Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrowsparing compound, exhibited a submicromolar IC 50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in activesite CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma.

A human colorectal cancer chip recapitulates aspects of CRC biology On-chip imaging and metabolomic effluent analyses offer insight into progression Organ-on-chip conditions induce phenotypic heterogeneity compared to 2D cultures Stromal cell cross talk and mechanical forces increase tumor cell intravasation

f CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and ␤-lactamase inhibitors in strains carrying bla CMY , novel ␤-lactam-␤-lactamase inhibitor combinations are sought to combat this significant threat to ␤-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-␤-lactam ␤-lactamase inhibitor in clinical development that reversibly inactivates serine ␤-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry bla CMY-2 or bla CMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC 90 , <0.5 mg/liter); in comparison, ceftazidime had a MIC 90 of >128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 ␤-lactamase, as demonstrated by a second-order onset of acylation rate constant (k 2 /K) of (4.9 ؎ 0.5) ؋ 10 4 M ؊1 s ؊1 and the off-rate constant (k off ) of (3.7 ؎ 0.4) ؋10 ؊4 s ؊1 . Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2-avibactam acylenzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69. Biochemical studies of avibactam have shown that this inhibitor inactivates ␤-lactamases with second-order acylation rate constants (k 2 /K) ranging from 1,400 to 160,000 M Ϫ1 s Ϫ1 (1-6). The highest acylation rates were those for class A enzymes, while the lowest were those for class D ␤-lactamases. Dissociation rate constants (k off ) ranged from (1.9 Ϯ 0.6) ϫ 10 Ϫ3 to (1.2 Ϯ 0.4) ϫ 10 Ϫ5 s Ϫ1 and were lowest for class D ␤-lactamases (6). Most importantly, avibactam combined with either ceftazidime, ceftaroline (the active metabolite of ceftaroline fosamil), or ceftarolinefosamil was effective in murine models of infection due to highly resistant extended-spectrum ␤-lactamase (ESBL)-producing, non-ESBL-producing, and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates (7-10).CMY-2 ␤-lactamase is the most prevalent and widely disseminated plasmid-borne cephalosporinase (11, 12). As such, Escherichia coli strains possessing bla CMY pose one of the most important challenges to the efficacy of ␤-lactam therapy in both community-and hospital-acquired infections. Inhibition studies with CMY-2 and tazobactam, for example, were important in helping to understand the path to the inhibition of...

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2′-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy.

Parkinson's disease (PD) is a movement disorder characterized by neuroinflammation, α-synuclein pathology, and neurodegeneration. Most cases of PD are non-hereditary, suggesting a strong role for environmental factors, and it has been speculated that disease may originate in peripheral tissues such as the gastrointestinal (GI) tract before affecting the brain. The gut microbiome is altered in PD and may impact motor and GI symptoms as indicated by animal studies, though mechanisms of gut-brain interactions remain incompletely defined. Intestinal bacteria ferment dietary fibers into short-chain fatty acids, with fecal levels of these molecules differing between PD and healthy controls and in mouse models. Among other effects, dietary microbial metabolites can modulate activation of microglia, brain-resident immune cells implicated in PD. We therefore investigated whether a fiber-rich diet influences microglial function in α-synuclein overexpressing (ASO) mice, a preclinical model with PD-like symptoms and pathology. Feeding a prebiotic high-fiber diet attenuates motor deficits and reduces α-synuclein aggregation in the substantia nigra of mice. Concomitantly, the gut microbiome of ASO mice adopts a profile correlated with health upon prebiotic treatment, which also reduces microglial activation. Single-cell RNA-seq analysis of microglia from the substantia nigra and striatum uncovers increased pro-inflammatory signaling and reduced homeostatic responses in ASO mice compared to wild-type counterparts on standard diets. However, prebiotic feeding reverses pathogenic microglial states in ASO mice and promotes expansion of protective disease-associated macrophage (DAM) subsets of microglia. Notably, depletion of microglia using a CSF1R inhibitor eliminates the beneficial effects of prebiotics by restoring motor deficits to ASO mice despite feeding a prebiotic diet. These studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.