Novel caffeine analogs as potential leaders on the cholinergic system

BISCOSSI, B; CORRADI, J.; MUNAFO, J.P.; MURRAY, A.P.; FABIANI, C.; SALVADOR, G.A.; ANTOLLINI, S.S.

Salta

Congreso; XIV Conference PABMB (Pan-American Association for Biochemistry and Molecular Biology. LV Reunión de SAIB (Sociedad Argentina de Investigación Bioquímica y Biología Molecular).; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Cholinergic deficit is regarded as an important factor responsible for Alzheimer?s disease symptoms. Two molecular targets for the treatment ofthis disease are acetylcholinesterase (AChE) and nicotinic receptor (nAChR). We previously demonstrated that caffeine has a dual effect onmuscle and a7 nAChRs, behaving as a weak agonist at low concentrations and as a negative modulator at high concentrations. Furthermore, it is well-known that caffeine also acts as an inhibitor of AChE. The aim of this work was to synthetize more potent caffeine analogs with a dualeffect on the cholinergic system by inhibiting AChE and potentiating nAChRs. With this objective, a theophylline fragment, resembled thecaffeine chemical structure, was connected with a pyrrole fragment, which is present in the nicotinic chemical structure, through homologationfrom 3 to 6 carbon atoms (Cn). We first tested the capacity of the different compounds to inhibit the AChE. We found that whether theophyllinealone inhibited the enzyme, pyrrolidine did not. With respect to Cn, they all can inhibit the AChE at concentrations of 100, 200 and 400 µM,having C6 the strongest effect. We then explored if theophylline, pyrrolidine and Cninfluence the nAChR conformational state. To this end, weused the AChR conformational-sensitive fluorescence probe crystal violet (CrV) and AChR-rich membranes from T. californica. We found thatwhether pyrrolidine induced changes in the KD values of CrV taking the nAChR to a state close to the desensitized one at concentrations of 200and 400 µM, theophylline did not show a significant change in the KD value. The combined analogues also produced changes in the KD values ofCrV. This effect was dependent on the length of homologation, being C5 and C6 the most potent analogues with effect at concentrations lowerthan 50 nM. To understand the molecular mechanism underlying the conformational changes of the nAChR, we expressed adult muscle nAChRin HEK293 cells, and performed single channel recordings with different Cn concentrations. We found that C5 activated muscle nAChR at verylow concentrations (from 0.01 pM). At the highest tested concentration (30 µM) we observed a decrease in the mean open duration, whichsuggest that C5 also acts as an open channel blocker. As a partial conclusion we can say that we have synthetized more potent caffeine analogsthrough the combination of caffeine and nicotinic structures. The effect of theophylline on AChE, the effect of pyrrol on AChR, and the effect ofCn on both proteins suggest different pharmacophore profiles for these target molecules. Our results bring new information about the mechanismof modulation of pharmacologic targets for the design of new therapies for the intervention in neurological diseases.