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PubMed1992

Differential cholinergic innervation within functional subdivisions of the human cerebral cortex: a choline acetyltransferase study.

The distribution of cholinergic fibers in the human brain was investigated with choline acetyltransferase immunocytochemistry in 35 cytoarchitectonic subdivisions of the cerebral cortex. All cortical areas and all cell layers contained cholinergic axons. These fibers displayed numerous varicosities and, on occasion, complex preterminal profiles arranged in the form of dense clusters. The density of cholinergic axons tended to be higher in the more superficial layers of the cerebral cortex. Several distinct patterns of lamination were identified. There were also major differences in the overall density of cholinergic axons from one cytoarchitectonic area to another. The cholinergic innervation of primary sensory, unimodal, and heteromodal association areas was lighter than that of paralimbic and limbic areas. Within unimodal association areas, the density of cholinergic axons and varicosities was significantly lower in the upstream (parasensory) sectors than in the downstream sectors. Within paralimbic regions, the non-isocortical sectors had a higher density of cholinergic innervation than the isocortical sectors. The highest density of cholinergic axons was encountered in core limbic structures such as the hippocampus and amygdala. These observations show that the cholinergic innervation of the human cerebral cortex displays regional variations that closely follow the organization of information processing systems.

Adult↗
PubMed1992

The structure of the diencephalic prepacemaker nucleus revisited: light microscopic and ultrastructural studies.

The prepacemaker nucleus (PPn), a bilateral cluster of neurons at the boundary of diencephalon and mesencephalon, controls frequency modulations of the electric organ discharge in weakly electric knifefish (Eigenmannia sp.). Previous light microscopic studies employing retrograde labelling with horseradish peroxidase suggested that the PPn is restricted to a small area, located approximately 400 microns laterally from the third ventricle and fusing at its medial edge with the thalamic central posterior nucleus (CP). In the present investigation we used Phaseolus vulgaris-leucoagglutinin and cholera toxin as highly sensitive markers. In contrast to the previous studies, these experiments yielded a large number of labelled cells not only in the region of the traditionally defined PPn but also in an area reaching far into the CP. Since the PPn has been defined by retrograde labelling rather than by topographic criteria, this result questions the traditional separation between PPn and CP. Such a notion is in agreement with observations of Nissl-stained sections at the light microscopic level and with a quantitative analysis of several morphological characteristics of the cell bodies in the PPn and CP at the ultrastructural level. Both sets of experiments failed to find differences between the two nuclei. Furthermore, autoradiographic studies have shown that, even in adulthood, cells are continuously born within the ventricular zone of the CP, and at least some of these newborn cells differentiate into CP cells and migrate laterally towards the PPn. Therefore, we postulate that CP and PPn form one large complex, with the medial CP providing precursors of neurons in the lateral CP and PPn.

Animals↗
PubMed1995

Spatial and temporal pattern of Purkinje cell degeneration in shaker mutant rats with hereditary cerebellar ataxia.

Temporal-spatial patterns of surviving Purkinje cells were studied quantitatively in a rat mutant (shaker) with differential hereditary cerebellar ataxia and Purkinje cell degeneration. Shaker rat mutants are characterized behaviorally as mild if they are ataxic or as strong if they have ataxia and tremor. Purkinje cells degenerate in both mild and strong shaker mutants, but the temporal and spatial patterns of cell death are strikingly different. In mild shaker mutants, Purkinje cell death is temporally restricted, with 31-46% of the Purkinje cells in lobules I-IX dying by 3 months of age. Very few Purkinje cells degenerate after this age. Purkinje cell death is spatially random. In lobules I-IX, every second, third, or fourth Purkinje cell degenerates. Purkinje cells in lobule X do not degenerate. In strong shaker mutants, Purkinje cell degeneration is temporally protracted and spatially restricted. By 3 months of age, most Purkinje cells in lobules I-VIa, -b, and -d have degenerated. Numerous Purkinje cells in the paravermis of lobules VIIb-VIII have also degenerated. Surviving Purkinje cells in the vermis and lateral hemisphere of lobules VIIb-VIII are aligned in parasagittally oriented stripes or transversely oriented bands. Purkinje cells continue to degenerate in localized areas of the posterior lobe such that, by 18 months of age, surviving Purkinje cells are limited primarily to lobules VIc, VIIa, IXd, and X. Quantitative analysis indicates that none of the Purkinje cells in these lobules degenerate.

