Application of SAMDI in Bioanalysis

The use of functionalized monolayers in monitoring enzymatic activities was tested using b-1,4-galactosyltransferase as model enzyme and immobilized N-acetylglucosamine as substrate. The enzyme solution was incubated on SAM-modified target surfaces. Subsequent to incubation, the target was rinsed, an appropriate matrix was applied, and the surface was analyzed by means of MALDI-MS. By varying the incubation times, time-resolved reaction profiles were obtained. The yield of the enzymatic...

MurF Lead Discovery

To efficiently identify compounds of interest for further study in biochemical or cell-based assays, the candidate ligands remaining after promiscuous compound filtering were deconvoluted in small non-mass-redundant mixtures. The eight deconvoluted hits are shown in Table 4.3, along with their enzyme inhibition values. The two best ligands (compounds 4 and 5) also are structurally related and were discovered in different initial screening mixtures. ASMS binding data is shown in Fig. 4.3. Both...

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Scheme 3.1 Isosteric competition diagram. Fig. 3.10 Examples of isosteric binding competition. (A) ALIS-MS results for the titration of 5 M Zap-70 by staurosporine in the presence of a 5 m concentration of its structural congener K252a and (B) titration of 5 M DHFR with the known DHFR inhibitor trimethoprim in the presence of ligand NGD-157 at 5 m concentration. Linear MS response ratios in these experiments are consistent with direct binding competition. (C) Compound structures. Fig. 3.10...

ApoCaM Interacting with Ca2B A 14 Protein Metal Ion Interaction

With the success in studying 1 1 protein metal ion binding, we applied PLIMSTEX to the more challenging 1 4 protein metal ion binding of calmodulin and Ca2+ binding. Calmodulin, a small ( 17 kDa), acidic, and highly conserved protein, is regulated by Ca2+ binding in most eukaryotic cells. When binding Ca2+, calmodulin undergoes conformational changes that enable it to bind to and activate other target proteins, an action that is critical to various aspects of cell metabolism 45, 46 . We wished...

Application of DIOS in Bioanalysis

Desorption ionization on porous silicon has been successfully applied to the direct mass analysis of a variety of analytes and analyte mixtures, e.g. from exocrine tissues as well as from single neurons. In forensic analysis, DIOS-MS was applied for the analysis of small molecular weight polymers from biological samples, e.g. spermicides or polyethylene glycol polymers. But it has also been applied in the fields of drug discovery 17 or fatty acid analysis 18 . DIOS-MS has early been used for...

Selfassociation of Insulin A Protein Protein Interaction

Protein-protein interactions mediate the majority of life processes. An understanding of these interactions is critical to understanding cell regulation 66 and to preventing human disease that can arise from errors in protein-protein interactions 67 . A clear understanding of these interactions points the way to developing new targets and discovering new drugs 68 . Insulin, a protein with 51 residues in two chains 69 , is a good model system for testing whether a PLIMSTEX-like approach can...

Discovery PK Assay Rules

While the rules that need to be followed when developing and using a bioanalyt-ical assay in a GLP setting are well documented 128, 129 , there is no standard set of rules to follow when one is developing or using a bioanalytical assay in a drug discovery setting. It is generally agreed that these nonGLP bioanalytical methods do not need to be validated before they can be used for the analysis of discovery (nonGLP) PK samples. This is important because the validation proce- Fig. 13.8...

DXMSguided Design of Small Molecules that Target Protein Protein Interaction Surfaces

Development of small molecule inhibitors of protein-protein binding interactions has been notoriously difficult. The tremendous investment that the pharmaceutical industry has made in the development and marketing of whole protein therapeutics such as monoclonal antibodies and recombinant proteins is testament to the strength of the belief that small molecule replacements for these ''expensive to produce and administer'' biologics will not soon be forthcoming. Multiple noncovalent interactions...

