Dissecting a ligand into smaller fragments can provide a strategy for analyzing the role of key functional groups in a protein-ligand interaction. Conversely, low-affinity ligand fragments that are able to occupy a receptor-binding site simultaneously, can often be linked together to form high-affinity inhibitors. The key challenge to such studies lies in measuring the thermodynamics of low-affinity interactions. This white paper will address the issues of studying low-affinity systems by isothermal titration calorimetry (ITC). Topics will include working with receptor concentrations below KD (i.e., low c-values), and how displacement techniques can be employed to measure very low affinities while still complying with the c-value rule. Illustrative examples will be drawn from studies with carbohydrate-binding proteins.
Conversely, low-affinity ligand fragments that are able to occupy a receptor-binding site simultaneously, can often be linked together to form high-affinity inhibitors. The key challenge to such studies lies in measuring the thermodynamics of low-affinity interactions. This white paper will address the issues of studying low-affinity systems by isothermal titration calorimetry (ITC). Topics will include working with receptor concentrations below KD (i.e., low c-values), and how displacement techniques can be employed to measure very low affinities while still complying with the c-value rule. Illustrative examples will be drawn from studies with carbohydrate-binding proteins.
Introduction
Molecular recognition is a fundamental prerequisite to all biological processes from enzymatic catalysis to signal transduction. The modern pharmaceutical industry is built upon the concept of selective intervention in such systems (1). Furthermore, similar concepts are being implemented, through supramolecular chemistry, to develop novel smart materials, components for nanotechnology and molecular electronics (2). If we are to harness the full potential of molecular recognition, it is essential that we understand the subtle interplay between structure and thermodynamics in complex formation.
ITC has rapidly become one of the most common techniques for studying molecular recognition processes, and the technique of choice for determining the enthalpic and entropic contributions to the overall free energy of binding, ∆G° (3–5).
As ITC only requires that there is a measurable enthalpy change upon complexation, it is generally applicable to most systems. The thermodynamic parameters obtained from an ITC experiment describe the sum of all processes occurring in solution — formation and breaking of hydrogen bonds, conformational changes, desolvation, etc. Therefore, a single experiment will not allow determination of the contribution of an individual hydroxyl group or phenyl ring to the overall binding enthalpy or free energy. Dissecting the binding contributions of individual fragments of a ligand is possible by studying the interactions of a given receptor for a series of ligands that display systematic variations in their structures. Assuming that all of the fragment ligands bind in an analogous fashion to their parent ligand, then it is possible to apportion thermodynamic contributions to each part of the key ligand (Fig 1A).
However, if a particular functional group or fragment is a major contributor to the total binding energy, then removal of that fragment will leave a ligand with very poor affinity for the receptor. Although one might conclude, qualitatively, that the deleted group is essential for activity, to achieve a more informative, quantitative picture of the system, it is necessary that even the lowest affinity interactions are measured.
If a high-affinity ligand can be broken down into low-affinity fragments, then the converse must also be true: if two or more, low-affinity fragments can be accommodated simultaneously in a receptor binding site, it should be possible to connect them together, via a suitable linker, to form a high-affinity ligand . In fact, this phenomenon is already widely known as the chelate effect (6). Once one half of a bivalent ligand has bound to its receptor, there is a very high local concentration of the second recognition element in the vicinity of the binding site, thus increasing the probability of the second interaction taking place. Alternatively, the chelate effect can be described in terms of the entropic penalty that must be paid for loss of independent rotational and translational degrees of freedom when two molecules interact to form a single particle (7). When two fragments of a bivalent ligand bind to their receptor, the penalty must be paid twice. However, the full bivalent ligand need pay this penalty only once, and thus may interact with a substantially improved affinity.
This fragment-based strategy for ligand design is gaining wider acceptance in the pharmaceutical industry (8,9), in particular in situations where high-throughput screening of existing compound libraries has failed to identify a suitable lead compound for a given target protein. Typically, NMR-based screening methods are employed to identify pairs of small molecule “fragments” (< 250 Da), and to provide structural data on their orientations in the receptor binding site (10,11). ITC can provide complementary information, for example, confirmation of fragment binding. Furthermore, selecting fragments that display the most favorable enthalpy changes may prove a useful strategy for maximizing binding selectivity for the final ligands (3). However, using ITC to determine the thermodynamic parameters for low-affinity systems is not always trivial.
Two strategies for studying weakly binding ligands will be outlined in this white paper, but first it is important to understand the specific problems associated with low-affinity systems.
Click here to read the full white paper
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