Allan Joaquim Lamontanara

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Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

Matteo Dal Peraro, Sandrine Madeleine Suzanne Georgeon, Oliver Hantschel, Tim Kükenshöner, Grégory La Sala, Allan Joaquim Lamontanara, Maria Josefina Marcaida Lopez, Daniel Pereira Duarte, Florence Pojer

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. Constitutive Btk signaling drives several B-cell cancers. Here the authors demonstrate key allosteric intramolecular interactions between the SH2 domain and the kinase domain of Btk, and propose an alternative approach for inhibition of both wild-type and tyrosine kinase inhibitor-resistant Btk.

NATURE PUBLISHING GROUP2020

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface

Barbara Gerig, Oliver Hantschel, Allan Joaquim Lamontanara

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

American Society for Biochemistry and Molecular Biology2016

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

ATP Site Ligands Determine the Assembly State of the Abelson Kinase Regulatory Core via the Activation Loop Conformation

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

ATP Site Ligands Determine the Assembly State of the Abelson Kinase Regulatory Core via the Activation Loop Conformation