Mass spectrometry for fragment screening

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening.

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Fragment-Based Screening for Inhibitors of PDE4A Using Enthalpy Arrays and X-ray Crystallography

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111430987 ◽

2012 ◽

Vol 17 (4) ◽

pp. 469-480 ◽

Cited By ~ 15

Author(s):

Michael I. Recht ◽

Vandana Sridhar ◽

John Badger ◽

Leslie Hernandez ◽

Barbara Chie-Leon ◽

...

Keyword(s):

Spectroscopic Properties ◽

Titration Calorimetry ◽

Label Free ◽

Lead Discovery ◽

High Resolution Data ◽

Affinity Ligands ◽

X Ray ◽

X Ray Crystallography ◽

Chemical Structures ◽

Fragment Library

Fragment-based screening has typically relied on X-ray or nuclear magnetic resonance methods to identify low-affinity ligands that bind to therapeutic targets. These techniques are expensive in terms of material and time, so it useful to have a higher throughput method to reliably prescreen a fragment library to identify a subset of compounds for structural analysis. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we have used enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 4A (PDE4A). Several inhibitors with K I <2 mM were identified and moved to X-ray crystallization trials. Although the co-crystals did not yield high-resolution data, evidence of binding was observed, and the chemical structures of the hits were consistent with motifs of known PDE4 inhibitors. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and provides a list of candidate fragments for inhibition of PDE4A.

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Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19017199 ◽

2020 ◽

Vol 76 (1) ◽

pp. 40-46

Author(s):

James H. Thorpe ◽

Ian D. Wall ◽

Robert H. Sinnamon ◽

Amy N. Taylor ◽

Robert A. Stavenger

Keyword(s):

Drug Discovery ◽

Acinetobacter Baumannii ◽

Structural Data ◽

X Ray ◽

Traditional Medicines ◽

X Ray Crystallography ◽

Fragment Screening ◽

Binding Pockets ◽

Antibacterial Target ◽

Robustness Check

Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines.

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Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113516493 ◽

2013 ◽

Vol 19 (4) ◽

pp. 497-507 ◽

Cited By ~ 15

Author(s):

Michael I. Recht ◽

Vandana Sridhar ◽

John Badger ◽

Pierre-Yves Bounaud ◽

Cheyenne Logan ◽

...

Keyword(s):

High Resolution ◽

Structural Information ◽

Spectroscopic Properties ◽

Low Complexity ◽

Titration Calorimetry ◽

Label Free ◽

Lead Discovery ◽

X Ray ◽

X Ray Crystallography ◽

Heavy Atoms

Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.

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Synthesis and X-ray structure of a new zinc(ii) coordination polymer: interaction with DNA and double stranded RNA and elucidation of the molecular aspects of the binding to bovine serum albumin

RSC Advances ◽

10.1039/c4ra06588c ◽

2014 ◽

Vol 4 (101) ◽

pp. 57855-57868 ◽

Cited By ~ 12

Author(s):

Swapan K. Jana ◽

Saikat K. Seth ◽

Horst Puschmann ◽

Maidul Hossain ◽

Sudipta Dalai

Keyword(s):

Bovine Serum Albumin ◽

Coordination Polymer ◽

Synthesis And Characterization ◽

Titration Calorimetry ◽

Double Stranded Rna ◽

X Ray ◽

Biophysical Techniques ◽

Molecular Aspects ◽

Polymer Interaction

Synthesis and characterization of [Zn(4-Me-5-CHOIm)2(HCOO)](ClO4) complex and binding with nucleic acids and BSA has been explored by different biophysical techniques with the combination of isothermal titration calorimetry (ITC).

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An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115618347 ◽

2015 ◽

Vol 21 (4) ◽

pp. 414-421 ◽

Cited By ~ 35

Author(s):

Pawel Linke ◽

Kwame Amaning ◽

Melanie Maschberger ◽

Francois Vallee ◽

Valerie Steier ◽

...