Multiple Passes Through Spin Columns Finding Strongest Binders

An application of GPC spin column ESI-MS methodology is to re-equilibrate the eluate of a spin column and pass it again through a second spin column to detect by ESI-MS the enriched tighter binding ligands at the expense of the weaker binding ligands 35 . This approach is applicable to mixtures of unknown structures and unknown concentrations for differentiating strong from weak binders. For these systems, the relative ESI-MS responses for different unknown compounds are not indicative of their...

Advantages of FPOP

One of the major advantages of using an irreversible reagent to probe protein interactions is that the sites of reaction can be readily determined by standard proteomic procedures. When the method is fully developed, one may be able to determine the Kd, binding stoichiometry, and the residues involved in ligand binding. As with PLIMSTEX, the hydroxyl radical method measures a change in the m z and, therefore, is not susceptible to variations in ESI efficiency. With the ability to use any enzyme...

Hplcmsms Overview

HPLC-MS MS has been described as the premier analytical tool for drug metabolism participation in the new drug discovery process and has been applied to a Fig. 13.2 Schematic diagram showing the various stages and the iterative steps involved in the lead optimization process from a DMPK perspective. This schematic represents the iterative process that is an important part of the lead optimization process. The in vitro and in vivo screens refer to DMPK assays. Reprinted from 12 , with permission...

References

Siegel Applications of NMR and MS to anticancer drug discovery, in Novel Anti-Cancer Agents Strategies for Discovery and Clinical Testing, ed. A. A. Adjei, J. K. Buolamwini, Elsevier Science, New York, 2006, pp 107-190. 2 M. M. Siegel Mass spectrometry-based drug screening assays for early phases in drug discovery, in Integrated Strategies for Drug Discovery Using Mass Spectrometry, ed. Mike S. Lee, John Wiley & Sons, 2005, pp 27-70. 3 M. M. Siegel Early discovery drug...

Multitarget Affinity Specificity Screening

We have integrated high throughput sampling robotics and a custom fluidics module to rapidly characterize noncovalent biological complexes in order to identify small molecules that bind RNA targets using ESI-FTICR 30 . The multitarget affinity specificity screening (MASS) assay takes advantage of the ''intrinsic mass'' label of each compound and target RNA by obtaining high resolution, high precision mass measurements of intact RNA-ligand complexes 13, 3133 . The identity of the small...

Principles of Samdims

Mrksich and co-workers developed a MALDI-based assay scheme making use of a target surface modification by self-assembled monolayers (SAMs) 22 . This combination of SAMs and MALDI is predominantly called SAMDI (self-assembled monolayers for MALDI). For SAMDI, a self-assembled monolayer with reactive end groups is used in order to covalently bind enzyme substrates to a surface. To Fig. 8.12 Scheme of the SAMDI principle. To a self-assembled monolayer (SAM) with reactive end groups, enzyme...

In Vivo Applications

While there are several high throughput in vitro screens as described above, there is still a significant need for in vivo assays as shown in Fig. 13.1. Several recent reports have discussed using HPLC-MS MS for the bioanalytical step in discovery PK studies 5, 6, 8, 23, 72-74 . In the following sections, various aspects of the strategies that can be utilized in these in vivo studies are discussed. While the focus of this section will be on PK studies, another aspect of the lead optimization...

ALIS An Affinity Selection Mass Spectrometry System based on Continuous SEC

Mass Spectrometry Pharmacology

The schematic shown in Fig. 3.1 describes an optimized, integrated SEC-RPC-MS-based affinity selection platform developed at NeoGenesis, dubbed the Fig. 3.1 Schematic of ALIS, an automated ligand identification system that uses continuous size-exclusion chromatography for the study of protein-ligand interactions. Fig. 3.1 Schematic of ALIS, an automated ligand identification system that uses continuous size-exclusion chromatography for the study of protein-ligand interactions. Automated Ligand...

