Previously, we observed that young-onset hypertension was independently associated with elevated plasma triglyceride(s) (TG) levels to a greater extent than other metabolic risk factors. Thus, focusing on the endophenotype--hypertension combined with elevated TG--we designed a family-based haplotype association study to explore its genetic connection with novel genetic variants of lipoprotein lipase gene (LPL), which encodes a major lipid metabolizing enzyme. Young-onset hypertension probands and their families were recruited, numbering 1,002 individuals from 345 families. Single-nucleotide polymorphism discovery for LPL, linkage disequilibrium (LD) analysis, transmission disequilibrium tests (TDT), bin construction, haplotype TDT association and logistic regression analysis were performed. We found that the CC- haplotype (i) spanning from intron 2 to intron 4 and the ACATT haplotype (ii) spanning from intron 5 to intron 6 were significantly associated with hypertension-related phenotypes: hypertension (ii, P=0.05), elevated TG (i, P=0.01), and hypertension combined with elevated TG (i, P=0.001; ii, P<0.0001), according to TDT. The risk of this hypertension subtype increased with the number of risk haplotypes in the two loci, using logistic regression model after adjusting within-family correlation. The relationships between LPL variants and hypertension-related disorders were also confirmed by an independent association study. Finally, we showed a trend that individuals with homozygous risk haplotypes had decreased LPL expression after a fatty meal, as opposed to those with protective haplotypes. In conclusion, this study strongly suggests that two LPL intronic variants may be associated with development of the hypertension endophenotype with elevated TG.
Dipeptidyl peptidase IV (DPP-IV) is a valid drug target for type-2 diabetes and DPP-IV inhibitors have been proven to efficiently improve glucose tolerance. In our study, 3D pharmacophore models were generated using a training set of 22 DPP-IV inhibitors. The best model consisted of important chemical features and mapped well into the active site of DPP-IV. The model gave high correlation coefficients of 0.97 and 0.84 for the training set and the test set, respectively, showing its good predictive ability for biological activity. Furthermore, the pharmacophore model demonstrated the capability to retrieve inhibitors from database with a high enrichment factor of 42.58. All results suggest that the model provides a useful tool for designing novel DPP-IV inhibitors.
Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1 site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies, it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of 2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was synthesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC(50) values of <50 nM with excellent selectivity over both DPP8 (IC(50) > 100 microM) and DPP-II (IC(50) > 30 microM). Compound 21a suppressed the blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo activities comparable to those of NVP-LAF237 (4), which is in clinical development.
To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that incorporated a bulky substituent at the first carbon position of benzylamine or isoquinoline showed over 30-fold selectivity for DPP-IV over both DPP8 and DPP-II. From structure-activity relationship studies, we speculate that the S2 site of DPP8 might be similar to that of DPP-IV, while DPP-IV inhibitor with N-substituted glycine in the P2 site and/or with a moiety involving in hydrophobic interaction with the side chain of Phe357 might provide a better selectivity for DPP-IV over DPP8.
Title: Subacute neurotoxicity induced in mice by potent organophosphorus neuropathy target esterase inhibitors Wu SY, Casida JE Ref: Toxicology & Applied Pharmacology, 139:195, 1996 : PubMed
The mouse is considered to be insensitive and the hen sensitive to clinical expression of organophosphorus-induced delayed neuropathy (OPIDN) which is associated with inhibition of neuropathy target esterase (NTE). This species difference is reevaluated with two optimized inhibitors of hen brain NTE by examining them for potential neurotoxic effects in mice. 2-Octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (OBDPO) and ethyl octylphosphonofluoridate (EOPF) inhibit mouse brain NTE in vitro by 50% at 0.12 and 0.02 nM and induce neurotoxic signs in mice at 10 and 5 mg/kg, respectively. The action of these compounds in both l- and 6-month-old mice, sometimes after early transient cholinergic signs, involves ataxia, paralysis, and death in 1 to 3 days and is accordingly referred to as subacute neurotoxicity. The neurotoxic signs are associated with brain edema and severe vacuolation in the grey matter of the brain and spinal cord, particularly the neuropile. Subacute neurotoxic signs are always associated with at least 80% inhibition of brain NTE activity 16-24 hr after treatment. Acetylcholinesterase and butyrylcholinesterase are much less sensitive than NTE to inhibition by OBDPO and EOPF both in vitro and in vivo. Selected carbamates, thiocarbamates, phosphinates, and sulfanyl fluorides are prophylactic agents and dipentyl 2,2-dichlorovinyl phosphate is a promoter for OBDPO-induced subacute neurotoxicity. Although this type of neurotoxicity in mice is similar to OPIDN in the correlation with NTE inhibition and the prophylactic action of reversible NTE inhibitors, it differs from OPIDN in the delay time prior to onset, the sensitivity of both young and old animals, and the high incidence of fatality. A full neuropathological study is desirable to further characterize this subacute neurotoxicity.
