. 3354:10.4155/fmc.14.152 2644: 2594:hydroxychloroquine 2494: 2388:structural analogs 2376: 2276: 2137:top-down proteomic 2039:, uses a combined 1799:structural analogs 1770:Affinity selection 1767: 1621:analysis of these 1555: 1472:after addition of 1449: 1267: 1180:Isocratic elution 1162:Can be fine tuned 1099: 1082:compound libraries 943:chemical libraries 924:immobilized enzyme 908: 890:directly on-bead. 830:can damage cells. 811: 712: 498: 413:Denatured proteins 371:mass spectrometers 246:medicinal chemists 220:The conclusion of 218: 52:biological targets 5373: 5372: 5309:Quantum mechanics 5274:Chemical compound 5257:Chemical reaction 5195:Materials science 5113:General chemistry 5108:Amateur chemistry 5036:Photogeochemistry 5021:Stellar chemistry 4991:Nuclear chemistry 4912:Molecular biology 4879:Polymer chemistry 4850:Organic synthesis 4845:Organic reactions 4810:Ceramic chemistry 4800:Cluster chemistry 4730:Chemical kinetics 4718:Molecular physics 4597:Quantum chemistry 4510:Mass spectrometry 3702:10.1021/cb100294v 3555:978-0-08-085812-8 3542:Turkov J (1978). 3451:(10): 1800–1813. 3147:978-0-12-821433-6 2897:Chemical genetics 2888: 2887: 2653:Bioactive Ligand 2558:phenotypic screen 2492: 2373: 2356:Molecular docking 2343:Molecular docking 2339:Molecular docking 2274: 2267: 2262:Drug and protein 2243: 2186:. Interestingly, 2168:mass spectrometry 2157:mass spectrometry 2015:of porous beads. 1976:mass spectrometry 1940:. A group at the 1900:formats, unbound 1765: 1750: 1679:mass spectrometry 1627:mass spectrometer 1591:hydrogen peroxide 1553: 1546: 1543:mass spectrometry 1527:Hydrogen peroxide 1518: 1447: 1440: 1437:mass spectrometry 1424: 1408:mass spectrometry 1370:thermal stability 1291:functional groups 1265: 1244: 1243: 1233:; high recovery. 995:superparamagnetic 906: 897:are analyzed via 808: 792:gel-based imaging 784:mass spectrometry 710: 686: 630:mass spectrometry 496: 481:serine hydrolases 311:understanding of 252:to generate high 216: 153:mass spectrometer 145:lead optimization 141:structural models 129:protein stability 5403: 5386:Chemical biology 5361: 5360: 5349: 5337: 5336: 5325: 5324: 5269:Chemical element 4924:Chemical biology 4783:Magnetochemistry 4760:Mechanochemistry 4713:Chemical physics 4654:Electrochemistry 4559:Characterization 4421: 4414: 4407: 4398: 4397: 4391: 4390: 4380: 4356: 4350: 4349: 4313: 4307: 4306: 4296: 4279:(2): 1133–1152. 4264: 4251: 4250: 4225:(7): 1801–1813. 4214: 4201: 4200: 4172: 4166: 4165: 4129: 4118: 4117: 4107: 4075: 4069: 4068: 4058: 4048: 4024: 4015: 4014: 3996: 3972: 3963: 3962: 3926: 3901: 3900: 3872: 3835: 3834: 3824: 3814: 3782: 3773: 3772: 3762: 3730: 3724: 3723: 3713: 3681: 3672: 3671: 3653: 3629: 3620: 3619: 3609: 3577: 3568: 3567: 3539: 3533: 3532: 3522: 3512: 3488: 3479: 3478: 3468: 3436: 3430: 3429: 3419: 3387: 3376: 3375: 3365: 3333: 3318: 3317: 3307: 3297: 3273: 3262: 3261: 3231: 3212: 3211: 3201: 3169: 3160: 3159: 3128:(1st ed.). 