Thinking outside the binding-pocket: in silico design of novel allosteric regulators of 14-3-3

MÜLLER, SERGIO; RIVERA, LAUTARO ; UHART, MARINA ; BARRERA GUISASOLA, EXEQUIEL E.; BUSTOS DIEGO M.

Conferencia; 12th international conference on structure and stability of biomacromolecules; 2023

The14-3-3 protein family, consisting of seven distinct mammalianparalogs, has emerged as a pivotal mediator in numerous cellularprocesses. Despite exhibiting some functional redundancy, recentevidence underscores their evolutionary and biochemical diversity.These proteins function by binding, either as a monomer or dimer, tophosphorylated Ser and Thr residues within disordered domains ofpartner proteins through a conserved amphipathic groove on theirsurface [1, 2, 3, 4, 5]. Consequently,14-3-3 proteins have garnered significant attention as attractivetargets for drug discovery. Various reports have explored theutilization of small molecules to achieve competitive inhibition orpartner stabilization, focusing on the well-defined ligand bindinggroove [6]. In order to detect distinct druggable hot-spots, wedecided to make a virtual screening of 2x10 6 compounds using ZINCdatabase over a monomer of 14-3-3. For this, we used Autodock 4.0 formolecular docking and excluded the region that belongs to thecanonical binding pocket. After manual selection of top dockingposes, we detected a list of 50 compounds that belong to a family ofβ-carbolineswith high affinity binding to a specific region located on theopposite face of the main binding groove of 14-3-3. We decided to useNorharmane (9H-Pyrido[3,4-B]indole) as a first approach for furtherin silico assays. Molecular dynamic simulations demonstrated that theamino acids that conform such cavity drive the dynamics of the wholeprotein. The presence of Norharmane induced a conformational shift,causing the protein to transition into a closed state. Thisconformational change obstructs partner accessibility to the primarygroove. Further on, to obtain paralog specific compounds, a set ofendogenous beta-carbolines were studied with a combination ofpharmacophore based docking and enhanced molecular dynamicssimulations, selectingcandidates by binding affinities and residence times. Collectively,our findings underscore the existence of small molecules like membersof the beta-carboline family with the potential to modulate 14-3-3proteins through a novel allosteric site. The convergence of insilico, in vitro, and in vivo methodologies holds promise for thediscovery and optimization of active agents that selectively targetthis site across distinct 14-3-3 paralogs. This novel regulatorymechanism adds a layer of complexity to the intricate network of14-3-3 protein interactions, fueling the quest for innovativetherapeutic interventions.p { margin-bottom: 0.1in; line-height: 115% }