The structures of salt-inducible kinase 3 in complex with inhibitors reveal determinants for binding and selectivity.

The salt-inducible kinases (SIKs) 1 to 3, belonging to the AMPK-related kinase family, serve as master regulators orchestrating a diverse set of physiological processes such as metabolism, bone formation, immune response, oncogenesis, and cardiac rhythm. Owing to its key regulatory role, the SIK kinases have emerged as compelling targets for pharmacological intervention across a diverse set of indications. Therefore, there is interest in developing SIK inhibitors with defined selectivity profiles both to further dissect the downstream biology and for treating disease. However, despite a large pharmaceutical interest in the SIKs, experimental structures of SIK kinases are scarce. This is likely due to the challenges associated with the generation of proteins suitable for structural studies. By adopting a rational approach to construct design and protein purification, we successfully crystallized and subsequently solved the structure of SIK3 in complex with HG-9-91-01, a potent SIK inhibitor. To enable further SIK3-inhibitor complex structures we identified an antibody fragment that facilitated crystallization and enabled a robust protocol suitable for structure-based drug design. The structures reveal SIK3 in an active conformation, where the ubiquitin-associated domain is shown to provide further stabilization to this active conformation. We present four pharmacologically relevant and distinct SIK3-inhibitor complexes. These detail the key interaction for each ligand and reveal how different regions of the ATP site are engaged by the different inhibitors to achieve high affinity. Notably, the structure of SIK3 in complex with a SIK3 specific inhibitor offers insights into isoform selectivity.

A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.