Summary Aberrant expression ratio of Cl − transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clinical trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl − transporter NKCC2, creating critical drug compliance issues and health concerns. Here, we report the discovery of a new chemical class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiological intracellular Cl − in murine Down syndrome neuronal cultures, has excellent solubility and metabolic stability, and displays no issues with off-target activity in vitro . ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclinical/manufacturing studies toward the first sustainable therapeutics for the neurological conditions characterized by impaired Cl − homeostasis.

The detection and characterization of binding pockets and allosteric communication in proteins is crucial for studying biological regulation and performing drug design. Nowadays, ever-longer molecular dynamics (MD) simulations are routinely used to investigate the spatiotemporal evolution of proteins. Yet, there is no computational tool that can automatically detect all the pockets and potential allosteric communication networks along these extended MD simulations. Here, we use a novel and fully automated algorithm that examines pocket formation, dynamics, and allosteric communication embedded in microsecond-long MD simulations of three pharmaceutically relevant proteins, namely, PNP, A2A, and Abl kinase. This dynamic analysis uses pocket crosstalk, defined as the temporal exchange of atoms between adjacent pockets, along the MD trajectories as a fingerprint of hidden allosteric communication networks. Importantly, this study indicates that dynamic pocket crosstalk analysis provides new mechanistic understandings on allosteric communication networks, enriching the available experimental data. Thus, our results suggest the prospective use of this unprecedented dynamic analysis to characterize transient binding pockets for structure-based drug design.

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Plinabulin is a novel tubulin-binding agent that is currently in phase 3 clinical trials for cancer treatment and prevention of chemotherapy-induced neutropenia. Plinabulin binds within a distinct tubulin pocket, which differentiates it from other tubulin binders. Aimed at disclosing structural and energetic details of plinabulin binding to tubulin, we combine X-ray crystallography and computational modeling. We compare the plinabulin residence time with that of colchicine and combretastatin-A4. Our study helps understand potential mechanisms underlying differential effects of this family of anti-tubulin drugs.

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A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.

Fyn is a member of the Src-family of nonreceptor protein-tyrosine kinases. Its abnormal activity has been shown to be related to various human cancers as well as to severe pathologies, such as Alzheimer's and Parkinson's diseases. Herein, a structure-based drug design protocol was employed aimed at identifying novel Fyn inhibitors. Two hits from commercial sources (1, 2) were found active against Fyn with K(i) of about 2 μM, while derivative 4a, derived from our internal library, showed a K(i) of 0.9 μM. A hit-to-lead optimization effort was then initiated on derivative 4a to improve its potency. Slightly modifications rapidly determine an increase in the binding affinity, with the best inhibitors 4c and 4d having K(i)s of 70 and 95 nM, respectively. Both compounds were found able to inhibit the phosphorylation of the protein Tau in an Alzheimer's model cell line and showed antiproliferative activities against different cancer cell lines.

Bruton’s tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.