Competition NMR for Detection of Hit/Lead Inhibitors of Protein–Protein Interactions

Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

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Competition NMR for Detection of Hit/Lead Inhibitors of Protein-Protein Interactions

10.1101/2020.06.12.148379 ◽

2020 ◽

Author(s):

Bogdan Musielak ◽

Weronika Janczyk ◽

Ismael Rodriguez ◽

Jacek Plewka ◽

Dominik Sala ◽

...

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Point Mutations ◽

Protein Protein Interactions ◽

Competition Assay ◽

Fragment Screening ◽

Different Types ◽

Lead Inhibitors

ABSTRACTScreening for small-molecule fragments that can lead to potent inhibitors of protein-protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM-mM affinities. Here, we describe an NMR competition assay - called w-AIDA-NMR (weak-Antagonist Induced Dissociation Assay-NMR) - that is sensitive to weak μM-mM ligand-protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

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Progress in discovery of small-molecule modulators of protein–protein interactions via fragment screening

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2015.04.089 ◽

2015 ◽

Vol 25 (12) ◽

pp. 2461-2468 ◽

Cited By ~ 22

Author(s):

Thomas V. Magee

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Fragment Screening ◽

Small Molecule Modulators

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ChemInform Abstract: Progress in Discovery of Small-Molecule Modulators of Protein-Protein Interactions via Fragment Screening

ChemInform ◽

10.1002/chin.201530293 ◽

2015 ◽

Vol 46 (30) ◽

pp. no-no

Author(s):

Thomas V. Magee

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Fragment Screening ◽

Small Molecule Modulators

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Faculty Opinions recommendation of Importance of Rigidity in Designing Small Molecule Drugs To Tackle Protein-Protein Interactions (PPIs) through Stabilization of Desired Conformers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732129727.793539183 ◽

2017 ◽

Author(s):

John Lowe

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Small Molecule Drugs

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Elucidating Protein-protein Interactions Through Computational Approaches and Designing Small Molecule Inhibitors Against them for Various Diseases

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666181025114903 ◽

2018 ◽

Vol 18 (20) ◽

pp. 1719-1736 ◽

Cited By ~ 3

Author(s):

Sharanya Sarkar ◽

Khushboo Gulati ◽

Manikyaprabhu Kairamkonda ◽

Amit Mishra ◽

Krishna Mohan Poluri

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Small Molecule Inhibitors ◽

Computational Techniques ◽

Protein Protein Interactions ◽

Computational Tools ◽

Computational Approaches ◽

Protein Protein Interaction ◽

Wide Range ◽

Therapeutic Measures

Background: To carry out wide range of cellular functionalities, proteins often associate with one or more proteins in a phenomenon known as Protein-Protein Interaction (PPI). Experimental and computational approaches were applied on PPIs in order to determine the interacting partners, and also to understand how an abnormality in such interactions can become the principle cause of a disease. Objective: This review aims to elucidate the case studies where PPIs involved in various human diseases have been proven or validated with computational techniques, and also to elucidate how small molecule inhibitors of PPIs have been designed computationally to act as effective therapeutic measures against certain diseases. Results: Computational techniques to predict PPIs are emerging rapidly in the modern day. They not only help in predicting new PPIs, but also generate outputs that substantiate the experimentally determined results. Moreover, computation has aided in the designing of novel inhibitor molecules disrupting the PPIs. Some of them are already being tested in the clinical trials. Conclusion: This review delineated the classification of computational tools that are essential to investigate PPIs. Furthermore, the review shed light on how indispensable computational tools have become in the field of medicine to analyze the interaction networks and to design novel inhibitors efficiently against dreadful diseases in a shorter time span.

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Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618087114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2146-E2155 ◽

Cited By ~ 7

Author(s):

Chi-Yun Lin ◽

Johan Both ◽

Keunbong Do ◽

Steven G. Boxer

Keyword(s):

Quantum Yield ◽

Protein Interactions ◽

Fluorescent Proteins ◽

Point Mutations ◽

Photoactive Yellow Protein ◽

Green Fluorescent Proteins ◽

Protein Protein Interactions ◽

Low Efficiency ◽

Green Fluorescent ◽

Rate Limiting

Split GFPs have been widely applied for monitoring protein–protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics–function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

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