Date Published: November 01, 2019
Publisher: International Union of Crystallography
Author(s): Jakub Luptak, Michal Bista, David Fisher, Liz Flavell, Ning Gao, Kate Wickson, Steven L. Kazmirski, Tina Howard, Philip B. Rawlins, David Hargreaves.
Mcl-1 is an important cancer target for drug therapy, through which normal apoptosis may be restored by inhibiting its protective function. An scFv and a Fab have been used to generate an apo Mcl-1 crystal system that is amenable to iterative structure-guided drug design.
Mcl-1 (myeloid cell leukaemia 1) is a member of the Bcl-2 (B-cell lymphoma 2) family of pro-survival proteins that regulate programmed cell death via apoptosis (Elmore, 2007 ▸). It comprises a C-terminal transmembrane helix anchor by which it associates with the mitochondrial membrane, an eight-helix bundle responsible for signalling via protein–protein interaction and a mostly disordered N-terminal region that includes a PEST-like domain (Day et al., 2004 ▸). Intrinsic or extrinsic cell signals are received through a complex cascade which eventually affect the balance of pro- and anti-apoptotic protein complexes at the mitochondrial membrane. The final effect is the permeation of the mitochondrial membrane, with the release of cytochrome c into the cytoplasm, resulting in cell death. Mcl-1 is an anti-apoptotic member of the Bcl-2 family which binds to pro-apoptotic proteins containing the BH3 domain, a short α-helical stretch of around 16 residues. Binding of Mcl-1 to the pro-apoptotic proteins prevents apoptosis. Normally, this process is finely regulated, but in certain types of cancers this regulation breaks down. A therapeutic approach has been pursued to bind and inhibit Mcl-1, promoting apoptosis in diseased tissues (Lee et al., 2008 ▸; Bruncko et al., 2015 ▸; Kotschy et al., 2016 ▸; Johannes et al., 2017 ▸; Tron et al., 2018 ▸).
Mcl-1 is an important oncology drug target but is only crystallizable with peptides or small molecules, as a fusion protein or, as described here, with antibody fragments. In recent years there has been increasing interest in using antibody fragments to enable the crystallization of structurally intractable targets (Griffin & Lawson, 2011 ▸). Over the last eight years AstraZeneca has made considerable efforts to facilitate a number of drug-target crystal systems using antibodies. Although all of the diverse targets pursued have resulted in scFvs or Fabs that appear to have the correct properties for crystallization, most have failed to produce crystals. The low success rate is most likely to be a product of the technique being used as a rescue strategy when all other attempts at crystallization had failed. In this light, a low success rate is an acceptable consequence.