3119: 3104: 3103: 3067: 3061: 3060: 3050: 3018: 3003: 3002: 2984: 2960: 2951: 2950: 2938: 2902:Chemical biology 2647: 2643: 2539:drug repurposing 2533:Drug repurposing 2475: 2448:Cytochrome P450s 2371: 2268: 2245: 2241: 1801:, ensuring that 1776:purified protein 1752: 1748: 1547: 1520: 1516: 1441: 1426: 1422: 1279:steric hindrance 1255: 1192:stationary phase 1139:solvent polarity 1106: 1098: 1090:stationary phase 1078:stationary phase 1070:stationary phase 1068:and acts as the 1054:false positives. 1042:steric hindrance 1007:chemical library 842: 806: 794:method, such as 788:fluorescent dyes 729:covalent bonding 688: 684: 586:covalent warhead 461:covalently bonds 447: 401:alkylating agent 388:, an agent that 238:knock-out models 183: 106:polypharmacology 76:drug promiscuity 46:, as opposed to 5411: 5410: 5406: 5405: 5404: 5402: 5401: 5400: 5376: 5375: 5374: 5369: 5313: 5216: 5210:Polymer science 5166:Click chemistry 5161:Green chemistry 5055:Ocean chemistry 5031:Biogeochemistry 4977: 4893: 4865:Total synthesis 4828:Stereochemistry 4814: 4764: 4681:Surface science 4671:Thermochemistry 4640: 4583: 4554:Crystallography 4459: 4431: 4425: 4395: 4394: 4357: 4353: 4314: 4310: 4265: 4254: 4215: 4204: 4173: 4169: 4130: 4121: 4076: 4072: 4025: 4018: 3973: 3966: 3927: 3904: 3873: 3838: 3783: 3776: 3731: 3727: 3682: 3675: 3630: 3623: 3578: 3571: 3556: 3540: 3536: 3489: 3482: 3437: 3433: 3388: 3379: 3334: 3321: 3274: 3265: 3258: 3232: 3215: 3190:10.1038/nrc2901 3170: 3163: 3148: 3120: 3107: 3068: 3064: 3019: 3006: 2961: 2954: 2939: 2935: 2930: 2893: 2880:natural product 2849:Merck & Co. 2828:natural product 2737:and discovered 2634: 2606: 2535: 2502:binding pockets 2469: 2464: 2423:structure-based 2419: 2364: 2335: 2330: 2285:chromatographic 2281: 2194:elucidation of 2184:high throughput 2153:small molecules 2109: 2037:Schering-Plough 1993:ultrafiltration 1954:Ultrafiltration 1934:small molecules 1922:Ultrafiltration 1918:small molecules 1914:ultrafiltration 1902:small molecules 1894:ultrafiltration 1878:ultrafiltration 1862:ultrafiltration 1858:small molecules 1854: 1831:stock solutions 1783:target proteins 1772: 1711:small molecules 1691:lead generation 1667:small molecules 1655:small molecules 1647: 1633:to concentrate 1560: 1454: 1435:or measured by 1350: 1249: 1061: 985:, which enable 932: 836: 780:click chemistry 757:click chemistry 753:fluorescent dye 717: 651:, and modified 554: 449:A prototypical 441: 393:disulfide bonds 365:high-resolution 355:, and recently 345:phosphorylation 321: 309:pharmacological 248:have leveraged 177: 172: 95:drug candidates 22:(also known as 20:Chemoproteomics 17: 12: 11: 5: 5409: 5399: 5398: 5393: 