**5. Drug optimization**

776 Thermodynamics – Interaction Studies – Solids, Liquids and Gases

(F/F0) - 1

<sup>Q</sup> - <sup>1</sup> (48)

(49)

[P] = [P]total - XL[L]total (50)

L + R + P ↔ PL + PR (52)

(53)

(54)

<sup>1</sup> <sup>+</sup> Ka[P] (51)

[PL] = [L]total

**4.3.3 Fluorescence anisotropy** 

be calculated from:

**4.4 Competition methods** 

ligand bound (XL=[PL]/[L]total) is determined from:

XL= r - r0

The binding constant can be determined from the hyperbola:

XL <sup>=</sup> Ka[P]

of the reference ligand (KR) is already known.

KL= KR

bound to its site (EC50). Thus KL is calculated from:

KL= <sup>1</sup> <sup>+</sup> [R]KR

Thus the binding constant can be determined from the Scatchard plot as described above.

Fluorescence anisotropy measures the rotational diffusion of a molecule. The effective size of a ligand bound to its target usually increases enormously, thus restricting its motion considerably. Changes in anisotropy are proportional to the fraction of ligand bound to its target. Using suitable polarizers at both sides of the sample cuvette, this property can be measured. In a tritation experiment similar to the ones described above, the fraction of

rmax - r0

where r is the anisotropy of ligand in the presence of the target molecule, r0 is the anisotropy of ligand in the absence of target and rmax is the anisotropy of ligand fully bound to its target (note that equation 49 can be used only in the case where ligand fluorescence intensity does not change, otherwise appropriate corrections should be done, see (Lakowicz 1999)). [P] can

The characterization of a ligand binding let us determine the binding constant of any other ligand competing for the same binding site. Measurements of ligand (L), target (P), reference ligand (R) and both complexes (PR and PL) concentrations in the equilibrium permit the calculation of the binding constant (KL) from equation 53 (see below) as the binding constant

> [PL][R] [L][PR]

In the case that the reference ligand has been characterized due to the change of a ligand physical property (i.e. fluorescence, absorbance, anisotropy) upon binding, would permit us also following the displacement of this reference ligand from its site by competition with a ligand "blind" to this signal (Diaz & Buey 2007). In this kind of experiment equimolar concentrations of the reference ligand and the target molecule are incubated, increasing concentrations of the problem ligand added and the appropiate signal measured. It is possible then to determine the concentration of ligand at which half the reference ligand is

EC50

Microtubule stabilizing agents (MSA) comprise a class of drugs that bind to microtubules and stabilize them against disassembly. During the last years, several of these compounds have been approved as anticancer agents or submitted to clinical trials. That is the case of taxanes (paclitaxel, docetaxel) or epothilones (ixabepilone) as well as discodermolide (reviewed in (Zhao *et al.* 2009)). Nevertheless, anticancer chemotherapy has still unsatisfactory clinical results, being one of the major reasons for it the development of drug resistance in treated patients (Kavallaris 2010). Thus one interesting issue in this field is drug optimization with the aim of improving the potential for their use in clinics: minimizing side-effects, overcoming resistances or enhancing their potency.

Our group has studied the influence of different chemical modifications on taxane and epothilone scaffolds in their binding affinities and the consequently modifications in ligand properties like citotoxicity. The results from these studies firmly suggest thermodynamic parameters as key clues for drug optimization.