Introduction

The early stages of new drug discovery in the pharmaceutical industry rely on many steps in the identification and optimization of small drug molecules. These include target identification, assay development, high throughput screening (HTS), hit characterization, and medicinal chemistry optimization. A current problem with this approach is that more funds are spent on drug discovery than those returned from the steadily decreasing number of drugs reaching the market. In order to continue down...

Experimental Results

The ALIS-based off-rate measurement method was applied to a proprietary series of Zap-70 Kinase inhibitors. First, an ACE50 experiment was conducted to demonstrate that the compounds bind the same site as the quench reagent staurospor-ine. As shown in Fig. 3.15, sigmoidal plots indicate that, with the exception of one compound, the ACE50 values were all very similar to one-another. Linear ratio plots of the same ACE50 data confirm that the compounds all bind isosteri-cally with respect to the...

Discovery of Ligands from Combinatorial Libraries

The ALIS platform has been used to successfully screen a variety of therapeuti-cally valuable proteins against combinatorial libraries of small, drug-like compounds, yielding novel ligands to a number of targets, including targets of unknown function identified by genomic and proteomic profiling, well established targets in the pharmaceutical industry, and popular but notoriously intractable (or ''hard'') targets for which the discovery of small molecule drugs has proven difficult 41 ....

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11 Quantification of Protein-Ligand Interactions in Solution by Hydrogen Deuterium Exchange (PLIMSTEX) 342 Mei M. Zhu, David Hambly, and Michael L. Gross 11.2.1 A General Protocol of H D Exchange and LC MS Analysis for PLIMSTEX 342 11.2.2 Determination and Interpretation of the Titration Curves 343 11.3 Applications of PLIMSTEX 345 11.3.1 Determination of Association Constant (Ka), Stoichiometry (n), and Protection (AD ) 345 11.3.2 Ras-GDP Interacting with Mg2+ A 1 1 Protein Metal Ion...

Modified Version of Plimstex for Insulin Selfassociation

To obtain data similar to that from PLIMSTEX, the concentration of insulin in solution is varied and amide exchange is initiated, followed by quenching the exchange, and injecting the ice-cold solution into the ESI source of a mass spectrometer. After the quench, the oligomers dissociate into monomers, but the increase in mass of the monomer (compared to the control) gives a weighted average of the increase in mass of the various oligomers. These data can be used to obtain a species-specific...

Affinity Ranking in Compound Mixtures

Advances in chemical synthesis have enabled considerable sophistication in the construction of diverse compound libraries to probe protein function 61, 62 . However, few general techniques exist that can directly assess binding mechanisms and evaluate ligand affinities in a multiplexed format. To realize the full potential of combinatorial chemistry in the drug discovery process, generic and efficient tools must be applied that combine mixture-based techniques to characterize protein-ligand...

MS Binding Assays Quantifying the Nonbound Marker

In radioligand binding assays, binding of the marker is always quantified by the amount of marker bound to the target. The practicality of a procedure that in contrast quantifies the nonbound marker to indirectly determine the amount of bound marker has been shown by applications which examined the binding of fluorescent markers to nicotinic acetylcholine and benzodiazepine receptors 37, 38 . Although very different from conventional binding assays with regard to the design of the binding...

Radioligand Binding Assays

Radioligand binding assays are a relatively simple but at the same time a very important and efficient tool to study target-ligand interactions. Since they are very widely used in drug discovery and have been described extensively, this section only discusses their fundamental aspects and those aspects that are important for the description of the MS binding assays below. More detailed information can be found in the relevant literature 6, 7, 16-21 . Radioligand binding assays depend on the use...

Matrix Effects

As scientists have shortened assay times by using shorter columns or higher mobile phase flow rates vide supra , a new problem has become more apparent. This problem is often referred to as ''matrix effects''. Matrix effects can be described as a component in the sample that is injected into the HPLC-MS MS system that results in a reduction of the analyte signal aka ion suppression or an increase in analyte signal. There have been multiple papers written on various aspects of matrix effects in...