Solution-Based Indirect Affinity Selection Mass Spectrometry—A General Tool For High-Throughput Screening Of Pharmaceutical Compound Libraries

O’Connell, Thomas N.; Ramsay, Jason T.; Rieth, Steven F.; Shapiro, Michael J.; Stroh, Justin G.

doi:10.1021/ac500938y

Cited by 69 publications

(67 citation statements)

References 25 publications

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“…Further, increasing the amount of heme nM at 25 o C and 5.0 nM at 37 o C. Figure S10). Affinity-Selection / Mass Spectrometry studies (AS/MS) [38][39] show that present in the reaction diminishes the signal of the small amount of GSK5628 that can bind holo-IDO1 and is consistent with shifting the equilibrium of IDO1 in solution towards an increased holo population, to which GSK5628 cannot bind (Figure S11B). Fluorescent Probe Studies.…”

Section: Gsk5628a Competes With the Ido1 Heme Cofactormentioning

confidence: 69%

Characterization of apo-form selective inhibition of indoleamine 2,3-dioxygenase

Ortiz‐Meoz

Wang

Matico

et al. 2018

Preprint

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Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism.As an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of established and novel IDO1 inhibitors remains of great interest. Comparison of a newly-developed IDO1 inhibitor (GSK5628) to the existing best-in-class compound, epacadostat (Incyte), allows us to report on a novel inhibition mechanism for IDO1. Here, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1) while epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, undescribed for other known IDO1 inhibitors. Detailed characterization of this novel binding mechanism for IDO1 inhibition may help design superior inhibitors or may confer a unique competitive advantage over other IDO1 inhibitors vis-àvis specificity and pharmacokinetic parameters.

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Section: Gsk5628a Competes With the Ido1 Heme Cofactormentioning

confidence: 69%

Characterization of apo-form selective inhibition of indoleamine 2,3-dioxygenase

Ortiz‐Meoz

Wang

Matico

et al. 2018

Preprint

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“…By using magnetic beads for protein immobilization, inhibitors with Ki’s in the micromolar range could also be assayed, using just 7 pmol of protein per compound screened. Finally, the method compares very favorably to the state-of-the-art MS-based assay (ASMS) [9], where the false-positive identification rate was nearly 10% for the ligands assayed herein.…”

Section: Resultsmentioning

confidence: 99%

“…This approach has shown to be highly amenable to automation, with a throughput of ~ 1*10 5 compounds per day [8–9]. However, it has some limiting disadvantages that we seek to address herein.…”

Section: Introductionmentioning

confidence: 99%

“…The method described herein not only picks out the tightest binding ligands, it can also be used to detect ligands with different binding affinities, by tuning the concentration of protein used in the assay. As an additional benefit of screening in this manner, the amount of protein required, and the amount of each binding ligand, can be substantially reduced compared to the state-of-the-art comparator method [9]. Reduction in the amount of protein required, in particular, is a strong asset in a high throughput screening method; it can substantially reduce assay cost because the quantity of the most expensive reagent, the protein, is reduced.…”

Section: Introductionmentioning

confidence: 99%

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HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

Imaduwage

Zhu

et al. 2016

J. Am. Soc. Mass Spectrom.

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A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM and 700 pM. These findings demonstrate that the approach described here compares favorably to traditional MS based screening methods.

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“…For example, AS-MS (affinity selection-mass spectrometry) provides evidence of compound-target interaction through separation of targetbound and free compound by size exclusion chromatography and subsequent identification of the ligand based on its molecular mass. [101] This approach has now even been applied for primary screening, [102][103][104] though functional testing is still required at a later point in the flowchart.…”

Section: Positive Proof Of Stoichiometric Bindingmentioning

confidence: 99%

Non-stoichiometric inhibition in integrated lead finding – a literature review

Klumpp

2015

Expert Opinion on Drug Discovery

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Over the last few years, awareness of non-stoichiometric inhibitors within screening libraries and HTS hit lists has considerably increased, not only in the pharmaceutical industry but also in the academic drug discovery community. This has resulted in a variety of methods to detect and handle such compounds. These range from in silico approaches to flag suspicious compounds, and counterassays to measure non-stoichiometric inhibition, to biophysical methods that positively demonstrate stoichiometric binding. In addition, novel technologies to verify target engagement within cells are becoming available. While still a time- and resource-consuming nuisance, non-stoichiometric inhibitors therefore do not fundamentally jeopardize the discovery of low molecular weight lead and drug candidates. Rather, they should be viewed as a manageable issue that with appropriate expertise can be overcome through integration of the above-mentioned approaches.

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Solution-Based Indirect Affinity Selection Mass Spectrometry—A General Tool For High-Throughput Screening Of Pharmaceutical Compound Libraries

Cited by 69 publications

References 25 publications

Characterization of apo-form selective inhibition of indoleamine 2,3-dioxygenase

Characterization of apo-form selective inhibition of indoleamine 2,3-dioxygenase

HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

Non-stoichiometric inhibition in integrated lead finding – a literature review

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