Promiscuous Aggregate-Based Inhibitors Promote Enzyme Unfolding

One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen-deuterium exchange mass spectrometry. For AmpC beta-lactamase, binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides, which covered 41% of the sequence of beta-lactamase. This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation. We then investigated whether enzyme-aggregate complexes were more susceptible to proteolysis than uninhibited enzyme. For five aggregators, trypsin degradation of beta-lactamase increased substantially when beta-lactamase was inhibited by aggregates, whereas uninhibited enzyme was generally stable to digestion. Combined, these results suggest that the mechanism of action of aggregate-based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle

Design and Synthesis of High Affinity Inhibitors of Plasmodium falciparum and Plasmodium vivax N-Myristoyltransferases Directed by Ligand Efficiency Dependent Lipophilicity (LELP)

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization

Inhibition of the Soluble Epoxide Hydrolase Promotes Albuminuria in Mice with Progressive Renal Disease

Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway

Qualitative prediction of blood–brain barrier permeability on a large and refined dataset

The prediction of blood–brain barrier permeation is vitally important for the optimization of drugs targeting the central nervous system as well as for avoiding side effects of peripheral drugs. Following a previously proposed model on blood–brain barrier penetration, we calculated the cross-sectional area perpendicular to the amphiphilic axis. We obtained a high correlation between calculated and experimental cross-sectional area (r = 0.898, n = 32). Based on these results, we examined a correlation of the calculated cross-sectional area with blood–brain barrier penetration given by logBB values. We combined various literature data sets to form a large-scale logBB dataset with 362 experimental logBB values. Quantitative models were calculated using bootstrap validated multiple linear regression. Qualitative models were built by a bootstrapped random forest algorithm. Both methods found similar descriptors such as polar surface area, pKa, logP, charges and number of positive ionisable groups to be predictive for logBB. In contrast to our initial assumption, we were not able to obtain models with the cross-sectional area chosen as relevant parameter for both approaches. Comparing those two different techniques, qualitative random forest models are better suited for blood-brain barrier permeability prediction, especially when reducing the number of descriptors and using a large dataset. A random forest prediction system (ntrees = 5) based on only four descriptors yields a validated accuracy of 88%

Validacija topokemijskih modela za predviđanje permeabilnosti kroz krvno-moždanu barijeru

Recently published topochemical models for permeability through the blood-brain barrier were validated and cross-validated in the present study. Five models based on three topochemical indices, Wiener’s topochemical index - a distance-based topochemical descriptor, molecular connectivity topochemical index - an adjacency-based topochemical descriptor and eccentric connectivity topochemical index - an adjacency-cum-distance based topochemical descriptor, for permeability of structurally and chemically diverse molecules through blood-brain barrier were used in the present investigation. A data set comprising 62 structurally and chemically diverse compounds was selected. This data set was divided into two sets of 31 compounds each - one to serve as the validation set and other as the cross-validation set. The values of all the three-topochemical indices in the original as well as in the normalized form for each of the 31 compounds of the validation set were computed using an in house computer program. Resultant data was analyzed and each compound was assigned a permeability characteristic using topochemical models, which was then compared with the reported permeability through the blood-brain barrier. Accuracy of prediction of these models was calculated. The same procedure was similarly followed for the cross-validation set. Studies revealed accuracy of prediction of the order of 7080% during validation. Surprisingly, very high predictability of the order of 7791% was observed during cross-validation. High predictability observed during validation as well as cross-validation authenticates topochemical models for prediction of permeability through the blood-brain barrier.U ovom radu su validirani i unakrsno validirani nedavno objavljeni topokemijski modeli za permeabilnost kroz krvno-moždanu barijeru. Predviđanje prolaska kroz krvno-moždanu barijeru strukturno i kemijski različitih molekula provedeno je na pet modela koji se temelje na tri topološka indeksa, Wienerovom topološkom indeksu, topološkom indeksu molekularne povezanosti i topološkom indeksu ekscentrične povezanosti. Ukupno 62 spoja podijeljena su u dva seta koji su sadržavali 31 spoj. Jedan set upotrebljen je za validaciju, a drugi za unakrsnu validaciju. Vrijednosti svih triju topoloških indeksa u početnom setu i u normaliziranom setu su računate pomoću kompjutorskog programa. Rezultati su analizirani i svakom spoju je pridružena teorijska vrijednost permeabilnosti, koja je zatim uspoređivana s objavljenim eksperimentalnim podacima za permeabilnost kroz krvno-moždanu barijeru. Točnost predviđanja bila je između 70 i 80%. Isti postupak je proveden za unakrsno validacijski set, a točnost je bila iznenađujeće velika (7791%), što ukazuje da se upotrebljeni topokemijski modeli mogu upotrijebiti za predviđanje permeabilnsot kroz krvno-moždanu barijeru

