Biased agonism at GPCRs highlights the potential for the discovery and design of pathway-selective ligands and may confer therapeutic advantages to ligands targeting the dopamine D2 receptor (D2R). We investigated the determinants of efficacy, affinity, and bias for three privileged structures for the D2R, exploring changes to linker length and incorporation of a heterocyclic unit. Profiling the compounds in two signaling assays (cAMP and pERK1/2) allowed us to identify and quantify determinants of biased agonism at the D2R. Substitution on the phenylpiperazine privileged structures (2-methoxy vs 2,3-dichloro) influenced bias when the thienopyridine heterocycle was absent. Upon inclusion of the thienopyridine unit, the substitution pattern (4,6-dimethyl vs 5-chloro-6-methoxy-4-methyl) had a significant effect on bias that overruled the effect of the phenylpiperazine substitution pattern. This latter observation could be reconciled with an extended binding mode for these compounds, whereby the interaction of the heterocycle with a secondary binding pocket may engender bias.

NATUre NeUrOSCieNCesignal monosynaptically and effectively to the LA and to a newly identified GABAergic amygdala population. By driving a complex intra-amygdala activity pattern, Calr + LT cells play an instrumental role in the establishment of CS+US signals and the formation of fear memories in the LA. Furthermore, these thalamic cells alter their activity during memory trace formation and can discriminate between US-paired and unpaired signals during retrieval. Together, our study indicates that the LT pathway to the amygdala, composed of Calr + LT neurons, provides associated and plastic signals for the acquisition of cue-related fear behavior. resultsCalr + LT cells project to the LA and are activated during fear learning. First, we identified which thalamic populations are connected to the LA. Thalamic cells retrogradely labeled from the LA with cholera toxin B subunit (CTB; Fig. 1a-c) were mainly located around the auditory thalamus (the MGN) in the PIL and the SG regions (Fig. 1b; Supplementary Fig. 1a-c). Since Calr + cells were specifically abundant in these lateral thalamic regions (Fig. 1b; Supplementary Fig. 1), we analyzed the Calr content of the CTBlabeled cells. The majority of them expressed Calr (Fig. 1c,d), which indicates that the thalamo-LA pathway is primarily formed by Calr + PIL and SG populations, hereafter collectively referred to as Calr + LT neurons (Supplementary Information).Next, we used an activity-dependent immediate early gene assay to investigate the involvement of the Calr + LT neurons in transferring CS and/or US signals during fear learning (Fig. 1e; Supplementary Fig. 2). Expression of the immediate early gene c-Fos was analyzed in four groups of mice that received either a conditioning tone (7.5 kHz, 30 s; CS+) or a US (foot shock, 1 mA, 1 s; US), or a US-associated tone (CS+US). Naive mice were used as controls. While CS+ markedly increased c-Fos expression in the PIL and SG areas, US and CS+US further increased the number of activated neurons in the PIL region relative to what was observed in control mice (Fig. 1f-j; Supplementary Table 1). Since the majority of c-Foslabeled cells were also Calr + (Fig. 1j; Supplementary Table 2), it was concluded that the LA can receive all relevant sensory information necessary for associative learning from Calr + LT cells.Calr + LT cells transfer short-latency signals related to fear learning. The expression of c-Fos provided spatially precise data about the cellular origin of CS, US and CS+US information (Fig. 1) for the LA. However, it did not reveal whether these neurons can convey and integrate a CS and a US within a short time window 19 and supply the LA with a salient cue within 20 ms 7 . Thus, we performed extracellular in vivo recordings from Calr + LT cells to investigate the time course of activation to short tone and footshock stimuli, as well as their possible potentiation to an associated footshock-coupled tone. Calb2-Cre mice (Calb2 encodes Calr; Supplementary Fig. 1k-n) were injected with conditional Cre-dependent recomb...

The Zika virus presents a serious risk for global health. Crystal structures of different constructs of the Zika virus NS2B-NS3 protease (NS2B-NS3pro) have been determined with the aim to provide a basis for rational drug discovery. In these structures, the C-terminal β-hairpin of NS2B, NS2Bc, was observed to be either disordered (open conformation) or bound to NS3pro complementing the substrate binding site (closed conformation). Enzymatically active constructs of flaviviral NS2B-NS3 proteases commonly used for inhibitor testing contain a covalent peptide linker between NS2B and NS3pro. Using a linked construct of Zika virus NS2B-NS3pro, we studied the location of NS2Bc relative to NS3pro in solution by pseudocontact shifts generated by a paramagnetic lanthanide tag attached to NS3pro. Both closed and open conformations were observed with different inhibitors. As the NS2B co-factor is involved in substrate binding of flaviviral NS2B-NS3 proteases, the destabilization of the closed conformation in the linked construct makes it an attractive tool to search for inhibitors that interfere with the formation of the enzymatically active, closed conformation.

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