5388: 5371: 5370: 5368: 5367: 5355: 5343: 5331: 5318: 5315: 5314: 5312: 5311: 5306: 5301: 5296: 5291: 5286: 5281: 5276: 5271: 5266: 5265: 5264: 5254: 5247: 5246: 5245: 5235: 5230: 5224: 5222: 5218: 5217: 5215: 5214: 5213: 5212: 5207: 5202: 5192: 5191: 5190: 5180: 5179: 5178: 5173: 5168: 5163: 5153: 5152: 5151: 5140: 5139: 5138: 5137: 5132: 5122: 5117: 5116: 5115: 5110: 5099: 5098: 5097: 5096: 5094:Soil chemistry 5086: 5085: 5084: 5079: 5072:Food chemistry 5069: 5067:Carbochemistry 5064: 5062:Clay chemistry 5059: 5058: 5057: 5052: 5041: 5040: 5039: 5038: 5033: 5023: 5017:Astrochemistry 5013:Cosmochemistry 5010: 5009: 5008: 5003: 4998: 4996:Radiochemistry 4987: 4985: 4979: 4978: 4976: 4975: 4970: 4965: 4960: 4955: 4953:Neurochemistry 4950: 4945: 4944: 4943: 4933: 4932: 4931: 4921: 4920: 4919: 4914: 4903: 4901: 4895: 4894: 4892: 4891: 4886: 4884:Petrochemistry 4881: 4876: 4871: 4862: 4857: 4852: 4847: 4842: 4837: 4836: 4835: 4824: 4822: 4816: 4815: 4813: 4812: 4807: 4802: 4797: 4796: 4795: 4785: 4780: 4774: 4772: 4766: 4765: 4763: 4762: 4757: 4752: 4747: 4745:Spin chemistry 4742: 4740:Photochemistry 4737: 4732: 4727: 4725:Femtochemistry 4722: 4721: 4720: 4710: 4705: 4700: 4695: 4694: 4693: 4683: 4678: 4673: 4668: 4667: 4666: 4661: 4650: 4648: 4642: 4641: 4639: 4638: 4637: 4636: 4626: 4621: 4616: 4611: 4610: 4609: 4599: 4593: 4591: 4585: 4584: 4582: 4581: 4576: 4571: 4566: 4561: 4556: 4551: 4550: 4549: 4544: 4537:Chromatography 4534: 4529: 4528: 4527: 4522: 4517: 4507: 4506: 4505: 4500: 4495: 4490: 4480: 4475: 4469: 4467: 4461: 4460: 4458: 4457: 4455:Periodic table 4452: 4447: 4442: 4436: 4433: 4432: 4424: 4423: 4416: 4409: 4401: 4393: 4392: 4371:(6): 411–422. 4351: 4308: 4252: 4202: 4183:(2): 187–197. 4167: 4119: 4090:(2): 146–157. 4070: 4016: 3987:(6): 498–505. 3964: 3902: 3883:(5): 518–526. 3836: 3774: 3725: 3673: 3644:(3): 394–423. 3621: 3569: 3554: 3534: 3480: 3431: 3377: 3348:(2): 159–183. 3319: 3263: 3256: 3213: 3184:(9): 630–638. 3161: 3146: 3105: 3078:(7): 495–496. 3062: 3004: 2975:(3): 371–393. 2952: 2932: 2931: 2929: 2926: 2925: 2924: 2919: 2914: 2909: 2907:Drug discovery 2904: 2899: 2892: 2889: 2886: 2885: 2883: 2869: 2866: 2863: 2859: 2858: 2856: 2846: 2841: 2838: 2834: 2833: 2831: 2830:Manassantin A 2825:antineoplastic 2817: 2812: 2811:Manassantin A 2809: 2805: 2804: 2802: 2791: 2786: 2781: 2777: 2776: 2774: 2763: 2758: 2753: 2745: 2744: 2742: 2731: 2722: 2719: 2713: 2712: 2710: 2700: 2697: 2692: 2686: 2685: 2683: 2676: 2671: 2668: 2664: 2663: 2660: 2657: 2654: 2651: 2637:, a difficult 2632: 2605: 2602: 2550:approved drugs 2534: 2531: 2519:covalent drugs 2489:ubiquitination 2468: 2465: 2463: 2460: 2440:pharmacophores 2430:modeling with 2418: 2415: 2363: 2360: 2334: 2331: 2329: 2326: 2313:ionic strength 2280: 2277: 2145:small molecule 2129:small