The ability to rapidly assess the preferred conformation of key fragments in a structure "by visual inspection" is a very useful starting point in the process of drug design. With the ability to do ...so, one could address questions like: "How could we avoid planarity in a molecule?", "Will a molecule change its conformational preference if we make it more or less basic?" or "How does this electronic repulsion affect the conformational preference in the system?" in timely fashion. In this paper, we describe how the conformational energy profile (CEP, plot of energy as a function of dihedral bond angle) of a fragment can be interpreted through the understanding the interplay between resonance stabilization, steric effects and electrostatic interactions. Fifty-nine biaryl and aryl carbonyl fragments present in oral drugs or which are close derivatives thereof were selected. Calculation of their CEPs using ab initio methodology allowed us to conclude the relative importance of these factors in the conformational preference of these fragments as follows: "steric repulsion > lone pair-lone pair repulsion > lone pair-fluorine repulsion > resonance stabilization" and to formulate "rules of thumb" that the practicing medicinal/organic chemist can apply when analysing molecules that contain these fragments.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although global ischemia induces troponin I (TnI) degradation, regional ischemia does not. We hypothesized that this disparity is related to preload-induced proteolysis, which varies as a function of ...the amount of myocardium at risk of ischemia.
Isolated rat hearts were buffer-perfused at controlled levels of preload. Increasing preload to 25 mm Hg in the absence of ischemia produced pronounced TnI degradation (27 kDa versus 31 kDa bands: 16.4 +/- 3.6% versus 4.7 +/- 1.9% in immediately excised controls, P<0.05). TnI degradation could be blocked by preventing the activation of endogenous calpains with 25 micromol/L calpeptin (4.3 +/- 0.6%). This improved function, with left ventricular systolic pressure increasing from 103 +/- 4 mm Hg to 137 +/- 7 mm Hg (P<0.05). Eliminating elevations in preload after global ischemia-induced stunning also prevented TnI degradation.
Calpain-mediated TnI proteolysis can be dissociated from stunning and arises from elevations in preload rather than ischemia. This raises the possibility that ongoing preload-induced TnI degradation could impair myocardial function long-term.
The purpose of this study was to assess the impact of MALDI-ToF identification and rapid short incubation MALDI-Tof identification protocol on patient care compared to conventional identification. By ...using a retrospective review we assessed the impact of a rapid Bruker MALDI-Tof identification protocol. Overall there was a 16.76-hour reduction in time to identification of the pathogen after the introduction of MALDI-TOF identification in 2013 (
P
<0.0001) and a further 15-hour reduction (
P
<9.37 E-05) after implementation of the short incubation MALDI-TOF identification protocol in 2014. Patients received appropriate therapy 20.25 hours earlier (
P
<0.002) in 2014 compared to the conventional identification group in 2012. Overall length in the patients needing optimization of antibiotic treatment was reduced by 6.87 days (
P
<0.042). In 2014 outcomes between the patients needing a change in their antibiotic compared to the patients where the empirical therapy was considered to be optimal were similar with respective difference in length of stay being reduced from 4.72 days (
P
<0.031) to 1.77 days (
P
<0.71) and an associated reduction in the absolute mortality risk of 3.79%. The all-cause mortality rate was twice as high in the group pre-implementation of the short incubation MALDI-TOF identification with an associated survival benefit in this patient population when 26 patients were treated. Rapid short incubation MALDI-ToF identification of bacterial pathogens in blood cultures is associated with a reduction in length of stay and mortality risk.
The binding site for DETQ 2-(2,6-dichlorophenyl)-1-((1
,3
)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1
)-yl)ethan-1-one, a positive allosteric modulator (PAM) of ...the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111
-(6-
-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide, a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a
2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state
2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.
DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all ...rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.
•The dopamine D1 potentiator DETQ was tested in humanized D1 mice and rhesus monkeys.•Actions of DETQ were dependent on endogenous dopaminergic tone.•DETQ displayed a behavioral profile consistent with central D1 receptor activation.•Neurochemical actions of DETQ support potential pro-cognitive effects.•D1 potentiators show promise for Parkinson's disease and other CNS disorders.
We recently demonstrated that (11)C-MePPEP, a PET ligand for CB(1) receptors, has such high uptake in the human brain that it can be imaged for 210 min and that receptor density can be quantified as ...distribution volume (V(T)) using the gold standard of compartmental modeling. However, (11)C-MePPEP had relatively poor retest and intersubject variabilities, which were likely caused by errors in the measurements of radioligand in plasma at low concentrations by 120 min. We sought to find an analog of (11)C-MePPEP that would provide more accurate plasma measurements. We evaluated several promising analogs in the monkey brain and chose the (18)F-di-deutero fluoromethoxy analog ((18)F-FMPEP-d(2)) to evaluate further in the human brain.
(11)C-FMePPEP, (18)F-FEPEP, (18)F-FMPEP, and (18)F-FMPEP-d(2) were studied in 5 monkeys with 10 PET scans. We calculated V(T) using compartmental modeling with serial measurements of unchanged parent radioligand in arterial plasma and radioactivity in the brain. Nonspecific binding was determined by administering a receptor-saturating dose of rimonabant, an inverse agonist at the CB(1) receptor. Nine healthy human subjects participated in 17 PET scans using (18)F-FMPEP-d(2), with 8 subjects having 2 PET scans to assess retest variability. To identify sources of error, we compared intersubject and retest variability of brain uptake, arterial plasma measurements, and V(T).
