NMR-Based Screening with Competition Water−Ligand Observed via Gradient Spectroscopy Experiments:  Detection of High-Affinity Ligands

Dalvit, Claudio; Fasolini, Marina; Flocco, Maria M.; Knapp, Stefan; Pevarello, Paolo; Veronesi, Marina

doi:10.1021/jm011122k

Cited by 112 publications

(109 citation statements)

References 28 publications

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“…In addition, an approximate value or a lower limit of the dissociation constant of the potential ligand can be derived from the reduction of the signal intensity of the STD indicator. 22,26 This is demonstrated next on the human serum albumin (HSA) model system. It is known that drugs such as diazepam and the endogenous amino acid tryptophan bind to site II of HSA.…”

Section: Resultsmentioning

confidence: 99%

“…22,26 The equations used to calculate the dissociation constants can be derived as follows. n T 1ρ filter g1 g1 g2 g2…”

Section: Methodsmentioning

confidence: 99%

“…21 In another approach, transverse and longitudinal relaxation parameters were utilized to probe high-affinity ligands. 22 Finally, competition binding WATERLOGSY experiments permits the detection of stronger binders. 23 STD NMR is based on the intermolecular transfer of magnetization from the protein to the bound small molecule ligands, which in turn, by fast chemical exchange between their bound and free states, is moved into solution where it is detected.…”

Section: Introductionmentioning

confidence: 99%

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Competition STD NMR for the detection of high‐affinity ligands and NMR‐based screening

Wang¹,

Liu²,

Wyss³

2004

Magnetic Reson in Chemistry

139

104

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The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (b-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both highand low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads.

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Section: Resultsmentioning

confidence: 99%

“…22,26 The equations used to calculate the dissociation constants can be derived as follows. n T 1ρ filter g1 g1 g2 g2…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Competition STD NMR for the detection of high‐affinity ligands and NMR‐based screening

Wang¹,

Liu²,

Wyss³

2004

Magnetic Reson in Chemistry

139

104

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“…The amide 7 was prepared (Scheme 5) as a reference ligand for a WaterLOGSY assay [50][51][52] to evaluate the de novo designed ligands.…”

Section: Evaluation Of the De Novo Designed Ligandsmentioning

confidence: 99%

Discovery of novel FabF ligands inspired by platensimycin by integrating structure-based design with diversity-oriented synthetic accessibility

et al. 2014

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Martin Fisher, [a,b] Ramkrishna Basak, [a,b] Arnout P. Kalverda, [b] Colin W. G. Fishwick, [a,b] W. Bruce Turnbull [a,b] and Adam Nelson* [a,b] , An approach for designing bioactive small molecules has been developed in which de novo structure-based ligand design (SBLD) was focused on regions of chemical space accessible using a diversity-oriented synthetic approach. The approach was exploited in the design and synthesis of a focused library of platensimycin analogues in which the com plex bridged ring system was replaced with a series of alternative ring systems. The affinity of the resulting compounds for the C163Q mutant of FabF was determined using a WaterLOGSY competition binding assay. Several compounds had significantly improved affinity for the protein relative to a reference ligand. The integration of synthetic accessibility with ligand design enabled focus to be placed on synthetically-accessible regions of chemical space that were relevant to the target protein under investigation.

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“…This approach is suitable for detecting binding affinity in the micromolar range (71), and thus it reveals more differences of the weak interactions. In the APC3 mutant WaterLOGSY experiment, the mutant of S584A/E616R directly affected the peaks 5 and 7, the interactions between the backbone of Arginine side chain and the binding groove.…”

Section: Validate Human Apc3 Interactions With the Ir-tails Through Mmentioning

confidence: 99%

Strengths and Weaknesses of Hybrid TPR Technology for Obtaining Structural and Mechanistic Insights into TPR Proteins

Yu¹

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DEDICATION ACKNOWLEDGEMENTSAbove all, I would like to thank my mentor, Brenda Schulman, for immersing me into the world of cutting-edge research and teaching me what it takes to keep doing a leading science. The training will serve me well always. I want to thank Brenda for creating a collaborative, informative and productive environment for doing sciencesboth in terms of recruiting fantastic people and providing great resources. Also, I would like to thank committee members: John Cox, Eric Enemark, Roderick Hori and Stephen White for their effort and the advices they put into guiding me through graduate school.I would like to thank the members of the Schulman lab for all that they have taught me and making this journey a fun one: Danny Scott for his insightful scientific questions and suggestions; Dave Duda for all the brilliant crystallography tips and tricks; David Miller for keeping the lab fully stocked and equipment always running; Hari Kamadurai for all the experiment guides over the years; Jing Wang and Masaya Yamaguchi for their help on projects we did together; Jeremy Frye for great advices towards experiments and structural softwares; Ryan VanderLinden for all the scientific discussion and suggestions; Nicholas Brown and Kuen-Phon Wu for the inspiring conversation and help; Randy Watson a fellow PhD candidate, for all the scientific (and nonscientific) discussion, advices sand being very helpful; Vladislav Sviderskiy for being helpful and for all the fun times. Manjeet Mukherjee and Yumei Zheng (a fellow graduate student), for sharing parts of the journey; Shelia Boezman for all the care, administrative support and activity organization (not forgetting the chocolates, candies and doughnuts); Jenny Olszewski, Shein Ei Cho and Omar Alsharif for being great lab mates and the fun time together.Additionally, my thanks are for: Billy Dye for the guidance with cloning and all the smart jokes; Steve Kaiser for all the scientific discussions and the insights to the career; Mathew "Matt-man" Calabrese for the taking the time to teach; Julie Monda, Alan "DDR" Deng and Steven Seyedin for spreading the joy and for being ever helpful. ABSTRACTTetratricopeptide (TPR) repeats are a 34-residue helix-turn-helix motif that when repeated pack into a superhelical structure. TPR domains are frequently found mediating protein-protein interactions, often through a central groove. One protein complex bearing numerous TPR repeats is the Anaphase Promoting Complex (APC). The anaphasepromoting complex (APC) is a multi-subunit complex, which orchestrates mitotic cell cycles. APC is an E3 ligase in the ubiquitin cascade, and directs the 26S proteosome degradation of cell-cycle regulators. Throughout mitotic progression, proteins that are key regulators of the cell cycle are assembled with polyubiquitin chains by APC.One domain of the human APC is comprised of four related TPR proteins, APC8, APC6, APC3, and APC7, with each found in pairs. Crystal structures of some of these indicate that each has an N-terminal dimerization domain...

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NMR-Based Screening with Competition Water−Ligand Observed via Gradient Spectroscopy Experiments: Detection of High-Affinity Ligands

Cited by 112 publications

References 28 publications

Competition STD NMR for the detection of high‐affinity ligands and NMR‐based screening

Competition STD NMR for the detection of high‐affinity ligands and NMR‐based screening

Discovery of novel FabF ligands inspired by platensimycin by integrating structure-based design with diversity-oriented synthetic accessibility

Strengths and Weaknesses of Hybrid TPR Technology for Obtaining Structural and Mechanistic Insights into TPR Proteins

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