Method for high throughput thermodynamic screening of ligands

Details Method for high throughput thermodynamic screening of ligands Method for high throughput thermodynamic screening of ligands

1999-12-01

2001-06-05

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S003000, C435S004000, C435S005000, C435S007100, C435S007900, C435S007920

Reexamination Certificate

active

06242190

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to the screening of ligands on the basis of their binding affinity and binding enthalpy. In particular, the invention provides a method to identify ligands that bind to a target macromolecule with favorable enthalpies by carrying out high throughput screening techniques at a minimum of two different temperatures.
2. Background of the Invention
The identification of drug candidates by screening large libraries of potential lead compounds and their optimization by structure-based design is an important component in the development of new pharmaceutical drugs. Modern high throughput screening procedures are able to process thousands of compounds and identify those that exhibit the highest binding affinity with relative accuracy. The binding affinity, Ka, is defined in terms of the free energy of binding:
Ka=e
−&Dgr;G/RT
  (1)
where R is the gas constant and T the absolute temperature. The free energy of binding is in turn defined by the enthalpy (&Dgr;H) and entropy (&Dgr;S) changes:
&Dgr;G=&Dgr;H−T&Dgr;S
  (2)
therefore,
Ka=e
−(&Dgr;H−T&Dgr;S)/RT
or,
Ka=e
−&Dgr;H/RT
×e
&Dgr;S/R
  (3)
It is evident that the binding affinity can be optimized by making &Dgr;H more negative, &Dgr;S more positive or by a combination of both. Even though compounds with many different combinations of &Dgr;H and &Dgr;S values will exhibit the same binding affinity (i.e. the same &Dgr;G and therefore the same Ka), the properties and the response of these compounds to changes in the environment or in the protein target are not the same. The binding enthalpy reflects the interactions of the ligand with the target protein (e.g., van der Waals, hydrogen bonds, etc.). The entropy change, on the other hand, reflects two main contributions: changes in solvation entropy and changes in conformational entropy. Upon binding, desolvation occurs, water is released and a gain in solvent entropy is observed. This gain is particularly important for hydrophobic groups. At the same time, the ligand (and certain groups in the protein) lose conformational freedom resulting in a negative change in conformational entropy. Accordingly, there are three main strategies for improving binding affinity: 1) Improving ligand protein interactions over those with the solvent in order to obtain a negative enthalpy change; 2) Making the ligand more hydrophobic in order to make the solvation entropy large and positive; and, 3) Pre-shaping the ligand to the geometry of the binding site in order to minimize the loss of conformational entropy upon binding. Of these three strategies the easier to implement in structure-based drug design have been 2) and 3). As a result, the majority of affinity-optimized drug candidates are entropy optimized and thus are highly hydrophobic and rigid (pre-shaped to the geometry of the binding site).
Highly hydrophobic, rigid molecules have several drawbacks as potential drugs. First, they are highly insoluble in aqueous solution making their administration difficult. Second, rigidity reduces their ability to accommodate to changes in the geometry of the binding site, increasing their susceptibility to drug resistant mutations in the target protein. The correlation between lack of flexibility and susceptibility to resistant mutants has been recently brought into light for HIV-I protease inhibitors and for HIV-I reverse transcriptase inhibitors.
It is evident that flexible ligands will be less susceptible to resistant mutations. However, introducing flexibility in existing ligands will lower their binding affinity because of the larger conformational entropy loss upon binding. The introduction of flexibility needs to be compensated by the introduction of additional favorable interactions. These interactions cannot be hydrophobic because flexible hydrophobic ligands will lack specificity and, in addition, ligand candidates in current databases already favor hydrophobic interactions. The solution is to improve their binding enthalpy. Since the favorable binding enthalpy originates from specific ligand/target interactions, an enthalpic optimization will provide the additional binding affinity and the necessary target specificity.
It is currently possible to obtain binding enthalpy data to be used in the selection of likely candidate ligands via isothermal titration calorimetry (ITC). However, such direct calorimetric titrations, while accurate, are extremely time consuming (~2 hours per compound) and thus cannot be incorporated into a fast screening protocol. Further, the instruments themselves are costly and require a high level of expertise to operate and maintain, making this technique unsuitable for rapid, high throughput ligand screening.
It would thus be highly desirable to have available a method and apparatus to screen multiple ligands rapidly and identify those with favorable enthalpies during the early stages of the screening procedure. These ligands would exhibit higher solubilities in aqueous solution, better specificity, and lower susceptibility to resistant mutations. Further, it would be advantageous if the method was readily adaptable to existing high throughput technologies.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a rapid method for screening multiple ligands on the basis of binding enthalpy. The method can be coupled to existing high throughput screening technologies, or used as a standalone technique. The method encompasses exposing a macromolecule to a variety of potential candidate ligands and measuring the binding affinities of the ligands at a minimum of two different temperatures. The binding affinities exhibited by the candidate ligands at the various temperatures are then compared, and ligands exhibiting favorable enthalpies may be selected for further optimization.
Potential macromolecules for which ligands may be selected in this manner include a wide array of naturally occurring and synthetic macromolecules, including proteins, peptides, nucleic acids, carbohydrates, and receptor molecules. Ligands which are encompassed by the present invention include a wide array of naturally occurring and synthetic molecules, including proteins, peptides, nucleic acids, carbohydrates, enzyme substrates, and enzyme inhibitors.
The method of the present invention is applicable to a wide variety of high throughput ligand screening protocols, including incubation of the macromolecule and ligands in multiwell plates, in etched channels, in capillary tubes, on absorbent solid supports, and the like. Further, the binding affinities may be measured by the detection of any parameter which changes in either the macromolecule or the ligand as a result of ligand binding, either directly or indirectly, including fluorescence, luminescence, emission of light, absorbance of light, radioactivity, fluorescence resonance energy transfer (FRET), pH, enzymatic activity, molecular weight, antibody binding, etc.
It is a further object of the present invention to provide an apparatus capable of carrying out the method of the present invention. Such an apparatus includes a means for exposing the macromolecule to the candidate ligands at a minimum of two different temperatures. In addition, the apparatus may include a means to measure the binding affinities of the various ligands at all temperatures at which the binding reaction is carried out, and a means to analyze the data, including a means to store, display and mathematically manipulate the binding affinities.


REFERENCES:
patent: 5518882 (1996-05-01), Lund et al.

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