FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing.

Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.

Multiple partners can kiss-and-run: Bax transfers between multiple membranes and permeabilizes those primed by tBid.

During apoptosis Bid and Bax are sufficient for mitochondrial outer membrane permeabilization, releasing pro-apoptotic proteins such as cytochrome c and Smac/Diablo into the cytoplasm. In most cells, both Bid and Bax are cytoplasmic but bind to mitochondrial outer membranes to exert pro-apoptotic functions. Binding to membranes is regulated by cleavage of Bid to truncated Bid (tBid), by conformation changes in tBid and Bax, and by interactions with other proteins. At least at the peripherally bound stage, binding is reversible. Therefore, regulation of apoptosis is closely linked with the interactions of tBid and Bax with mitochondria. Here we use fluorescence techniques and cell-free systems containing mitochondria or liposomes that faithfully mimic tBid/Bax-dependent membrane permeabilization to study the dynamic interactions of the proteins with membranes. We confirm that the binding of both proteins to the membrane is reversible by quantifying the binding affinity of proteins for the membrane. For Bax, both peripherally bound (inactive) and oligomerized (active) proteins migrate between membranes but much slower than and independent of tBid. When re-localized to a new membrane, Bax inserts into and permeabilizes it only if primed by an activator. In the case of tBid, the process of transfer is synergetic with Bax in the sense that tBid 'runs' faster if it has been 'kissed' by Bax. Furthermore, Mtch2 accelerates the re-localization of tBid at the mitochondria. In contrast, binding to Bcl-XL dramatically impedes tBid re-localization by lowering the off-rate threefold. Our results suggest that the transfer of activated tBid and Bax to different mitochondria is governed by dynamic equilibria and potentially contributes more than previously anticipated to the dissemination of the permeabilization signal within the cell.

Bid: a Bax-like BH3 protein.

Bid, a pro-apoptotic member of the Bcl-2 family, was initially discovered through binding to both pro-apoptotic Bax and anti-apoptotic Bcl-2. During apoptosis, Bid can be cleaved not only by caspase-8 during death receptor apoptotic signaling, but also by other caspases, granzyme B, calpains and cathepsins. Protease-cleaved Bid migrates to mitochondria where it induces permeabilization of the outer mitochondrial membrane that is dependent on the pro-apoptotic proteins Bax and/or Bak, and thus Bid acts as a sentinel for protease-mediated death signals. Although sequence analysis suggests that Bid belongs to the BH3-only subgroup of the Bcl-2 family, structural and phylogenetic analysis suggests that Bid may be more related to multi-BH region proteins such as pro-apoptotic Bax. Analysis of membrane binding by protease-cleaved Bid reveals mechanistic similarities with the membrane binding of Bax. For both proteins, membrane binding is characterized by relief of N-terminal inhibition of sequences promoting migration to membranes, insertion into the bilayer of the central hydrophobic hairpin helices and exposure of the BH3 region. These findings implicate Bid as a BH3-only protein that is both structurally and functionally related to multi-BH region Bcl-2 family proteins such as Bax.