(18)F-FMPEP-d(2) had high uptake in the monkey brain, with greater than 80% specific binding, and yielded less radioactivity uptake in bone than did (18)F-FMPEP. High brain uptake with (18)F-FMPEP-d(2) was also observed in humans, in whom V(T) was well identified within approximately 60 min. Retest variability of plasma measurements was good (16%); consequently, V(T) had a good retest variability (14%), intersubject variability (26%), and intraclass correlation coefficient (0.89). V(T) increased after 120 min, suggesting an accumulation of radiometabolites in the brain. Radioactivity accumulated in the skull throughout the entire scan but was thought to be an insignificant source of data contamination.
Studies in monkeys facilitated our development and selection of (18)F-FMPEP-d(2), compared with (18)F-FMPEP, as a radioligand demonstrating high brain uptake, high percentage of specific binding, and reduced uptake in bone. Retest analysis in human subjects showed that (18)F-FMPEP-d(2) has greater precision and accuracy than (11)C-MePPEP, allowing smaller sample sizes to detect a significant difference between groups.
Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor ...potentiator DETQ 2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a K
of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.
11CMePPEP is a high affinity, CB1 receptor-selective, inverse agonist that has been studied in rodents and monkeys. We examined the ability of 11CMePPEP to quantify CB1 receptors in human brain as ...distribution volume calculated with the “gold standard” method of compartmental modeling and compared results with the simple measure of brain uptake. A total of 17 healthy subjects participated in 26 positron emission tomography (PET) scans, with 8 having two PET scans to assess retest variability. After injection of 11CMePPEP, brain uptake of radioactivity was high (e.g., 3.6 SUV in putamen at ∼60 min) and washed out very slowly. A two-tissue compartment model yielded values of distribution volume (which is proportional to receptor density) that were both well identified (SE 5%) and stable between 60 and 210 min. The simple measure of brain uptake (average concentration of radioactivity between 40 and 80 min) had good retest variability (∼8%) and moderate intersubject variability (16%, coefficient of variation). In contrast, distribution volume had two-fold greater retest variability (∼15%) and, thus, less precision. In addition, distribution volume had three-fold greater intersubject variability (∼52%). The decreased precision of distribution volume compared to brain uptake was likely due to the slow washout of radioactivity from brain and to noise in measurements of the low concentrations of 11CMePPEP in plasma. These results suggest that brain uptake can be used for within subject studies (e.g., to measure receptor occupancy by medications) but that distribution volume remains the gold standard for accurate measurements between groups.
The in-vitro potency and selectivity, in-vivo binding affinity and effect of the 5-HT6R antagonist Lu AE58054 (2-(6-fluoro-1H-indol-3-yl)-ethyl-3-(2,2,3,3-tetrafluoropropoxy)-benzyl-amine) on ...impaired cognition were evaluated. Lu AE58054 displayed high affinity to the human 5-HT6 receptor (5-HT6R) with a Ki of 0.83 nm. In a 5-HT6 GTPγS efficacy assay Lu AE58054 showed no agonist activity, but demonstrated potent inhibition of 5-HT-mediated activation. Besides medium affinity to adrenergic α1A- and α1B-adrenoreceptors, Lu AE58054 demonstrated >50-fold selectivity for more than 70 targets examined. Orally administered Lu AE58054 potently inhibited striatal in-vivo binding of the 5-HT6 antagonist radioligand 3HLu AE60157 (3H8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline), with an ED50 of 2.7 mg/kg. Steady-state modelling of an acute pharmacokinetic/5-HT6R occupancy time-course experiment indicated a plasma EC50 value of 20 ng/ml. Administration of Lu AE58054 in a dose range (5–20 mg/kg p.o.) leading to above 65% striatal 5-HT6R binding occupancy in vivo, reversed cognitive impairment in a rat novel object recognition task induced after subchronic treatment for 7 d with phencyclidine (PCP 2 mg/kg b.i.d., i.p. for 7 d, followed by 7 d drug free). The results indicate that Lu AE58054 is a selective antagonist of 5-HT6Rs with good oral bioavailability and robust efficacy in a rat model of cognitive impairment in schizophrenia. Lu AE58054 may be useful for the pharmacotherapy of cognitive dysfunction in disease states such as schizophrenia and Alzheimer's disease.
We have reported that methyl-11C (3R,5R)-5-(3-methoxyphenyl)-3-(R)-1-phenylethylamino-1-(4-trifluoromethylphenyl)pyrrolidin-2-one (11C8, 11CMePPEP) binds with high selectivity to cannabinoid type-1 ...(CB1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely, the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy-d 2 (21, FMPEP-d 2), and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high, selective potency at CB1 receptors in vitro (K b < 1 nM). Each ligand (30 μg/kg, iv) was injected into rats under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand uptakes were high (22.6−48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced (6.5−17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB1 receptors, we then established and here describe methods for producing 11C8, 11C16, and 18F20−22 in adequate activities for evaluation as candidate PET radioligands in vivo.
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