Carles Gil’s research while affiliated with Autonomous University of Barcelona and other places

CHIR99021, also known as laduviglusib or CT99021, is a Glycogen-synthase kinase 3 α/β inhibitor, which has been reported as a promising drug for cardiomyocyte regeneration or treatment of sensorial hearing loss, among other pathologies. Since the activation of dopamine (DA) receptors regulate dopamine synthesis itself and use glycogen-synthase kinase 3β (GSK3β) as an element in the β-arrestin pathway, we decided to check the effect of GSK3β inhibitors (CHIR99021, SB216763 and lithium ion) in the control of DA synthesis. Using ex vivo experiments with minces from rat brain striatum, we observed that CHIR99021, but not SB216763 nor lithium, causes a complete abrogation of DA synthesis and accumulation, pointing to off-target effects of CHIR99021. This decrease can be attributed to tyrosine hydroxylase (TH) inhibition since the accumulation of L-DOPA in the presence of a DOPA decarboxylase inhibitor was similarly decreased. On the other hand, CHIR99021 caused an exponential increase in the DOPAC / DA ratio, an indicator of DA metabolization, and decreased the incorporation of extracellular DA into striatum minces. In addition, CHIR99021 or SB216763, but not lithium, decreased TH phosphorylation in Ser19. These results demonstrate that CHIR99021 can lead to TH inactivation and DA decrease in brain striatum, opening the possibility of its use in DA-related disorders, and shows effects to be considered in future clinical trials.

Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.

Evidences from different techniques suggest that GPCRs oligomerize into homo- and hetero-oligomers. Oligomerization adds an extra level of complexity to receptor functionality because the allosteric interactions between protomers modify the intrinsic functional capacity of the single protomers. Reliable mathematical models are needed to quantify and mechanistically explain the observed functional effects. The operational model of agonism of Black and Leff (1983) constitutes a cornerstone in the description of agonism by providing parameter estimates for the pharmacological properties of affinity and efficacy. In the present study we revisit the operational model of agonism through the description of various adaptations and extensions to the model developed in our group. In particular, models for homo- and heterodimer receptors are discussed. Inclusion in the models of constitutive receptor activity and explicit parameters for cooperativity are useful for the interpretation of experimental data. Moreover, these quantitative approaches may clarify complex experimental relationships related to biased signaling. Finally, it is expected that the combined application of mathematical modeling and structural techniques will represent a powerful strategy to identify, visualize and quantify the allosteric interactions responsible for the observed functional effects dependent on receptor aggregation. This information is fundamental in the design and development of drug discovery programs. We believe that further efforts in this field are necessary for the construction of a robust and flexible modern pharmacology.

Lipids are becoming known as essential allosteric modulators of G protein-coupled receptor (GPCRs). However, how they exert their effects on GPCR conformation at the atomic level is still unclear. In light of recent experimental data, we have performed several long-timescale molecular dynamics (MD) simulations, totalling 24 μs, to rigorously map allosteric modulation and conformational changes in the β2adrenergic receptor (β2AR) that occur as a result of interactions with three different phospholipids. In particular, we identify different sequential mechanisms behind receptor activation and deactivation, respectively, mediated by specific lipid interactions with key receptor regions. We show that net negatively charged lipids stabilize an active-like state of β2AR that is able to dock Gsα protein. Clustering of anionic lipids around the receptor with local distortion of membrane thickness is also apparent. On the other hand, net-neutral zwitterionic lipids inactivate the receptor, generating either fully inactive or intermediate states, with kinetics depending on lipid headgroup charge distribution and hydrophobicity. These chemical differences alter membrane thickness and density, which differentially destabilize the β2AR active state through lateral compression effects.