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

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...

Info

In atmospheric pressure ionization sources (API) the ions are first formed at atmospheric pressure and then transferred into the vacuum. In addition, some API sources are capable of ionizing neutral molecules in solution or in the gas phase prior to ion transfer to the mass spectrometer. Because no liquid is introduced into the mass spectrometer these sources are particularly attractive for the coupling of liquid chromatography with mass spectrometry. Pneumatically assisted electrospray (ESI),...

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 ....

Ms 339

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...

References

Using Mass Spectrometry for Drug Metabolism-Studies, CRC Press, Boca Raton, 2005, 370 pp. 1 Frank, R. Hargreaves, R. Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2003, 2, 566-580. 6 Korfmacher, W. Bioanalytical assays in a drug discovery environment, in Using Mass Spectrometry for Drug Metabolism Studies, CRC Press, Boca Raton, 2005, pp 1-34. 3 Lee, M. S. Integrated Strategies for Drug Discovery using Mass Spectrometry, John Wiley amp Sons,...

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...