A novel small molecule approach for the treatment of propionic and methylmalonic acidemias.

Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.

A transgenic mouse for imaging caspase-dependent apoptosis within the skin.

Apoptosis is an essential process for the maintenance of normal physiology. The ability to noninvasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta-galactosidase gene flanked by two estrogen receptor regulatory domains and intervening Asp-Glu-Val-Glu sequences was constructed to serve as a tool for in vivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable beta-galactosidase activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of beta-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled noninvasive measurement of beta-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose- and time-dependent manner. Using a near-infrared fluorescent substrate of beta-galactosidase (9H-{1,3-dichloro-9,9-dimethylacridin-2-one-7-yl} beta-D-galactopyranoside), noninvasive in vivo imaging of apoptosis was achieved in a xenograft tumor model in response to proapoptotic therapy. Finally, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals, especially in the context of skin biology.

Evaluation of D-methionine as a novel oral radiation protector for prevention of mucositis.

PURPOSE: Oral mucositis is a common acute morbidity associated with radiation and/or chemotherapy treatment for cancer. D-Methionine (D-Met), the dextro-isomer of the common amino acid l-methionine, has been documented to protect normal tissues from a diverse array of oxidative insults. EXPERIMENTAL DESIGN: We evaluated if D-Met could selectively prevent radiation-induced oral mucositis using in vitro cell culture models as well as an in vivo model of radiation injury to the oral mucosa in C3H mice. RESULTS: Unlike free-radical scavengers, which protected both normal and transformed tumor cells in vitro from radiation-induced cell death, treatment with d-Met in culture protected nontransformed primary human cells from radiation-induced cell death (protective factor between 1.2 and 1.6; P<0.05) whereas it did not confer a similar protection on transformed tumor cells. D-Met treatment also provided significant protection to normal human fibroblasts, but not to tumor cell lines, from radiation-induced loss of clonogenicity (protection factor, 1.6+/-0.15). D-Met treatment did not alter DNA damage (as measured by histone phosphorylation) following irradiation but seemed to selectively mitigate the loss of mitochondrial membrane potential in nontransformed cells, whereas it did not provide a similar protection to tumor cells. Tumor control of implanted xenografts treated with radiation or concurrent cisplatin and radiation was not altered by D-Met treatment. Pharmacokinetics following administration of a liquid suspension of D-Met in rats showed 68% bioavailability relative to i.v. administration. Finally, in a murine model of mucositis, a dose-dependent increase in protection was observed with the protective factor increasing from 1.6 to 2.6 over a range of oral D-Met doses between 200 and 500 mg/kg (P<0.0003). CONCLUSIONS: D-Met protected normal tissues, but not tumor cells, in culture from radiation-induced cell death; it also protected normal cells from radiation-induced mucosal injury in a murine model but did not alter tumor response to therapy. Further studies on the use of D-Met to protect from oral mucositis are warranted.

Radiosynthesis and evaluation of 5-[125I]iodoindol-3-yl-beta-D-galactopyranoside as a beta-galactosidase imaging radioligand.

The synthesis and investigation of 5-[125I]iodoindol-3-yl-beta-d-galactopyranoside ([125I]IBDG) as a radioligand for single-photon emission computed tomography (SPECT) imaging of b-galactosidase expression are described. No-carrier-added [125I]IBDG was synthesized by a radioiododestannylation approach in > 75% overall radiochemical yield and > 99% radiochemical purity. [125I]IBDG was evaluated as a substrate using beta-galactosidase-expressing (D54L) and nonexpressing (D54) human glioma cell lines. A 24-hour incubation of this substrate with cultured cells revealed a 6.5-fold greater intracellular trapping of radioactivity in D54L cells compared with D54 cells. Systemic delivery of [125I]IBDG in nude mice bearing D54L tumors failed to show significant trapping of radioactivity within these tumors by SPECT imaging. In contrast, intratumoral injection of the substrate resulted in efficient trapping of radioactivity in D54L tumors but not D54 tumors, resulting in clear SPECT visualization of the former tumor. Based on dynamic SPECT imaging and blood metabolite analysis, we conclude that although [125I]IBDG is an efficient in vivo substrate for beta-galactosidase, its rapid renal clearance hampers its intratumoral availability on systemic administration.

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.