Optimization of a "bump-and-hole" approach to allele-selective BET bromodomain inhibition.

Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins - BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin via their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound - 9-ME-1 - shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of in vitro and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and in vivo target validation studies.

The concept behind sugammadex.

Sugammadex is the first selective relaxant binding agent. It allows rapid reversal of any degree of neuromuscular blockade induced by steroidal neuromuscular blocking agents. Sugammadex acts by encapsulation of the neuromuscular blocking agent. This prevents the drug from acting on prejunctional and postjunctional nicotinic receptors, allowing acetylcholine to activate these receptors, and resulting in reversal of the neuromuscular blockade. Objective monitoring of the degree of neuromuscular blockade is strongly recommended to determine the optimal dose of sugammadex. A good understanding of the concept behind sugammadex is essential in order to use this reversal agent in clinical practice.

The pharmacokinetics of 1,2-diethyl-3-hydroxypyridin-4-one (CP94) in rats.

The pharmacokinetics of 1,2-diethyl-3-hydroxypyridin-4-one (CP94) and its 2-(1-hydroxyethyl) metabolite (metabolite A) were examined in male Wistar rats using a chronically cannulated conscious-rat model. Serial blood samples were assayed by a reversed phase HPLC method with UV detection. Following iv doses of 25, 50, and 100 mg/kg, the parent compound was eliminated from blood in a biexponential fashion with an average systemic clearance of 1.5 liters/hr/kg. The mean terminal elimination half-life was 2.02 hr and the mean volume of distribution at steady state was 2.69 liters/kg. The areas under the curve (AUCs) for the 25, 50, and 100 mg/kg iv doses were 15, 36, and 72 micrograms/ml/hr, respectively, suggesting that the disposition of CP94 in rats obeys linear kinetics. The oral bioavailability of CP94 (100 mg/kg) was about 53%. Peak blood concentration occurred at about 0.5 hr after oral administration. Following iv doses of CP94 at 25, 50, and 100 mg/kg, metabolite A peaked at about 0.75 hr.

Comparison of the platelet pro-aggregatory effect of conventional unfractionated heparins and a low molecular weight heparin fraction (CY 222).

Two unfractionated heparins (UH), a porcine intestinal mucosal heparin (PIM), a bovine lung heparin (BLH) and a low molecular weight heparin (LMWH), CY 222, were assessed for their capacity to enhance platelet aggregation in vitro. Their potential proaggregatory effect was investigated in four systems: (a) enhancement of submaximal platelet aggregation induced by conventional agonists in platelet rich plasma and (b) in whole blood; (c) reversal of inhibition of platelet aggregation induced by iloprost, a stable analogue of prostacyclin; (d) the direct aggregatory effect of these anticoagulants on hyperactive platelets prepared from patients with severe peripheral vascular disease or anorexia nervosa. Whereas BLH and PIM, at therapeutic concentrations, had a proaggregatory effect in all four systems, CY 222 had no significant effect when compared with the controls. BLH was more potent than PIM in three of the four systems studied. These observations confirm that conventional UH are more proaggregatory than LMWH, and thus the latter may be potentially safer. These observations are also consistent with the fact that BLH administration causes thrombocytopenia more frequently than PIM. The direct activation by UH of platelets from patients not previously exposed to heparin also challenges the hypothesis that heparin-induced platelet activation and thrombocytopenia is solely mediated by classical heparin-dependent immune mechanisms.

Ethanol ingestion inhibits human whole blood platelet impedance aggregation.

The authors investigated the effect of ethanol on platelet impedance aggregation in whole blood (WB-PIA). Healthy moderate drinkers were given ethanol, 1 mL/kg body weight, to drink. Thirty minutes after ingestion of ethanol, WB-PIA was significantly inhibited when compared with baseline values. There was no significant inhibition when the same volunteers ingested water instead of ethanol. These observations suggest that WB-PIA is a sensitive technic for the detection of the effect of ethanol on platelets. These findings also support the view that blood ethanol levels achievable during social drinking impair platelet function, thus possibly accounting, at least in part, for the reported "protection" from ischemic heart disease in moderate drinkers. The sensitivity of human platelets to the inhibitory effect of ethanol suggests that continued drinking will adversely influence the incidence of initial bleeds and of rebleeding in gastrointestinal hemorrhage associated with alcoholism.