Development of peptide epoxyketone-based proteasome inhibitors

SY18-01 The proteasome is a multicatalytic protease complex that is responsible for the ubiquitin-dependent turnover of many cellular proteins including key components of signaling cascades that regulate cell proliferation and survival pathways. The catalytic core of the proteasome consists of four heptameric rings made up of distinct α- and β-subunits. Three of the seven β-subunits are catalytically active and are commonly described by their substrate selectivities: chymotrypsin-like - β5 subunit; trypsin-like - β2 subunit; and post-acidic or caspase-like - β1 subunit. Each proteasome active site utilizes the side-chain hydroxyl group of an N-terminal threonine as the catalytic nucleophile, a mechanism that distinguishes the proteasome from other cellular proteases. Clinical validation of the proteasome as a therapeutic target in oncology has been provided by the dipeptide boronic acid bortezomib (also known as PS-341 or VelcadeTM), a proteasome inhibitor that has proven efficacious as a single agent in multiple myeloma and non-Hodgkin’s lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing to a recommended twice weekly schedule (Day1/Day4), a schedule that allows full proteasome recovery between doses. To further evaluate and potentially expand upon the clinical utility of proteasome inhibitors, we have generated a series of novel inhibitors related to the natural product epoxomicin. First identified based on its in vivo anti-tumor activity, epoxomicin was subsequently shown to be a potent and selective inhibitor of the proteasome. Structurally, epoxomicin is comprised of two key elements: a peptide portion that selectively binds in the substrate binding pocket(s) of the proteasome with high affinity and an epoxyketone pharmacophore that stereospecifically interacts with the catalytic threonine residue to inhibit enzyme activity. X-ray crystallography has demonstrated that epoxyketone pharmacophore forms a dual covalent morpholino adduct with the proteasome that requires the close juxtaposition of both the side-chain hydroxyl and α-amino groups of the active site threonine residue. This unique mechanism imparts a high degree of specificity to the proteasome relative to the active sites of cysteine, metallo, serine, or aspartyl proteases. PR-171, a peptide epoxyketone-based proteasome inhibitor that we have developed, has recently entered clinical trials. Distinctions between PR-171 and bortezomib include their mechanisms of action (irreversible for PR-171 vs. slowly reversible for bortezomib) and target selectivity (greater with PR-171 both for the chymotrypsin-like active site within the proteasome and across other protease classes). In preclinical animal studies, intravenous administration of PR-171 results in dose-dependent inhibition of the chymotrypsin-like proteasome activity in all tissues examined with the exception of brain. Despite its irreversible mechanism of action, proteasome activity recovers in all tissues except whole blood with a half-life of approximately 24 hr following PR-171 treatment, presumably due to de novo proteasome synthesis. Importantly, daily doses of PR-171 that achieve >80% proteasome inhibition in blood and most tissues are well tolerated (for up to five consecutive days, QDx5). Furthermore, dosing of PR-171 twice weekly on two consecutive days (QDx2) elicits an improved anti-tumor response in human tumor xenograft models compared to either once weekly or twice weekly (Day1/Day4) schedules. These preclinical studies demonstrate the tolerability and efficacy of PR-171 delivered on repetitive schedules that preclude full proteasome recovery between doses. Supported by these results, two dose-intensive Phase I clinical trials have been initiated. Both are dose-escalation studies aimed at determining the safety, tolerability, and clinical response to PR-171 in patients with hematological malignancies. The two dosing schedules being tested are: i) QDx5 with nine days rest (two week cycle), and ii) QDx2 weekly for three weeks with 12 days rest (4 week cycle). Thus far, PR-171 has been found to be well tolerated in patients at doses that suppress chymotrypsin-like proteasome activity by >80% in whole blood. Furthermore, partial responses in multiple myeloma patients have been observed on both schedules, as measured by reductions in malignant plasma cell-derived monoclonal protein levels. To further explore the versatility of the epoxyketone pharmacophore, medicinal chemistry and preclinical pharmacology efforts have focused on the development of proteasome inhibitors with distinct biochemical and pharmacological properties. We have identified orally-bioavailable peptide epoxyketone proteasome inhibitors that are both well tolerated on multi-dose schedules and efficacious in human tumor xenograft models. Such inhibitors promise greater dosing flexibility and patient convenience. In addition, peptide epoxyketone analogs have been generated with varying levels of selectivity among the different proteasome active sites. A total of six catalytically active proteasome subunits have been described: the three β-subunits noted above within the ubiquitously-expressed or “constitutive” form of the proteasome and three distinct but highly homologous β-subunits within a form of the proteasome known as the immunoproteasome. The immunoproteasome is expressed primarily by normal and transformed cells of hematopoietic origin and contributes to class I major histocompatibility complex restricted antigen processing. We have identified inhibitors that exhibit selectivity for individual subunits of the constitutive and immunoproteasomes as well as inhibitors that display activity across multiple proteasome active sites. This collection of inhibitors provides a valuable tool for dissecting the relative contribution of the different active sites to proteasome functions. For example, we have found that inhibitors selective for the chymotrypsin-like active sites of either the constitutive proteasome (β5) or the immunoproteasome (LMP7) have minimal impact on the viability of hematologic tumor cell lines that express predominantly (>70%) immunoproteasome. However, in combination, β5- and LMP7-selective inhibitors exhibit strong synergy suggesting that both forms of the proteasome contribute to tumor cell viability. Further in vitro and in vivo evaluation of peptide epoxyketone proteasome inhibitors with distinct active site selectivities will establish their potential as therapeutic agents.