Altered body composition and metabolism in the male offspring of high fat-fed rats

An intrauterine environment may play a role in predisposing a developing fetus to metabolic diseases during adulthood. We investigated the hypothesis that a maternal diet high in omega-6 polyunsaturated fat can modify the programming of an offspring's glucose tolerance, insulin sensitivity, body composition, lipid metabolism, and insulin signaling. High omega-6 polyunsaturated fat diets were fed to female rats 4 weeks before mating and throughout the gestation period. The offspring were maintained on chow diet. At 3 months of age, indirect calorimetry, oral glucose tolerance tests, and dual x-ray absorptiometry measurements were performed. Triglyceride content and beta-hydroxyacyl coenzyme A dehydrogenase activity were determined in the liver and quadriceps muscle. Expression levels of key insulin signaling pathway proteins were measured in the liver and quadriceps muscle of the 3-month-old offspring. Offspring from the fat-fed dams had significantly increased proportions of both total body fat and abdominal fat. All offspring displayed normal insulin sensitivity and glucose tolerance, although the offspring from the fat-fed dams were significantly more hyperinsulinemic 15 minutes after an oral glucose challenge. Whole body fuel oxidation was not altered. The offspring of fat-fed dams had significantly elevated liver triglyceride content. Insulin signaling protein expression levels in the offspring of fat-fed dams were consistent with reduced hepatic insulin sensitivity but increased quadriceps insulin sensitivity. A maternal diet high in omega-6 polyunsaturated fat evokes programming within the metabolic processes of the offspring that may predispose the offspring to the development of metabolic diseases.Alexandra J. Buckley, Benjamin Keserü, Julie Briody, Madeline Thompson, Susan E. Ozanne, Campbell H. Thompso

Seasonal variability in spontaneous cervical artery dissection

We examined the seasonal variability of spontaneous cervical artery dissection (sCAD) by analysing prospectively collected data from 352 patients with 380 sCAD (361 symptomatic sCAD; 305 carotid and 75 vertebral artery dissections) admitted to two university hospitals with a catchment area of 2 200 000 inhabitants between 1985 and 2004. Presenting symptoms and signs of the 380 sCAD were ischaemic stroke in 241 (63%), transient ischaemic attack in 40 (11%), retinal ischemia in seven (2%), and non‐ischaemic in 73 (19%) cases; 19 (5%) were asymptomatic sCAD. A seasonal pattern, with higher frequency of sCAD in winter (31.3%; 95% confidence interval (CI): 26.5 to 36.4; p = 0.021) compared to spring (25.5%; 95% CI: 21.1 to 30.3), summer (23.5%; 95% CI: 19.3 to 28.3), and autumn (19.7%; 95% CI: 15.7 to 24.1) was observed. Although the cause of seasonality in sCAD is unclear, the winter peaks of infection, hypertension, and aortic dissection suggest common underlying mechanisms

Hypoxia-induced pulmonary hypertension: comparison of soluble epoxide hydrolase deletion vs. inhibition

AIMS: The C-terminal domain of the soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active diols, while the N-terminal domain demonstrates lipid phosphatase activity. As EETs are potent vasoconstrictors in the pulmonary circulation, we assessed the development of pulmonary hypertension induced by exposure to hypoxia (10% O(2)) for 21 days in wild-type (WT) and sEH(-/-) mice and compared the effects with chronic (4 months) sEH inhibition. METHODS AND RESULTS: In isolated lungs from WT mice, acute hypoxic vasoconstriction (HPV) was potentiated by sEH inhibition and attenuated by an EET antagonist. After prolonged hypoxia, the acute HPV and sensitivity to the EET antagonist were increased, but potentiation of vasoconstriction following sEH inhibition was not evident. Chronic hypoxia also stimulated the muscularization of pulmonary arteries and decreased sEH expression in WT mice. In normoxic sEH(-/-) mice, acute HPV and small artery muscularization were greater than that in WT lungs and enhanced muscularization was accompanied with decreased voluntary exercise capacity. Acute HPV in sEH(-/-) mice was insensitive to sEH inhibition but inhibited by the EET antagonist and chronic hypoxia induced an exaggerated pulmonary vascular remodelling. In WT mice, chronic sEH inhibition increased serum EET levels but failed to affect acute HPV, right ventricle weight, pulmonary artery muscularization, or voluntary running distance. In human donor lungs, the sEH was expressed in the wall of pulmonary arteries, however, sEH expression was absent in samples from patients with pulmonary hypertension. CONCLUSION: These data suggest that a decrease in sEH expression is intimately linked to pathophysiology of hypoxia-induced pulmonary remodelling and hypertension. However, as sEH inhibitors do not promote the development of pulmonary hypertension it seems likely that the N-terminal lipid phosphatase may play a role in the development of this disease