Keyword(s):

Protein Denaturation ◽

Lead Discovery ◽

X Ray ◽

X Ray Crystallography ◽

Fragment Screening ◽

Lead Compounds ◽

Microscale Thermophoresis ◽

Biophysical Techniques ◽

Orthogonal Methods ◽

Induced Aggregation

Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

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Perspectives on Fragment-based Drug Discovery: A Strategy Applicable to Diverse Targets

Current Topics in Medicinal Chemistry ◽

10.2174/1568026621666210804115700 ◽

2021 ◽

Vol 21 ◽

Author(s):

Qingxin Li ◽

Congbao Kang

Keyword(s):

Clinical Trials ◽

Drug Discovery ◽

Nmr Spectroscopy ◽

Binding Modes ◽

X Ray ◽

X Ray Crystallography ◽

Fragment Screening ◽

Hit Rate ◽

Powerful Strategy ◽

Fragment Based Drug Discovery

: Fragment-based drug discovery (FBDD) is a strategy to develop potent lead molecules and is frequently used in drug discovery projects of the pharmaceutical industry. This method starts from identifying a small-molecule fragment which usually binds weakly to the target and follows with a hit-to-lead step in which the fragment is grown into potent molecules that bind tightly to the target to affect its function. Quite a few drugs and compounds in clinical trials are developed using this approach, making FBDD a powerful strategy in drug discovery. FBDD can be applied to multiple targets and the hit rate in screening can be used in target druggability assessment. In this mini-review, we provide a summary for the development of FBDD. In addition to giving a brief summary of the methods used in fragment screening, we highlight some methods that are critical in the fragment growth. Biophysical methods and carefully chemical modification of the fragments are the key elements in FBDD. We show several strategies that can be utilized in FBDD. We emphasize that NMR spectroscopy such as 19F-NMR and 1H-15N-HSQC experiment and X-ray crystallography are important in FBDD due to their roles in fragment screening and understanding the binding modes of the fragment hits, which provides a strategy for fragment growth.

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Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Journal of Visualized Experiments ◽

10.3791/57750 ◽

2018 ◽

Cited By ~ 1

Author(s):

June Ereño-Orbea ◽

Taylor Sicard ◽

Hong Cui ◽

Indira Akula ◽

Jean-Philippe Julien

Keyword(s):

X Ray ◽

X Ray Crystallography ◽

Biophysical Techniques ◽

Immunoglobulin Fold

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Structural Characterization of Heterodinuclear ZnII-LnIII Complexes (Ln = Pr, Nd) with a Ring-Contracted H2valdien-Derived Schiff Base Ligand

Journal of Chemical Crystallography ◽

10.1007/s10870-021-00891-4 ◽

2021 ◽

Author(s):

Shabana Noor ◽

Richard Goddard ◽

Fehmeeda Khatoon ◽

Sarvendra Kumar ◽

Rüdiger W. Seidel

Keyword(s):

Molecular Structure ◽

Schiff Base ◽

Structural Characterization ◽

Schiff Base Ligand ◽

Crystal And Molecular Structure ◽

X Ray ◽

X Ray Crystallography ◽

Imidazoline Ring

AbstractSynthesis and structural characterization of two heterodinuclear ZnII-LnIII complexes with the formula [ZnLn(HL)(µ-OAc)(NO3)2(H2O)x(MeOH)1-x]NO3 · n H2O · n MeOH [Ln = Pr (1), Nd (2)] and the crystal and molecular structure of [ZnNd(HL)(µ-OAc)(NO3)2(H2O)] [ZnNd(HL)(OAc)(NO3)2(H2O)](NO3)2 · n H2O · n MeOH (3) are reported. The asymmetrical compartmental ligand (E)-2-(1-(2-((2-hydroxy-3-methoxybenzylidene)amino)-ethyl)imidazolidin-2-yl)-6-methoxyphenol (H2L) is formed from N1,N3-bis(3-methoxysalicylidene)diethylenetriamine (H2valdien) through intramolecular aminal formation, resulting in a peripheral imidazoline ring. The structures of 1–3 were revealed by X-ray crystallography. The smaller ZnII ion occupies the inner N2O2 compartment of the ligand, whereas the larger and more oxophilic LnIII ions are found in the outer O2O2’ site. Graphic Abstract Synthesis and structural characterization of two heterodinuclear ZnII-LnIII complexes (Ln = Pr, Nd) bearing an asymmetrical compartmental ligand formed in situ from N1,N3-bis(3-methoxysalicylidene)diethylenetriamine (H2valdien) through intramolecular aminal formation are reported.

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