Efficient Crystallization of Apo Sirt2 for Small-Molecule Soaking and Structural Analysis of Ligand Interactions
J. Med. Chem. 2024, 67, 15, 12461–12462
Sirtuin proteins (Silent Information Regulators, Sirt1-7) are NAD+-dependent enzymes that have a range of important functions as metabolic regulators, depending on their subcellular localisation. This family of enzymes is relevant to the discovery of molecular drivers of aging and may provide useful targets for the pharmacological reversal/retardation of the same. Sirt2 resides primarily in the cytoplasm and has de-acetylase activity across a wide substrate scope. Activation of Sirt2 in-vitro has increased the longevity of yeast cells.
Sirt2 is “structurally enabled”, meaning that it is possible to obtain 3D structural information about the target with- and without ligands bound thanks to X-Ray Crystallography or other methods. Classically, to obtain a ligand-bound crystal structure, the ligand and target protein are co-crystallised. This is a robust method for obtaining structures, but is time consuming and can be hit-and-miss due to the need for a new crystal system each time. Another approach to obtaining ligand-bound structures is soaking, where solutions of the ligand are soaked into pre-formed crystals of the target protein. Soaking is much more amenable to high-throughput experimentation, provided suitable crystals are available.
Accessing soakable crystal systems of a target protein can be a game-changing moment in a drug discovery campaign. It provides a foothold in the high-throughput 3D structural analysis of test compounds bound to the target. This facilitates a deeper, molecular understanding of compound potency which could be significant for the development of not only binders/inhibitors, but activators, stabilisers, degraders and other means of interacting with natural catalytic cycles. Soaking is also an excellent method for the structural analysis of weakly-binding ligands (such as screening hits) that are not easy to co-crystallise with the target, this has the potential to massively accelerate the hit-to-lead stage of discovery.
In this paper, the authors articulate the challenges found with existing soaking methods for obtaining ligand-bound Sirt2 structures. Specifically, existing soakable Sirt2-ADPR crystals present unfavourable crystal contacts that partially block the ligand entry channel, making this system unsuitable as a broad Sirt2 soaking platform. They employed an inhibitor compound: SirReal2, which binds in a cryptic pocket (“selectivity pocket”) that isn’t seen in previously solved apo (empty) structures, to produce Sirt2-SirReal2 co-crystals. These are then used as seeds to obtain apo crystals of Sirt2 without the template compound bound.
With their Sirt2 soakable crystal system in hand, they screened a fragment library against Sirt2 in a fluorescence polarisation assay and soaked all the hits. Numerous new Sirt2 structures were solved and deposited into the pdb, including Sirt2-fragment-NAD+ tricomplexes, providing fresh insight into the binding of ligands to Sirt2.
This paper was published as part of a special issue of J. Med. Chem., focusing on structural biology in drug discovery. See the editorial at the following link:
https://doi.org/10.1021/acs.jmedchem.4c01492