A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC_(50) value of 0.25 μM. It was confirmed that APPB inhibits N-glycosylation of β-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC_(50) > 200 μM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, 60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay

Mapping isoprene emissions over North America using formaldehyde column observations from space

We present a methodology for deriving emissions of volatile organic compounds (VOC) using space-based column observations of formaldehyde (HCHO) and apply it to data from the Global Ozone Monitoring Experiment (GOME) satellite instrument over North America during July 1996. The HCHO column is related to local VOC emissions, with a spatial smearing that increases with the VOC lifetime. Isoprene is the dominant HCHO precursor over North America in summer, and its lifetime (≃1 hour) is sufficiently short that the smearing can be neglected. We use the Goddard Earth Observing System global 3-D model of tropospheric chemistry (GEOS-CHEM) to derive the relationship between isoprene emissions and HCHO columns over North America and use these relationships to convert the GOME HCHO columns to isoprene emissions. We also use the GEOS-CHEM model as an intermediary to validate the GOME HCHO column measurements by comparison with in situ observations. The GEOS-CHEM model including the Global Emissions Inventory Activity (GEIA) isoprene emission inventory provides a good simulation of both the GOME data (r2 = 0.69, n = 756, bias = +11%) and the in situ summertime HCHO measurements over North America (r2 = 0.47, n = 10, bias = −3%). The GOME observations show high values over regions of known high isoprene emissions and a day-to-day variability that is consistent with the temperature dependence of isoprene emission. Isoprene emissions inferred from the GOME data are 20% less than GEIA on average over North America and twice those from the U.S. EPA Biogenic Emissions Inventory System (BEIS2) inventory. The GOME isoprene inventory when implemented in the GEOS-CHEM model provides a better simulation of the HCHO in situ measurements than either GEIA or BEIS2 (r2 = 0.71, n = 10, bias = −10%)

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration

Gait Balance Disorder by Thalamic Infarction with the Disorder of Interstitial Nucleus of Cajal

The interstitial nucleus of Cajal (INC) is thought to play an important role in torsional/vertical eye position and head posture, and disorders of the INC induce abnormal ocular movements and head tilt. Our patients with ocular tilt reactions simultaneously also had disturbances in ambulatory balance, yet no reports address the loss of balance control induced by disorders of the INC. We examined the ambulatory disturbances induced by INC lesion. We experienced three patients with ocular movement disorders and abnormal head tilt due to thalamic infarction. We performed ophthalmic examinations on and checked the balance of them. With funduscopy, abnormal cycloduction was seen in the unaffected side and normal cycloduction was observed in the affected side. Nevertheless, Hess charts showed distortions in the visual image of both eyes. They all had disorders of balance control. We tried to treat them using the Bobath approach for improving their ambulatory balance. With subsequent improvements in balance control it was possible for them to take short walks, but it was difficult to make any improvements in their ocular movement. The INC is related to balance control of ambulation and disorders of the INC induce ambulatory disturbances. Cycloduction was only observed in the unaffected side, but Hess charts showed distortions of the visual image in both eyes. Ambulation was briefly improved, but diplopia persisted in these patients

Characteristics of tropospheric ozone depletion events in the Arctic spring: analysis of the ARCTAS, ARCPAC, and ARCIONS measurements and satellite BrO observations

Arctic ozone depletion events (ODEs) are caused by halogen catalyzed ozone loss. In situ chemistry, advection of ozone-poor air mass, and vertical mixing in the lower troposphere are important factors affecting ODEs. To better characterize the ODEs, we analyze the combined set of surface, ozonesonde, and aircraft in situ measurements of ozone and bromine compounds during the Arctic Research of the Composition of the Troposphere from Aircraft and Satellites (ARCTAS), the Aerosol, Radiation, and Cloud Processes affecting Arctic Climate (ARCPAC), and the Arctic Intensive Ozonesonde Network Study (ARCIONS) experiments (April 2008). Tropospheric BrO columns retrieved from satellite measurements and back trajectory calculations are also used to investigate the characteristics of observed ODEs. In situ observations from these field experiments are inadequate to validate tropospheric BrO columns derived from satellite measurements. In view of this difficulty, we construct an ensemble of tropospheric column BrO estimates from two satellite (OMI and GOME-2) measurements and with three independent methods of calculating stratospheric BrO columns. Furthermore, we select analysis methods that do not depend on the absolute magnitude of column BrO, such as time-lagged correlation analysis of ozone and tropospheric column BrO, to understand characteristics of ODEs. Time-lagged correlation analysis between in situ (surface and ozonesonde) measurements of ozone and satellite derived tropospheric BrO columns indicates that the ODEs are due to either local halogen-driven ozone loss or short-range (∼1 day) transport from nearby regions with ozone depletion. The effect of in situ ozone loss is also evident in the diurnal variation difference between low (10th and 25th percentiles) and higher percentiles of surface ozone concentrations at Alert, Canada. Aircraft observations indicate low-ozone air mass transported from adjacent high-BrO regions. Correlation analyses of ozone with potential temperature and time-lagged tropospheric BrO column show that the vertical extent of local ozone loss is surprisingly deep (1–2 km) at Resolute and Churchill, Canada. The unstable boundary layer during ODEs at Churchill could potentially provide a source of free-tropospheric BrO through convective transport and explain the significant negative correlation between free-tropospheric ozone and tropospheric BrO column at this site

Chemoenzymatic syntheses of water-soluble lipid I fluorescent probes

Peptidoglycan (PG) is unique to bacteria, and thus, the enzymes responsible for its biosynthesis are promising antibacterial drug targets. The membrane-embedded enzymes in PG remain significant challenges in studying their mechanisms due to the fact that preparations of suitable enzymatic substrates require time-consuming biological transformations or chemical synthesis. Lipid I (MurNAc(pentapeptide)-pyrophosphoryl prenol) is an important PG biosynthesis intermediate to study the central enzymes, translocase I (MraY/MurX) and MurG. Lipid I isolated from nature contains the C_(50)- or C_(55)-prenyl unit that shows extremely poor water-solubility that renders studies of translocase I and MurG enzymes difficult. We have studied biological transformation of water soluble lipid I fluorescent probes using bacterial membrane fractions and purified MraY enzymes. In our investigation of the minimum structural requirements of the prenyl phosphates in MraY-catalyzed lipid I synthesis, we found that (2Z,6E)-farnesyl phosphate (C_(15)-phosphate) can be recognized by Escherichia coli MraY to generate the water-soluble lipid I fluorescent probe in high-yields. Under the optimized conditions, the same reaction was performed by using the purified MraY from Hydrogenivirga spp. to afford the lipid I analog with high-yields in a short reaction time

Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)_2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)_2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield

A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC_(50) value of 0.25 μM. It was confirmed that APPB inhibits N-glycosylation of β-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC_(50) > 200 μM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, 60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration