Anti-cancer Drug Development - Structural Biology
Carfilzomib is an anti-cancer drug developed by Onyx Pharmaceuticals, now a subsidiary of Amgen. It is sold under the brand name of Kyprolis. In July 2012, carfilzomib was approved by the United States Food and Drug Administration (FDA) for the treatment of multiple myeloma. Its target in the cell is the proteasome which is a large macromolecular complex containing multiple proteases.
Proteases are a group of enzymes that break the long chainlike molecules of proteins into shorter fragments called peptides. Early view considered the protease essentially as a destructive enzyme involved in protein catabolism and generation of amino acids in an organism. Nevertheless, extensive scientific investigations over more than a century have established their meaningful proteolytic role in the control of multiple biological processes in all living organisms.
One of the ways proteases are activated is through the formation of large protein complexes, such as the proteasome. and compartmentalization of the proteolytic subunits. Multiple proteasome forms are usually present in a single cell type. The various forms differ in their substrate preferences as well as proteolytic products. Nevertheless, all proteasome forms are built on a common 20S core particle (20S is its Svedberg sedimentation coefficient).
The 20S core particle (CP) is about 750 kDa; its overall architecture is defined by four coaxially stacked heteroheptameric rings. Each of the two outer rings is composed of seven a subunits (a1-7), whereas each of the inner two is formed by seven b subunits (b1-7). The proteolytic activity of the complex is due to three b subunits – b1, b2, and b5 – present in each of the inner rings. The three catalytically active subunits have distinct cleavage specificities. Based on the target amino acid residues, b1 has been referred to as caspase-like, b2 as tryptic-like while b5 as chymotryptic-like.
Proteasome as drug target
Whereas the constitutive proteosome (cCP) is present in most normal tissues, the major proteosome form in antigen-presenting cell in the immune system is the immunoproteosome (iCP). The corresponding catalytic subunits in iCP are b1i, b2i, and b5i, each of which differs from its constitutive counterpart by a few crucial amino acid residues.
The highly regulated ubiquitin/proteasome pathway (UPP) is responsible for the degradation of approximately 80% of intracellular proteins. Ubiquitin is a small regulatory protein found “ubiquitously” in most tissues of eukaryotic organisms. The addition of ubiquitin to a substrate protein, ubiquitylation, marks the substrate for degradation by the proteasome.
Uncontrolled gene transcription in cancer cells leads to overproduction of proteins, particularly misfolded and mutated proteins – such malformed proteins must be degraded for cell survival. Therefore, cancer cells are heavily dependent on UPP and this makes them more vulnerable to proteosome inhibition compared to normal cells. Quite reasonably, proteosome has been considered to be a potent therapeutic target.
Carfilzomib – second-generation proteasome inhibitor
Bortezomib has been the first drug approved by the FDA for the treatment of multiple myeloma. Although it is a potent proteasome inhibitor, its significant toxicity has restricted its usage. Subsequently, carfilzomib was developed as a second-generation proteasome inhibitor with improved efficacy and safety profiles.
The improved efficacy and safety profile of carfilzomib is due to its remarkable specificity towards the proteosome’s chymotrypsin-like activity and the speedy extrahepatic clearance of its free form. It interacts effectively with b5 and b5i subunits of the proteasome and immunoproteasome respectively, and not with other subunits. Furthermore, it shows minimum effect to non-proteasome substrates. The inhibition of other proteasome and/or non-proteasome substrates could have been detrimental to normal cells.
Proteasome inhibition is dependent on specificity pockets (S) and an N-terminal threonine in its substrate binding channel. Most proteasome inhibitors contain peptide-like side chains (P sites) whose interaction with the S pockets determine subunit selectivity.
Carfilzomib is a tetrapeptide with four side chains (P1-4). The specificity for the catalytic subunit b5 is determined by van der Waals interactions of its hydrophobic P1 leucyl group in the S1 pocket. The side chains of amino acid residues Ala20, Val31, Met45 and Ala49 are involved in these interactions.
Although carfilzomib has a much-improved safety profile in comparison with bortezomib, yet it is associated with some cardiovascular toxicity and renal toxicity. These side-effects pose a challenge to the treatment of multiple myeloma requiring careful optimization of carfilzomib treatment.
References:
Cromm, P. M. and Crews, C. M. 2017. The proteasome in modern drug discovery: second life of a highly valuable drug target. ACS Central Science 3:830-838.
Harshbarger, W. et al. 2015. Crystal structure of the human 20S proteasome in complex with carfilzomib. Structure 23:418-424.
* For the basic concept of van der Waals interactions the readers are referred to Fundamentals of Molecular Structural Biology (Chapter 4, Elsevier publication, 2019)