molecule 2108: 2105: 2093:centrifugation 1997:Size-exclusion 1989:drug discovery 1908:that excludes 1868:, making only 1853: 1850: 1813:and to inform 1771: 1768: 1755:gel filtration 1723:derivatization 1719:derivatization 1646: 1643: 1559: 1556: 1453: 1450: 1349: 1346: 1295:Derivatization 1281:. Immobilized 1271:derivatization 1248: 1245: 1242: 1241: 1234: 1227: 1221: 1210: 1206: 1205: 1198: 1195: 1188: 1185:elution buffer 1181: 1177: 1176: 1173: 1160: 1153:ionic strength 1146: 1127: 1123: 1122: 1121:Disadvantages 1119: 1116: 1113: 1110: 1060: 1057: 971:functionalized 949:properties of 931: 928: 868:derivatization 846:In all cases, 835: 832: 716: 713: 647:, derivatized 584:site-directed 553: 550: 506:transcriptomic 440: 437: 397:dithiothreitol 357:ubiquitination 320: 317: 281:overexpression 269:drug discovery 265:drug discovery 234:Drug discovery 176: 173: 171: 168: 164:drug discovery 160:chemical probe 133:chromatography 121:Derivatization 83:drug discovery 80:small molecule 40:lead compounds 32:small molecule 15: 9: 6: 4: 3: 2: 5408: 5397: 5394: 5392: 5389: 5387: 5384: 5383: 5381: 5366: 5365: 5356: 5354: 5353: 5348: 5344: 5342: 5341: 5332: 5330: 5329: 5320: 5319: 5316: 5310: 5307: 5305: 5302: 5300: 5299:Chemical bond 5297: 5295: 5292: 5290: 5287: 5285: 5282: 5280: 5277: 5275: 5272: 5270: 5267: 5263: 5260: 5259: 5258: 5255: 5252: 5248: 5244: 5241: 5240: 5239: 5236: 5234: 5231: 5229: 5226: 5225: 5223: 5219: 5211: 5208: 5206: 5203: 5201: 5198: 5197: 5196: 5193: 5189: 5188:Stoichiometry 5186: 5185: 5184: 5181: 5177: 5174: 5172: 5169: 5167: 5164: 5162: 5159: 5158: 5157: 5154: 5150: 5147: 5146: 5145: 5144:Nanochemistry 5142: 5141: 5136: 5133: 5131: 5128: 5127: 5126: 5123: 5121: 5118: 5114: 5111: 5109: 5106: 5105: 5104: 5101: 5100: 5095: 5092: 5091: 5090: 5087: 5083: 5080: 5078: 5075: 5074: 5073: 5070: 5068: 5065: 5063: 5060: 5056: 5053: 5051: 5048: 5047: 5046: 5043: 5042: 5037: 5034: 5032: 5029: 5028: 5027: 5024: 5022: 5018: 5014: 5011: 5007: 5004: 5002: 4999: 4997: 4994: 4993: 4992: 4989: 4988: 4986: 4984: 4980: 4974: 4971: 4969: 4966: 4964: 4961: 4959: 4956: 4954: 4951: 4949: 4946: 4942: 4939: 4938: 4937: 4934: 4930: 4927: 4926: 4925: 4922: 4918: 4915: 4913: 4910: 4909: 4908: 4905: 4904: 4902: 4900: 4896: 4890: 4887: 4885: 4882: 4880: 4877: 4875: 4872: 4870: 4869:Semisynthesis 4866: 4863: 4861: 4858: 4856: 4853: 4851: 4848: 4846: 4843: 4841: 4838: 4834: 4831: 4830: 4829: 4826: 4825: 4823: 4821: 4817: 4811: 4808: 4806: 4803: 4801: 4798: 4794: 4791: 4790: 4789: 4786: 4784: 4781: 4779: 4776: 4775: 4773: 4771: 4767: 4761: 4758: 4756: 4753: 4751: 4748: 4746: 4743: 4741: 4738: 4736: 4733: 4731: 4728: 4726: 4723: 4719: 4716: 4715: 4714: 4711: 4709: 4706: 4704: 4703:Sonochemistry 4701: 4699: 4698:Cryochemistry 4696: 4692: 4691:Micromeritics 4689: 4688: 4687: 4684: 4682: 