Title: Neuropathy target esterase of hen brain: active site reactions with 2-[octyl-3H]octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide and 2-octyl-4H-1,3,2-[aryl-3H]benzodioxaphosphorin 2-oxide Yoshida M, Tomizawa M, Wu SY, Quistad GB, Casida JE Ref: Journal of Neurochemistry, 64:1680, 1995 : PubMed
2-Octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (octyl-BDPO) is one of the most potent inhibitors known for neuropathy target esterase (NTE) of hen brain with 50% inhibition at 0.2 nM. Two NTE-like proteins, i.e., resistant to paraoxon and sensitive to mipafox, of approximately 155 and approximately 119 kDa (designated NTE-155 and NTE-119, respectively) are labeled by [octyl-3H]octyl-BDPO and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [aryl-3H]octyl-BDPO is only approximately 15% of that with [octyl-3H]octyl-BDPO, indicating that the majority of the phosphorylated NTE undergoes aging with only a small proportion of nonaged target or intramolecular group transfer ("alkylation"). NTE-155 and NTE-119 have the same kinetic constants and maximal number of phosphorylation sites, equivalent for each of them to 26 fmol/mg of protein and totaling at least 0.44-1.2 micrograms of NTE protein/g of brain. Structure-activity investigations involving 17 combinations of organophosphorus (OP) compounds of varied chemical type, stereochemistry, and concentration establish an excellent correlation (r = 0.95) between inhibition of NTE activity and protein labeling and thereby the toxicological relevance of these assays, which equally implicate NTE-155 and NTE-119 (probably an autolysis product of NTE-155) as target in OP-induced delayed neuropathy. [octyl-3H]-Octyl-BDPO is an improved probe for NTE in terms of its potency, reactivity, selectivity, and the formation of 3H-labeled NTE with a stable phosphorus-carbon bond.
Title: Neuropathy target esterase inhibitors: enantiomeric separation and stereospecificity of 2-substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides Wu SY, Casida JE Ref: Chemical Research in Toxicology, 7:77, 1994 : PubMed
2-Substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides (2-substituted-BDPOs) are known to be potent neuropathy target esterase (NTE) inhibitors (I50s for the racemates of 0.2-3 nM) when the 2-substituents are n-alkyl (C5-C12), N-alkoxy (C7-C10), or p-n-alkylbenzyl (C3 and C4). The list of potent inhibitors (I50s < 3 nM) is expanded by the new n-alkylamino (C9) and n-alkylthio (C5, C7, and C9) analogs reported here. The optimal chain length of the 2-substituent is about 10 atoms in the alkylamino and alkylthio series as in our previous study on alkyl and alkoxy moieties. In contrast, an I50 of 60 nM is reported for o-methylphenoxy-BDPO, the neuropathic metabolite of tri-o-cresyl phosphate (TOCP). In addition to substituent effects, each of these compounds contains two enantiomers of unknown stereospecificity as NTE inhibitors. Separation by chiral HPLC with the CHIRALCEL OC column and hexane-2-propanol eluent gives individual enantiomers of > 98% e.e. and a stereospecificity for NTE inhibition depending on the type and chain length of the 2-substituent; e.g., the ratio for inhibitory potency of the individual enantiomers is 1.7-fold for nonylthio, 1255-fold for nonylamino, and 9-fold for the TOCP metabolite. In comparing enantiomeric pairs of BDPOs with alkyl, alkoxy, alkylamino, alkylthio, benzyl, p-butylbenzyl, o-methylphenoxy, or phenyl as the 2-substituent, the more retained enantiomer in HPLC is always the better NTE inhibitor (in a series of twenty-two pairs) and housefly toxicant (based on two pairs) than the less retained one.(ABSTRACT TRUNCATED AT 250 WORDS)
Title: Reactivity and stereospecificity of neuropathy target esterase and alpha-chymotrypsin with 2-substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides Yoshida M, Wu SY, Casida JE Ref: Toxicol Lett, 74:167, 1994 : PubMed
2-Substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides (2-substituted-BDPOs) are of special interest as neuropathy target esterase (NTE) inhibitors because they include not only the neuropathic metabolite of tri-o-cresyl phosphate (the 2-methylphenoxy analog) but also the most potent NTE inhibitors known. These compounds react much faster with NTE than 2 standard inhibitors, O,O-diisopropyl fluorophosphonate (DFP) and mipafox. alpha-Chymotrypsin is similar to NTE in undergoing rapid inhibition by BDPOs which is known to involve phosphorylation followed by aging. NTE and alpha-chymotrypsin were compared for reaction rates with BDPOs varying in the 2-substituent as follows: 4-methyl-, 4-propyl-, and 4-hexylphenoxy; butyl, octyl and dodecyl; (S)- and (R)-butyl. The active site of NTE differs from that of alpha-chymotrypsin in preference for long-chain substituents and in stereospecificity.
