Pharmacophore-guided lead optimization: The rational design of a non-zinc coordinating,sub-micromolar inhibitor of the botulinum neurotoxin serotype a metalloprotease

Botulinum neurotoxins, responsible for the neuroparalytic syndrome botulism, are the deadliest of known biological toxins. The work described in this study was based on a three-zone pharmacophore model for botulinum neurotoxin serotype A light chain inhibition. Specifically, the pharmacophore defined a separation between the overlaps of several different, non-zinc(II)-coordinating small molecule chemotypes, enabling the design and synthesis of a new structural hybrid possessing a K_i = 600 nM (±100 nM).     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

Muscle fiber type characteristics in females with chronic obstructive pulmonary disease. A preliminary study

Chronic obstructive pulmonary disease (COPD) is known to elicit intrinsic abnormalities in male skeletal muscle. However, it is unclear to what extent these changes occur in women and whether they are fiber-type specific. We investigated fiber-type specific differences in selected histochemical properties in muscle obtained from women with moderate to severe COPD compared to healthy control (CON) women. Tissue was obtained from the vastus lateralis in five COPD patients (age 66.9 ± 2.6 years; FEV₁ = 43 ± 7%) and eight CON (age 68 ± 4.9 years; FEV₁ = 113 ± 4.2%). Compared to CON, the distribution (30.6 ± 5.2 vs. 57.9 ± 4.6%) and cross sectional area of type I (CSA, 5660 ± 329 vs. 3586 ± 257 μm²) and type IIA (2770 ± 302 vs. 2099 ± 206 μm²) were lower (P < 0.05) and higher (P < 0.05), respectively, in COPD. Disease state did not alter either the distribution or CSA of the IIA, IIAX or type X subtypes. Although differences were found between fiber types in the number of capillary contacts (n) (I > IIAX, IIX; IIA > IIX) and the capillaries per CSA (μm²10⁻³) (I < IIA, IIAX, IIX), no differences were found between CON and COPD. Succinic dehydrogenase activity and sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity, measured photometrically (OD units), were higher (P < 0.05), and lower (P < 0.05), respectively, in type I compared to the type II fiber subtypes. These properties were not altered with COPD. COPD in females is accompanied by a higher percent of type II fibers, a larger CSA of type I and type IIA fibers, both of which occur in the absence of differences in oxidative potential and the potential for SR Ca²⁺-sequestration.     

Design and synthesis of orally efficacious benzimidazoles as melanin-concentrating hormone receptor 1 antagonists

Biaryl urea lead compound 1 was discovered earlier in our MCH antagonist program. Novel benzimidazole analogues with increased chemical stability, devoid of the potential carcinogenic liability associated with a biarylamine moiety, were synthesized and evaluated to be potent MCH R1 antagonists. Two compounds in this series have demonstrated in vivo efficacy in a rodent obesity model.     

Hydrazides of clozapine: a new class of D1 dopamine receptor subtype selective antagonists

Acylated and aroylated hydrazinoclozapines are highly potent dopamine D(1) antagonists that show remarkable selectivity over other dopamine receptors. The most potent compound in this series is the 2,6-dimethoxybenzhydrazide 33 with a D(1)K(i) of 1.6 nM and 212-fold selectivity over D(2) receptor.     

Segregation of a single outboard left-end origin is essential for the viability of parvovirus minute virus of mice

During DNA replication, the hairpin telomeres of Minute Virus of Mice (MVM) are extended and copied to create imperfectly palindromic duplex junction sequences that bridge adjacent genomes in concatameric replicative-form DNA. These are resolved by the viral initiator protein, NS1, but mechanisms employed at the two telomeres differ. Left-end:left-end junctions are resolved asymmetrically at a single site, OriLTC, by NS1 acting in concert with a host factor, parvovirus initiation factor (PIF). Replication segregates doublet and triplet sequences, initially present as unpaired nucleotides in the bubble region of the left-end hairpin stem, to either side of the junction. These act as spacers between the NS1 and PIF binding sites, and their asymmetric distribution sets up active (OriLTC) and inactive (OriLGAA) forms of OriL. We used a reverse genetic approach to disrupt this asymmetry and found that neither opposing doublets nor triplets in the hairpin bubble were tolerated. Viable mutants were isolated at low frequency and found to contain second-site mutations that either restored the asymmetry or crippled one PIF binding site. These mutations either inactivated the inboard or activated the outboard form of OriL, a polarity that strongly suggests that, in the genus Parvovirus, an active inboard OriL is lethal.     

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K_ATP) channels. K_ATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K_ATP channel subunit were compared with littermate controls. Evaluation of K_ATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80–85% reduction in cardiac K_ATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K_ATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.     

Identification of presenilin 1-selective γ-secretase inhibitors with reconstituted γ-secretase complexes

Accumulation of the β-amyloid (Aβ) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. Aβ is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by β- and γ-secretases. Inhibition of Aβ production by γ-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, γ-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce Aβ production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce Aβ production and have less Notch-mediated side effect liability is therefore an important goal. γ-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four γ-secretase complexes in Sf9 cells containing PS1--aph-1a, PS1--aph-1b, PS2--aph-1a, and PS2--aph-1b complexes. While PS1--aph-1a, PS1--aph-1b, and PS2--aph-1a complexes displayed robust γ-secretase activity, the reconstituted PS2--aph-1b complex was devoid of detectable γ-secretase activity. γ-Secretase complexes containing PS1 produced a higher proportion of the toxic species Aβ42 than γ-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 γ-secretase activity. These findings may provide important insights into developing a new generation of γ-secretase inhibitors with improved side effect profiles.     

Association of peak aerobic power with capillary density but not oxidative potential in human vastus lateralis muscle

In this study, we have postulated that in healthy males, peak aerobic power ([Formula: see text]) would associate with muscle capillary density rather than oxidative potential, regardless of fibre type or subtype. To test this, active but untrained volunteers (n = 11) were separated into high (HI) and low (LO) groups based on [Formula: see text] obtained during a progressive cycle task to fatigue. The 26% higher (P?< 0.05) [Formula: see text] observed in HI (40.8?± 1.5?mL·kg(-1)·min(-1), mean?± SE) compared with LO ( 51.4?± 0.90?mL·kg(-1)·min(-1), mean?± SE) was not accompanied by differences in age (21.3?± 1.2 compared with 21.1?± 0.63?years, respectively) or body mass (72.4?± 4.6 compared with 71.6?± 1.9?kg, respectively). Tissue samples obtained from the vastus lateralis indicated greater (P?< 0.05) capillary counts per fibre (CC;?+24%) in HI compared with LO, regardless of fibre type (I, IIA, IIX, IIAX). Capillary density (CD) as measured in a field of defined area was also elevated (+22%; P?< 0.05), as was the number of capillaries per fibre (+22%; P?< 0.05). No differences were observed between the 2 groups in the distribution, area, and the CC/fibre area ratio in the different fibre types and subtypes. Similarly, there was no difference between the HI and LO groups in oxidative potential, as measured by succinic dehydrogenase activity in the different fibre types. It is concluded that the higher capillary density may contribute to improved vascular conductance and the elevated [Formula: see text] observed in the untrained participants.