Development of new mono- and multivalent ligands for medically relevant proteins and peptides

Abstract

Among all biomolecules, proteins are the main effectors of biological functions in living organisms. Thanks to their high diversity in term of structure, shape and composition, proteins have been exploited by Nature for a wide range of purposes acting as enzymes, receptors, messengers, structural fibres and transporters. Unfortunately, when genes codifying for them are mutated, their expression might lead to afunctional proteins or to unusual concentration levels for those proteins. For this reason, proteins can be used as convenient targets for limiting or even stopping the incoming disease. In this context, this PhD project has dealt with the development of new synthetic molecules for the targeting of medically relevant peptides/proteins, in particular Carbonic Anhydrases, Transthyretin, Aβ16-22, Mcl-1 and p300 . Looking for selectivity and efficiency in Carbonic Anhydrases (CAs) inhibition, a wide set of monovalent and multivalent potential inhibitors bearing ammonium and sulfonamide moieties were designed, synthetized and tested in stopped-flow assay towards a small library of CA isoforms. Monovalent sulfonamide-bearing molecules showed to be very potent and selective inhibitors (Ki in low-nanomolar range) towards human CAs (in particular, hCAI and hCAII), whereas dimeric analogues were pretty inactive towards all tested isoforms. On the contrary, almost all the series of calixarene-based derivatives exposing ammonium or benzensulfonamide units showed very good selectivity towards fungine Can2 CA, and significant efficiency (Ki in micromolar range) towards fungine MgCA, even better than the reference compound acetazolamide (nowadays, used for the topic treatment of glaucoma). Remarkably, the structure at the solid state of the complexes between hCAII and four ligands were determined by X-Ray diffraction. Even if no multivalent effect was observed with the calixarene-based inhibitors, however, for some of them, the efficiency shown suggests the involvement of the macrocyclic cavity in establishing additional interactions with the protein. This aspect is currently still under investigation. Transthyretin (TTR), when mutated, is prone to unfold its tetrameric structure and produce amyloidogenic fibrils responsible of Alzeheimer’s disease. In order to find molecules able to prevent the tetramer disaggregation, we synthetized new “kinetic stabilizers” based on well-known Tolcapone and Biochanin A structures with the aim to improve their binding affinities. By Western Blot assays in plasma samples it turned out in particular that 3-deoxytolcapone is more selective towards Wild-Type TTR with respect to Tafamidis and Diflunisal, but less than Tolcapone. By the way since metabolic transformations modify Tolcapone at 3-OH group impairing its activity, the synthetized 3-deoxytolcapone represents a promising alternative in the treatment of amyloidogenic diseases. Another project of this PhD work has dealt with the study of polarity and aggregation of Aβ16-22, the smallest model peptide fragment of β-amyloid (Aβ42), which is another etiological factor of the formation of amyloidogenic fibrils. In this context, during a secondment at University of Leeds two classes of more hydrophobic Aβ16-22 sequences were synthetized with iodo-, chloro- and methoxy-phenylalanine in position 19 and 20, one capped at N-terminus with acetyl moiety, one capped with TAMRA-AhX moiety The former was employed for the study of morphology and topology of fibrils and nanotubes with Electron Microscopy (TEM), the latter was employed for kinetic aggregation studies in Fluorescence Quenching Assay (FQA). From these studies it came out that these new peptides are still able to make fibrils and nanotubes and that increasing the apolarity, in particular in position 19, the aggregation is slightly enhanced. In the same period at University of Leeds, the attention was focused also on the inhibition of protein-protein interactions (PPIs) in systems like p300/HIF1α and Mcl-1/Noxa, often involved in tumour development. Since their reciprocal recognition passes through interactions involving either Leu or Val side chains, a library was synthetized of calix[4]arenes with isopropyl or isobutyl groups at the upper rim for the recognition and ethoxyethyl or methylencarboxy moieties at the lower rim to increase the solubility in water. Mainly for solubility issues, in Fluorescence Anisotropy assays only the derivative with methylencarboxy groups at lower rim showed to be active towards Mcl-1 and p300 (low micromolar Ki). To understand more about the binding of this molecule with biological counterparts, NMR and cocrystallization assays are currently in progress.