Title: Putative transmitter systems of mammalian sympathetic preganglionic neurons Dun NJ, Karczmar AG, Wu SY, Shen E Ref: Acta Neurobiologiae Experimentalis, 53:53, 1993 : PubMed
The sympathetic nervous system evokes complex effects at multiple target organs in response to external, internal as well as mental stimuli. This output involves an interplay between the actions of a number of transmitters and modulators and at the postsynaptic and presynaptic sites of the autonomic ganglia and the sympathetic preganglionic neurons (SPNs). This review concerns particularly the SPNs of the cat and neonatal rat, studied by means of electrophysiological and immunohistochemical methods. Four types of responses may be elicited, the fast EPSP and IPSP, and their currents, and the slow EPSPs and IPSPs, and their currents. Glutamate and glycine appear to mediate the fast excitatory and inhibitory responses, respectively; peptides and amines seem to be responsible for generating the slow excitatory response, while the slow inhibitory response, found so far only in the cat, appears to be mediated by norepinephrine. Finally, glutamate, enkephalin and GABA, but not glycine attenuate the release of the inhibitory and excitatory transmitters from the nerve terminals abutting on the SPNs. The supraspinal efferent and afferent projections which may release the transmitters and modulators in question are discussed, as well the mechanisms that ensure appropriate programming and moment-to-moment regulation of the autonomic output.
Title: Neuropathy target esterase inhibitors: 2-alkyl-, 2-alkoxy-, and 2-(aryloxy)-4H-1,3,2-benzodioxaphosphorin 2-oxides Wu SY, Casida JE Ref: Chemical Research in Toxicology, 5:680, 1992 : PubMed
The standard probes used earlier to study neuropathy target esterase (NTE) are N,N'-diisopropyl phosphorofluorodiamidate (mipafox), diisopropyl phosphorofluoridate (DFP), 2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin 2-oxide (2-CH3C6H4O-BDPO) (the neurotoxic metabolite of tri-o-cresyl phosphate), and dipentyl 2,2-dichlorovinyl phosphate (DDP) with I50s for hen brain enzyme of 7000, 700, 29, and 3 nM, respectively. NTE phosphorylated by DFP and DDP is proposed to undergo alkylation on aging, and this probably also occurs with 2-CH3C6H4O-BDPO. Optimized probes for NTE should meet the following specifications: highest potency achievable; rapid aging perhaps associated with alkylation; preferably a phosphonate so there are only two leaving groups. An attempt was made to achieve these goals in the 4H-1,3,2-benzodioxaphosphorin 2-oxide series by synthesis of 49 analogs systematically varied in the 2-alkyl, 2-alkoxy, or 2-(aryloxy) substituent. Special precautions are required in synthesis of BDPO derivatives because of their potential hazard on human exposure. Thirty of these compounds had NTE I50s lower than 3 nM. Representative high-potency NTE inhibitors in each series are [2-substituent,I50 (nM) for hen and human brain NTE, respectively]: octyl, 0.25 and 0.18; nonyloxy, 0.89 and 0.98; 4-propylphenoxy, 0.82 and 0.77. In comparing these compounds, although the octyl analog is the most potent in vitro NTE inhibitor, the propylphenoxy compound is the most effective in vivo NTE inhibitor and delayed neurotoxicant in hens. These benzodioxaphosphorins are improved probes for investigations on NTE phosphorylation and alkylation in relation to delayed neurotoxicity.