4679: 4677: 4674: 4672: 4669: 4665: 4662: 4660: 4657: 4656: 4655: 4652: 4651: 4649: 4647: 4643: 4635: 4632: 4631: 4630: 4627: 4625: 4622: 4620: 4617: 4615: 4612: 4608: 4605: 4604: 4603: 4600: 4598: 4595: 4594: 4592: 4590: 4586: 4580: 4577: 4575: 4572: 4570: 4569:Wet chemistry 4567: 4565: 4562: 4560: 4557: 4555: 4552: 4548: 4545: 4543: 4540: 4539: 4538: 4535: 4533: 4530: 4526: 4523: 4521: 4518: 4516: 4513: 4512: 4511: 4508: 4504: 4501: 4499: 4496: 4494: 4491: 4489: 4486: 4485: 4484: 4481: 4479: 4476: 4474: 4471: 4470: 4468: 4466: 4462: 4456: 4453: 4451: 4448: 4446: 4443: 4441: 4438: 4437: 4434: 4430: 4422: 4417: 4415: 4410: 4408: 4403: 4402: 4399: 4388: 4384: 4379: 4374: 4370: 4366: 4362: 4355: 4347: 4343: 4339: 4335: 4331: 4327: 4323: 4319: 4312: 4304: 4300: 4295: 4290: 4286: 4282: 4278: 4274: 4270: 4263: 4261: 4259: 4257: 4248: 4244: 4240: 4236: 4232: 4228: 4224: 4220: 4213: 4211: 4209: 4207: 4198: 4194: 4190: 4186: 4182: 4178: 4171: 4163: 4159: 4155: 4151: 4147: 4143: 4139: 4135: 4128: 4126: 4124: 4115: 4111: 4106: 4101: 4097: 4093: 4089: 4085: 4081: 4074: 4066: 4062: 4057: 4052: 4047: 4042: 4038: 4034: 4030: 4023: 4021: 4012: 4008: 4004: 4000: 3995: 3990: 3986: 3982: 3978: 3971: 3969: 3960: 3956: 3952: 3948: 3944: 3940: 3936: 3932: 3925: 3923: 3921: 3919: 3917: 3915: 3913: 3911: 3909: 3907: 3898: 3894: 3890: 3886: 3882: 3878: 3871: 3869: 3867: 3865: 3863: 3861: 3859: 3857: 3855: 3853: 3851: 3849: 3847: 3845: 3843: 3841: 3832: 3828: 3823: 3818: 3813: 3808: 3804: 3800: 3796: 3792: 3788: 3781: 3779: 3770: 3766: 3761: 3756: 3752: 3748: 3744: 3740: 3736: 3729: 3721: 3717: 3712: 3707: 3703: 3699: 3695: 3691: 3687: 3680: 3678: 3669: 3665: 3661: 3657: 3652: 3647: 3643: 3639: 3635: 3628: 3626: 3617: 3613: 3608: 3603: 3599: 3595: 3591: 3587: 3583: 3576: 3574: 3565: 3561: 3557: 3551: 3547: 3546: 3538: 3530: 3526: 3521: 3516: 3511: 3506: 3502: 3498: 3494: 3487: 3485: 3476: 3472: 3467: 3462: 3458: 3454: 3450: 3446: 3442: 3435: 3427: 3423: 3418: 3413: 3409: 3405: 3401: 3397: 3393: 3386: 3384: 3382: 3373: 3369: 3364: 3359: 3355: 3351: 3347: 3343: 3339: 3332: 3330: 3328: 3326: 3324: 3315: 3311: 3306: 3301: 3296: 3291: 3287: 3283: 3279: 3272: 3270: 3268: 3259: 3257:9780123809025 3253: 3249: 3245: 3241: 3237: 3230: 3228: 3226: 3224: 3222: 3220: 3218: 3209: 3205: 3200: 3195: 3191: 3187: 3183: 3179: 3175: 3168: 3166: 3157: 3153: 3149: 3143: 3139: 3135: 3131: 3127: 3126: 3118: 3116: 3114: 3112: 3110: 3101: 3097: 3093: 3089: 3085: 3081: 3077: 3073: 3066: 3058: 3054: 3049: 3044: 3040: 3036: 3032: 3028: 3024: 3017: 3015: 3013: 3011: 3009: 3000: 2996: 2992: 2988: 2983: 2978: 2974: 2970: 2966: 2959: 2957: 2948: 2944: 2937: 2933: 2923: 2920: 2918: 2915: 2913: 2910: 2908: 2905: 2903: 2900: 2898: 2895: 2894: 2884: 2881: 2878: 2874: 2870: 2867: 2864: 2861: 2860: 2857: 2854: 2850: 2847: 2845: 2842: 2839: 2836: 2835: 2832: 2829: 2826: 2822: 2818: 2816: 2813: 2810: 2807: 2806: 2803: 2800: 2796: 2792: 2790: 2787: 2785: 2782: 2779: 2778: 2775: 2772: 2768: 2764: 2762: 2759: 2757: 2754: 2751: 2747: 2746: 2743: 2740: 2736: 2732: 2730: 2726: 2723: 2720: 2718: 2715: 2714: 2711: 2708: 2705: 2701: 2698: 2696: 2693: 2691: 2688: 2687: 2684: 2681: 2677: 2675: 2672: 2669: 2666: 2665: 2661: 2658: 2655: 2652: 2649: 2648: 2642: 2640: 2636: 2628: 2624: 2620: 2616: 2611: 2601: 2599: 2595: 2591: 2588: 2584: 2580: 2577: 2573: 2569: 2565: 2564: 2559: 2555: 2551: 2548: 2544: 2540: 2530: 2528: 2524: 2520: 2515: 2511: 2510:pharmacopoeia 2507: 2503: 2499: 2490: 2486: 2482: 2478: 2473: 2459: 2457: 2453: 2452:pharmacophore 2450:. Therefore, 2449: 2445: 2441: 2437: 2433: 2429: 2428:pharmacophore 2424: 2414: 2411: 2410:Pharmacophore 2407: 2402: 2401:pharmacophore 2397: 2393: 2389: 2385: 2384:Pharmacophore 2381: 2374: 2368: 2359: 2357: 2353: 2349: 2344: 2340: 2325: 2322: 2318: 2314: 2310: 2306: 2302: 2298: 2294: 2290: 2286: 2272: 2271:BioRender.com 2265: 2261: 2257: 2253: 2249: 2244: 2238: 2234: 2232: 2228: 2224: 2220: 2217: 2213: 2209: 2205: 2201: 2197: 2193: 2189: 2185: 2181: 2177: 2173: 2169: 2166: 2162: 2158: 2154: 2150: 2146: 2142: 2138: 2134: 2130: 2126: 2122: 2118: 2114: 2113:bioanalytical 2104: 2102: 2098: 2094: 2090: 2086: 2082: 2078: 2074: 2070: 2066: 2062: 2058: 2054: 2050: 2046: 2042: 2038: 2034: 2030: 2026: 2022: 2018: 2014: 2010: 2006: 2002: 1998: 1994: 1990: 1987: 1983: 1979: 1977: 1973: 1969: 1965: 1962: 1959: 1955: 1951: 1947: 1943: 1939: 1935: 1931: 1927: 1923: 1919: 1915: 1911: 1907: 1903: 1899: 1895: 1891: 1890:precipitation 1887: 1883: 1879: 1875: 1871: 1867: 1863: 1859: 1849: 1847: 1843: 1839: 1836: 1832: 1828: 1824: 1820: 1816: 1812: 1808: 1804: 1800: 1796: 1792: 1788: 1784: 1780: 1777: 1763: 1762:BioRender.com 1759: 1756: 1751: 1745: 1741: 1739: 1736: 1735:heterogeneous 1732: 1728: 1724: 1720: 1716: 1712: 1708: 1704: 1700: 1696: 1692: 1688: 1684: 1680: 1676: 1672: 1668: 1664: 1660: 1656: 1652: 1642: 1640: 1636: 1632: 1631:fractionation 1628: 1624: 1620: 1616: 1612: 1608: 1603: 1599: 1596: 1592: 1588: 1584: 1581: 1577: 1573: 1569: 1565: 1551: 1550:BioRender.com 1544: 1540: 1536: 1532: 1528: 1524: 1519: 1513: 1509: 1507: 1503: 1499: 1495: 1491: 1487: 1483: 1479: 1475: 1471: 1467: 1463: 1459: 1445: 1444:BioRender.com 1438: 1434: 1430: 1425: 1419: 1415: 1413: 1409: 1405: 1404: 1399: 1397: 1393: 1388: 1384: 1380: 1375: 1371: 1367: 1363: 1359: 1355: 1345: 1343: 1339: 1335: 1331: 1327: 1323: 1319: 1318:conformations 1315: 1311: 1307: 1302: 1300: 1296: 1292: 1288: 1284: 1280: 1276: 1272: 1263: 1262:BioRender.com 1258: 1253: 1239: 1235: 1232: 1228: 1225: 1222: 1218: 1214: 1211: 1208: 1207: 1203: 1199: 1196: 1193: 1189: 1186: 1182: 1179: 1178: 1174: 1172: 1169: 