Researchers say they have identified a new class of protein disulfide isomerase (PDI) inhibitors that sensitize multiple myeloma (MM) cells to proteasome inhibitors (PIs).
The investigators screened approximately 20,000 compounds spanning multiple chemical libraries and found the compound E61 to be a “striking hit,” with a six-fold increase in bortezomib cytotoxicity and the ability to re-sensitize PI activity at low micromolar concentrations.
The researchers then synthesized and evaluated 150 E61 derivatives and discovered the lead candidate, E64FC26, which was highly synergistic with PIs at concentrations as low as 200 nM.
They reported that E64FC26 has “several advantages over previously reported PDI inhibitors, including superior potency and a pan-style mode of inhibition.”
“PDI is an attractive target in oncology, but good PDI inhibitors have been hard to find,” said Nathan G. Dolloff, PhD, of the Medical University of South Carolina (MUSC) in Charleston.
“The compounds we discovered have a lot of advantages, including high potency and good drug-like properties. We hope that those strengths translate into an effective new drug that can ultimately help patients.”
Dr. Dolloff and his colleagues reported their discovery in Leukemia.
The investigators detected the synergistic effects of E61 in combination with next-generation PIs, including carfilzomib, ixazomib, and oprozomib in both PI-sensitive and -resistant MM cell lines.
On the other hand, E61 had no effect on dexamethasone activity in dexamethasone-resistant cells. And E61 did not affect lenalidomide or doxorubicin cytotoxicity in PI-resistant cells.
The researchers also determined that E61 was only active in MM cells. E61 did not enhance the cytotoxic effects of PIs in normal cells.
This selective toxicity suggests that E61 may have “a wide therapeutic index in vivo,” the investigators wrote.
In vivo activity
To investigate the anti-MM activity and tolerability of E61 in vivo, the researchers treated a NOD-SCID IL2Rγ−/− mouse model with E61 at a continuous dose of 50 mg/kg/day.
E61 prolonged survival by 11 days in the treated mice (P=0.0007), and four of the 11 treated mice survived to the experiment’s end.
In another experiment, two of eight mice achieved a complete response.
After continuous dosing for 40 or more days, E61 was well tolerated, the investigators reported. The mice showed no overt signs of distress and did not lose weight.
Molecular target of E61
Using click (Cu(I)-catalyzed azide-alkyne cycloaddition) chemistry and a proteomics approach, the researchers then confirmed that PDI family members are the molecular target of E61.
Functional studies indicated that E61 inhibited PDI reductase activity in vitro. E61 also enhanced the accumulation of ubiquitinylated proteins and produced strong endoplasmic reticulum (ER) and oxidative stress responses when combined with PIs.
Anti-MM activity of E64FC26
The investigators used a structure activity relationship program to narrow the candidate molecules down to E64FC26.
E64FC26 demonstrated pan-inhibition in that it inhibited all members of the PDI family tested, including PDIA3, PDIA4, TXNDC5, and PDIA6.
E64FC26 also had greater in vitro potency against PDIA1 and the other PDI isoforms. It was the only compound to sensitize MM cells to PIs, with an average increase in PI sensitivity ranging from six- to seven-fold.
The researchers also noted that E64FC26 was superior to other PDI inhibitors they tested in activating ER stress.
The investigators tested the activity of E64FC26 in vivo using Vk*MYC transgenic mice, a model that closely resembles human MM.
Mice treated with E64FC26 had an immediate anti-MM response. Serum M-protein decreased in all mice by an average of 33 ± 7.9% (P=0.0135).
The investigators observed similar effects in a human xenotransplant MM model.
These mice were randomized to receive treatment with vehicle, E64FC26 (2 mg/kg for 3 days/week), bortezomib (0.25 mg/kg for 2 days/week), or a combination of the two agents.
E64FC26 increased median survival by 2 weeks compared with vehicle-treated mice (P<0.0001). By day 36, no vehicle-treated mouse survived, compared with 100% of the E64FC26-treated mice.
Single-agent bortezomib increased survival by 6 days (P=0.0007).
And the combination produced the greatest improvement in median survival, increasing it by 20 days (P<0.0001).
The investigators reported no overt toxicity or body weight fluctuation for mice treated with E64FC26 or the combination.
“These results provide preclinical proof of concept for the strategy of targeting PDI with this new class of compound for the treatment of MM,” the researchers concluded.
“One of the strengths of this study is that we spanned almost the entire drug discovery process,” Dr. Dolloff said. “We screened thousands of compounds, found an exciting molecule, deconvoluted what its binding target was, synthesized hundreds of derivatives to make it better, and then conducted animal studies.”
“The study has everything from biochemistry and cell biology to medicinal chemistry and animal pharmacology in it. There is still a lot of work to be done before this drug is ready for clinical trials in humans, but it has been a rewarding project, and I’m looking forward to the next steps.”
Dr. Dolloff is founder of Leukogene Therapeutics, Inc., which has licensed patents from MUSC, and a second study author is an inventor on patents. The other authors declared no conflicts of interest.
The research was supported by the National Institutes of Health/National Cancer Institute, the South Carolina Clinical & Translational Research Institute, the MUSC Hollings Cancer Center, and by the Hollings Cancer Center T32 Ruth L. Kirschstein National Research Service Award Training Program.