Publication

Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

Roberto Buratto
2015
EPFL thesis
Abstract

The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. Nowadays, the screening of drug candidates is routinely done by NMR in pharmaceutical industry. We have proposed to use the relaxation of Long-Lived States (LLS) for drug screening by NMR. Long-lived states are nuclear spin states whose decay time constant Tlls can be much longer than the longitudinal relaxation time T1. LLS can be used to screen and determine the dissociation constant KD of molecular fragments that bind weakly to protein targets. The use of LLS for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with KD in the millimolar range, which is difficult to achieve by other methods such as ITC. By exploiting the LLS behavior of a spy molecule, we experimentally demonstrate that it is possible to measure dissociation constants KD as large as 12 mM, corresponding to very weak binding, where most other biophysical techniques fail, including other NMR methods based on the observation of ligands. Furthermore, we have combined LLS for screening for improved contrast with 1H dissolution-DNP to enhance the sensitivity. DNP-enhanced screening for measuring LLS signals of a weak ligand allows one to use very low concentrations of ligands and proteins. We observed dramatic differences between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, we have explored LLS involving pairs of 19F nuclei to study binding phenomena. Indeed, fluorine detection is quite interesting because it offers the possibility to perform screening experiments without any problems due to overlapping signals. In a customdesigned fluorinated ligand that binds trypsin, we have observed a promising ratio Tlls / T1 > 4. This fluorinated ligand has been used as spy molecule in competition experiments, which allowed us to rank the affinities and estimate dissociation constants of arbitrary ligands that do not contain any fluorine.

About this result
This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.
Related concepts (37)
Ligand
In coordination chemistry, a ligand is an ion or molecule with a functional group that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's electron pairs, often through Lewis bases. The nature of metal–ligand bonding can range from covalent to ionic. Furthermore, the metal–ligand bond order can range from one to three. Ligands are viewed as Lewis bases, although rare cases are known to involve Lewis acidic "ligands".
Virtual screening
Virtual screening (VS) is a computational technique used in drug discovery to search libraries of small molecules in order to identify those structures which are most likely to bind to a drug target, typically a protein receptor or enzyme. Virtual screening has been defined as "automatically evaluating very large libraries of compounds" using computer programs. As this definition suggests, VS has largely been a numbers game focusing on how the enormous chemical space of over 1060 conceivable compounds can be filtered to a manageable number that can be synthesized, purchased, and tested.
Bridging ligand
In coordination chemistry, a bridging ligand is a ligand that connects two or more atoms, usually metal ions. The ligand may be atomic or polyatomic. Virtually all complex organic compounds can serve as bridging ligands, so the term is usually restricted to small ligands such as pseudohalides or to ligands that are specifically designed to link two metals. In naming a complex wherein a single atom bridges two metals, the bridging ligand is preceded by the Greek letter mu, μ, with a subscript number denoting the number of metals bound to the bridging ligand.
Show more
Related publications (120)

Improved microfluidic transcriptome based screening methods

Christoph Merten, Xiaoli Ma, Leonie Kolmar, Hongxing Hu

The invention provides microfluidic screening systems for the identification of compounds or compositions influencing cellular transcriptomes. The invention is predicated upon taking into account differences in newly synthesized mRNA in contrast to so call ...
2024

Methods for high-throughput synthesis and screening of peptide libraries

Zsolt Bognár

Macrocycles provide an attractive modality for drug development but the identification of ligands to targets of interest is hindered by the lack of large macrocyclic compound libraries for high-throughput screening. A strategy to efficiently synthesize lar ...
EPFL2024

Antibodies expand the scope of angiotensin receptor pharmacology

Patrick Daniel Barth, Shuhao Zhang

G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often l ...
2024
Show more
Related MOOCs (6)
Basic Steps in Magnetic Resonance
A MOOC to discover basic concepts and a wide range of intriguing applications of magnetic resonance to physics, chemistry, and biology
Fundamentals of Biomedical Imaging: Magnetic Resonance Imaging (MRI)
Learn about magnetic resonance, from the physical principles of Nuclear Magnetic Resonance (NMR) to the basic concepts of image reconstruction (MRI).
Fundamentals of Biomedical Imaging: Magnetic Resonance Imaging (MRI)
Learn about magnetic resonance, from the physical principles of Nuclear Magnetic Resonance (NMR) to the basic concepts of image reconstruction (MRI).
Show more

Graph Chatbot

Chat with Graph Search

Ask any question about EPFL courses, lectures, exercises, research, news, etc. or try the example questions below.

DISCLAIMER: The Graph Chatbot is not programmed to provide explicit or categorical answers to your questions. Rather, it transforms your questions into API requests that are distributed across the various IT services officially administered by EPFL. Its purpose is solely to collect and recommend relevant references to content that you can explore to help you answer your questions.