Design and synthesis of a new FP probe

The first step in the establishment of a new FP-based competition binding assay consisted in the design of a suitable fluorescent probe for the target enzymes. Compound 1 (Figure 2.1 and 3.1B) was discovered in a previous in-house study as a potent dual JNK3/p38α

MAPK inhibitor, with IC50 values in the low nM range on both enzymes and was therefore

selected as a potential precursor for the FP fluoroprobe.

An extremely important aspect in projecting an FP-probe consists in the position of the labelling as the introduction of a bulky fluorescent tag might result in a loss of affinity of the probe for its targets. Munoz et al. reported in 2010 an FP probe for the p38α MAPK

derived from the pyridinylimidazole-based reference compound SB203580.145 _{In such}

molecule, the fluorescent tag, represented by a fluorescein moiety, was linked at the imidazole-C2 position of the scaffold (compound 2, Figure 3.1A). Nevertheless, when planning a new probe suitable for both JNK3 and p38α MAPK, the aniline moiety at the pyridine-C2 position of compound 1 was assumed to be optimal for the installation of the fluorescent label. The reason for this lies in the well-known binding mode of pyridinylimidazole molecules, wherein the substituent at the pyridine-C2 position is located in the solvent-exposed HR II (a more detailed explanation of the binding mode of pyridinylimidazole-base inhibitors will be provided in section 3.2.1). Due to its opening on the outside of the protein, this area is presumably more prone to tolerate the large fluorescent group without substantial effects on the binding

A second crucial setting concerns the choice of the fluorophore and the method of labeling. Fluorescein was selected as a fluorophore due to the affordable cost and to the availability of a broad range of derivatives, allowing the labeling of diverse functional groups.

42 3. RESULTS AND DISCUSSION

Furthermore, virtually all plate readers are provided with the excitation/emission filters necessary for this fluorophore (approximately 490/525 nm). Finally, the short lifetime τ of this fluorescent marker (≈ 4 ns) is well-suited for the analysis of small ligands interacting with proteins heavier than 10 kDa.140 _{These features made fluorescein the first choice}

fluorescent dye employed in FP assays, as also recently reviewed by Hall and coworkers.135

In particular, the selected dye was the fluorescein isothiocyanate (FITC) isomer 5’, which would easily react with the terminal aniline of compound 1 through the formation of a thiourea bridge (Figure 3.1B).

Figure 3.1. Design of FP probes; A) probe 2 reported from Munoz et al.145 _{derived from standard}

inhibitor SB203580; B) probe 3 designed for a new FP-based competition binding assay derived from compound 1.

An initial choice made consisted in avoiding the introduction of a spacer between the core scaffold and the fluorescent moiety, which was therefore directly linked to the terminal amino group of compound 1. The rationale behind this decision lies in the fact that although a spacer might increase the flexibility and reduce the impact of the bulky fluorophore on the binding interactions, it could also determine the rotational freedom of the fluorescent tag even when the probe is bound to the target. This phenomenon is named “propeller effect” and can have detrimental outcomes on the FP measurement as it reduces the signal

3. RESULTS AND DISCUSSION 43

Before synthesis, the suitability of the designed probe 3 was confirmed by docking studies (Figure 3.2). Both in the case of JNK3 and p38α MAPK reasonable poses were generated wherein the fluorescent marker was positioned outside of the binding site, therefore seeming not to hinder the interactions of the core scaffold.

Figure 3.2. Docking poses of probe 3 with JNK3 (left, PDB entry: 3FI3) and p38α MAPK (right, PDB

entry: 1OUK).

Besides the preparation of probe 3, four additional scaffolds and corresponding fluorescent probes were synthesized varying the substitution pattern around the imidazole ring (probes 3 and 8-11, Figure 3.3). This was carried out in order to broaden the subset of potential probes and therefore select the one displaying the best features in terms of applicability. All probes were tested in the aforementioned ELISA activity assay on both JNK3 and p38α MAPK and the results were compared with their precursors, aimed at observing the influence of the labelling on the inhibition potency (Figure 3.3). Among all the synthesized probes, compounds 3 and 10 seemed to display optimal features to serve as FP probes as they showed a high potency on the two enzymes which appeared to be only negligibly affected by the installation of the fluorescent dye. For this reason, such compounds were selected as candidates for the following assay optimization steps and the introduction of a spacer was not considered necessary. On the other hand, probes 8 and 9 are characterized by a lower potency on JNK3 and by scarce or no inhibition of p38α MAPK and are therefore not suitable to be employed as dual probes. Moreover, in case of these two compounds the labelling with fluorescein seemed to have a considerable effect on the inhibitory activity, which decreased significantly on both kinases. Analogously to its

precursor, probe 11 showed instead a moderate inhibition of both enzymes, with IC50 values

44 3. RESULTS AND DISCUSSION

Figure 3.3. Structures and biological activity of the synthesized probe candidates (compounds 3 and 8-11) and their precursors (compounds 1 and 4-7). The synthetic schemes of compounds 1 and 4-7

will be discussed in the second part of the thesis, section 3.2 and detailed procedures as well as analytical data can be found in Publication I (compounds 1 and 6), Publication IV (compound 4), or in the experimental section of this thesis (compounds 5 and 7); experimental procedures for the preparation of the fluorescent probes together with analytical data can be found in Publication I (probes 3 and 10), Publication VI (probe 11) or in the experimental section of this thesis (probes 8 and 9).

3. RESULTS AND DISCUSSION 45