Comparative Mechanisms of Action of Proteasome Inhibitors
Comparative Mechanisms of Action of Proteasome Inhibitors
The proteasome has emerged as an important target for therapeutic intervention. In preclinical studies, proteasome inhibitors (PIs) induced apoptosis and inhibited tumor growth, supporting their potential role in the treatment of various tumor types, especially hematologic malignancies. Bortezomib (Velcade), the first clinically validated PI, reversibly binds to the chymotrypsin-like (ChT-L) active sites in the 20S proteasome and potently inhibits cell growth and proliferation in human tumor cell lines and in multiple myeloma (MM) and mantle cell lymphoma. However, the adverse event profile and intravenous administration of bortezomib have underscored the need for the development of PIs with selective actions on different proteasome subunits, which would have different binding kinetics and routes of administration. The most advanced next-generation PI is carfilzomib, an epoxyketone that differs structurally and mechanistically from bortezomib. In preclinical studies, carfilzomib demonstrates sustained inhibition of proteasomal ChT-L activity and greater selectivity than bortezomib. It is thought that the selectivity of carfilzomib for the β5 subunit contributes to its greater cytotoxic response and improved tolerability profile relative to bortezomib. Furthermore, in preclinical studies, carfilzomib did not exhibit the same magnitude of off-target activity against non-proteasomal proteases that is observed with bortezomib. Variations in the binding profiles of some of the next-generation PIs may translate into key differences in pharmacokinetic and toxicity profiles, and thus may be clinically relevant in the treatment of MM.
The proteasome is an important therapeutic target in the treatment of a small but increasing number of diseases— most notably, B cell malignancies. Multiple myeloma (MM) is one of the first diseases for which proteasome inhibitors (PIs) have been validated and in which they have found widespread use. Following the launch of bortezomib (Velcade) in 2003, other PIs have been developed that have different chemical structures and proteasome-binding profiles. This article reviews the biology of the proteasome and its involvement in MM and compares the physicochemical profiles of various PIs, examining how specific differences in their mechanisms of action and binding characteristics may affect their clinical profiles.
Proteasome: Biology and Normal Function
The ubiquitin proteasome system (UPS) plays a key role in maintaining normal cellular homeostasis in eukaryotic cells. The majority of intracellular proteins are regulated by ubiquitin-mediated proteolysis, including those controlling cell-cycle progression, apoptosis, DNA repair, and the stress response.[2,3] A key step in this process is the "tagging" of proteins targeted for degradation with multiple molecules of ubiquitin, a 76–amino acid protein whose primary sequence is highly conserved in organisms ranging from yeast to mammals. Once polyubiquitinated, proteins targeted for degradation bind to the 26S proteasome.
The 26S eukaryotic proteasome is a large ATP-dependent complex consisting of a barrel-shaped 20S proteolytic core, the ends of which are covered by two 19S regulatory cap complexes (Figure 1).[5-8] The 19S regulatory complex is an additional feature of the eukaryotic proteasome consisting of a lid and a base that binds to the 20S particle to form the 26S proteasome holoenzyme. The 19S regulatory complex opens a gated channel into the 20S proteasome core, access to which is blocked in the free 20S proteasome. While the lid recognizes ubiquitinated protein substrates with high fidelity, the base (which caps the end of the 20S proteasome core) unfolds protein substrates and threads them into the catalytic chamber of the 20S particle. The structure of the 20S core is well conserved in virtually all organisms ranging from archaebacteria and yeast to humans; it is essentially a hollow barrel shape consisting of four stacked heptameric rings. The two outer rings, composed of α-subunits (α1 through α7), and two inner rings, composed of β-subunits (β1 through β7), form a central chamber within the stack that runs from top to bottom.
Unlike typical proteases, the 20S proteasome in eukaryotic cells exhibits multiple proteolytic activities, resulting in cleavage of protein targets at multiple sites. Distinct β-subunits are associated with the specific catalytic activities of the proteasome, which can be characterized as chymotrypsin-like (ChT-L; β5), trypsin-like (T-L; β2), and peptidylglutamyl-peptide hydrolyzing (also known as caspase-like [C-L; β1]).[9,12] All three types of activities contribute significantly to proteolysis, although their relative importance varies widely according to the specific primary amino acid sequence of the protein substrate. Of the three catalytically active β-subunits, particular interest has focused on the ChT-L activity of the β5 subunit. Increased levels of ChT-L activity and the resultant protein breakdown have been implicated in various disease states, including malignancies. Inhibition of ChT-L activity results in accumulation of ubiquitin-protein conjugates and induction of apoptosis, particularly in transformed cell lines. Two major isoforms of the proteasome include the constitutive form, which is present in most cells, and the immunoproteasome, which is primarily expressed in cells of lymphoid origin.
The Proteasome as a Therapeutic Target in Multiple Myeloma
Because of its involvement in the regulation of key functions necessary for cell growth and viability in both normal and transformed cells, protein degradation appeared to be an unlikely and somewhat counterintuitive target for pharmaceutical intervention. Nonetheless, there are a number of reasons why the proteasome presents such an important target for therapeutic intervention. The UPS controls the abundance and activity of oncogene and tumor suppressor gene products, transcription factors, and other signaling molecules, such as nuclear factor kappa B (NFκB) and the tumor suppressor protein p53, and it has been shown to play an important role in the formation of metastases. Cancer cells generally have higher levels of proteasome activity compared with normal well-differentiated cells—possibly due to higher levels of oxidative stress and the secretion of cytokines or growth factors—making the proteasome a potential therapeutic target in oncology. Preclinical research with PIs demonstrated their ability to induce apoptosis and inhibit tumor growth, supporting the potential role of PIs in the treatment of various tumor types, especially hematologic malignancies.[7,14,15]
Chemical Classes of Proteasome Inhibitors
Because of evidence demonstrating the key role of proteasomes in regulating a variety of critical proliferative pathways in transformed cells, both synthetic and naturally derived PIs have been intensely pursued as therapeutic agents. PIs may have varying specificities, affinities, and avidities for the different catalytic sites within the proteasome core and can be broadly classified into three distinct categories based on their chemical structures (Figure 2): boronates, epoxyketones, and lactams (cyclic amides). The synthetic PIs include a peptide moiety with a reactive pharmacophore group (eg, boronate, aldehyde).
Bortezomib was the first clinically validated boronate-based dipeptide PI approved for use in cancer—specifically, relapsed/refractory MM (R/R MM). Bortezomib was approved in the United States in 2005 for the treatment of patients with MM who have received at least one prior therapy, and in 2008 for frontline treatment of patients with MM in combination with melphalan and prednisone. Two other boronate-based analogs (CEP-18770 and MLN9708) are currently in the early phases of clinical development.[5,9,16]
The synthetic epoxyketone inhibitors are related to the natural product epoxomicin and include carfilzomib (previously PR-171; currently in phase III clinical trials in MM) and its orally bioavailable analog ONX 0912 (formerly PR-047). Carfilzomib is structurally similar to epoxomicin but differs structurally and mechanistically from the boronate-based PI bortezomib.
Lactacystin is a Streptomyces lactacystinaeus metabolite that was discovered because of its ability to induce neurite outgrowth in a murine neuroblastoma cell line. It was subsequently shown to target the 20S proteasome via irreversible modification of the N-terminal threonine of β-subunits. The naturally occurring lactacystin analog salinosporamide A (NPI-0052; marizomib), a β-lactone derived from the marine bacteria Salinospora tropica, is currently in phase I clinical development for treatment of various hematologic malignancies and solid tumors.