1167: 1161: 1158: 1154: 1150: 1147: 1144: 1140: 1136: 1132: 1128: 1125: 1124: 1120: 1117: 1115:Mechanism(s) 1114: 1111: 1108: 1107: 1103: 1097: 1095: 1091: 1087: 1083: 1079: 1075: 1071: 1067: 1056: 1055: 1051: 1047: 1043: 1039: 1035: 1031: 1026: 1024: 1020: 1016: 1012: 1008: 1004: 1000: 996: 992: 988: 984: 980: 976: 972: 968: 964: 960: 956: 952: 948: 944: 940: 936: 927: 925: 921: 917: 913: 904: 903:BioRender.com 900: 896: 893: 889: 885: 881: 880:cross-linking 877: 873: 869: 865: 861: 857: 853: 849: 845: 840: 831: 829: 825: 822:, at various 821: 816: 809: 803: 799: 797: 793: 789: 785: 781: 777: 773: 770: 766: 762: 758: 754: 750: 746: 743:, such as an 742: 738: 734: 730: 726: 722: 708: 707:BioRender.com 704: 700: 696: 692: 687: 681: 677: 674: 670: 666: 661: 660:physiological 656: 654: 650: 646: 642: 639: 635: 631: 627: 623: 622:affinity tags 619: 614: 611: 607: 603: 599: 595: 591: 587: 582: 581:covalent bond 578: 574: 571: 567: 563: 559: 549: 547: 543: 539: 535: 531: 527: 523: 520:. Given that 519: 515: 511: 507: 503: 494: 493:BioRender.com 490: 486: 482: 478: 474: 470: 466: 462: 458: 454: 452: 445: 436: 434: 430: 426: 422: 419:, often with 418: 414: 410: 409:iodoacetamide 406: 402: 398: 394: 391: 387: 383: 380: 376: 372: 369: 366: 362: 358: 354: 350: 349:glycosylation 346: 342: 338: 334: 330: 326: 316: 314: 310: 306: 302: 298: 294: 290: 286: 282: 278: 274: 270: 266: 262: 261:drug approval 259: 255: 251: 247: 243: 239: 235: 231: 227: 223: 214: 213:BioRender.com 210: 209:mass spectrum 206: 202: 198: 194: 190: 186: 181: 167: 165: 161: 157: 154: 150: 146: 142: 138: 134: 130: 126: 122: 118: 114: 109: 107: 103: 100: 96: 92: 88: 84: 81: 77: 73: 69: 65: 61: 57: 53: 49: 45: 41: 37: 33: 29: 25: 21: 5362: 5350: 5338: 5326: 5176:Biosynthesis 5026:Geochemistry 4941:Pharmacology 4917:Cell biology 4907:Biochemistry 4735:Spectroscopy 4634:VSEPR theory 4483:Spectroscopy 4427:Branches of 4368: 4364: 4354: 4321: 4317: 4311: 4276: 4272: 4222: 4218: 4180: 4176: 4170: 4137: 4133: 4087: 4083: 4073: 4036: 4032: 3984: 3980: 3937:(1): 62–71. 3934: 3930: 3880: 3876: 3794: 3790: 3745:(3): e9232. 3742: 3738: 3728: 3696:(1): 34–46. 3693: 3689: 3641: 3637: 3589: 3585: 3548:. Elsevier. 3544: 3537: 3503:(11): 1516. 3500: 3496: 3448: 3444: 3434: 3399: 3395: 3345: 3341: 3285: 3281: 3239: 3235: 3181: 3177: 3124: 3075: 3071: 3065: 3033:(1): 11–22. 3030: 3026: 2972: 2968: 2946: 2936: 2659:Description 2607: 2561: 2536: 2514:nucleophilic 2495: 2476: 2467:Druggability 2462:Applications 2420: 2392:lock-and-key 2377: 2370: 2336: 2305:fractionated 2282: 2252:fractionated 2246:Drug-spiked 2240: 2221:and release 2141:denaturation 2110: 1980: 1970:, while the 1964:amyloid-beta 1956:was used to 1946:amyloid-beta 1924:can also be 1855: 1773: 1747: 1681:. 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