Animals↗
PubMed1992

Supravital staining of synovial fluid with Testsimplets.

We explored the use of Testsimplet (TS) in synovial fluid (SF) analysis. TS is a glass slide coated with a dry mixture of methylene blue and cresyl violet, which in contact with one drop of SF provides a stained fresh preparation. We applied the TS to the study of 159 SFs of patients with different rheumatic diseases. In those SFs of patients with crystal-associated diseases, the crystal search was performed both on unstained preparations and with TS. TS was as good as the Wright's and Papanicolaou stain in characterizing SF cells, lupus erythematosus cells, and detection of occasional bacteria. TS allowed a better visualization of Reiter's cells, cartilage fragments, synovial villi, fat droplets, and fibrin. Crystals were identified in every TS of those patients with crystal-associated diseases. TS is a rapid and reproducible method of SF supravital staining. Crystals are well preserved for simultaneous examination with compensated polarized light.

Benzoxazines↗
PubMed2003

Cannabinoids ablate release of TNFalpha in rat microglial cells stimulated with lypopolysaccharide.

Upon activation, brain microglial cells release proinflammatory mediators, such as TNFalpha, which may play an important role in eliciting neuroinflammatory processes causing brain damage. As cannabinoids have been reported to exert anti-inflammatory and neuroprotective actions in the brain, we here examined the effect of both synthetic and endogenous cannabinoids on TNFalpha release elicited by bacterial endotoxin lypopolysaccharide (LPS) in cultured microglia. Exposure of primary cultures of rat cortical microglial cells to LPS significantly stimulated TNFalpha mRNA expression and release. The endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG), as well as the synthetic cannabinoids (+)WIN 55,212-2, CP 55,940, and HU210, inhibited in a concentration-dependent manner (1-10 microM) the LPS-induced TNFalpha release. Unlike the high-affinity cannabinoid receptor agonist (+)WIN 55,212-2, the low-affinity stereoisomer (-)WIN 55,212-2 did not exert any significant inhibition on TNFalpha release. Given this stereoselectivity, the ability of (+)WIN 55,212-2 to inhibit LPS-induced TNFalpha release from microglia is most likely receptor-mediated. By RT-PCR we found that the two G(i/o) protein-coupled cannabinoid receptors (type 1 and 2) are both expressed in microglial cultures. However, selective antagonists of type 1 (SR141716A and AM251) and type 2 (SR144528) cannabinoid receptors did not affect the effect of (+)WIN 55,212-2. Consistent with this finding is the observation that the ablative effect of (+)WIN 55,212-2 on LPS-evoked release of TNFalpha was not sensitive to the G(i/o) protein inactivator pertussis toxin. In addition, the cAMP elevating agents dibutyryl cAMP and forskolin both abolished LPS-induced TNFalpha release, thus rendering unlikely the possibility that (+)WIN 55,212-2 could ablate TNFalpha release through the inhibition of adenylate cyclase via the G(i)-coupled cannabinoid receptors type 1 and 2. In summary, our data indicate that both synthetic and endogenous cannabinoids inhibit LPS-induced release of TNFalpha from microglial cells. By showing that such effect does not appear to be mediated by either CB receptor type 1 or 2, we provide evidence suggestive of the existence of yet unidentified cannabinoid receptor(s) in brain microglia.

Animals↗
PubMed2005

Synthetic cannabinoid WIN55,212-2 inhibits generation of inflammatory mediators by IL-1beta-stimulated human astrocytes.

Activated glial cells have been implicated in the neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators have been proposed to play a role in glial cell-related brain damage; e.g., free radicals such as nitric oxide (NO), cytokines, and chemokines. Our laboratory has been interested in the effect of psychoactive drugs and their derivatives on the production of these mediators. Cannabinoids have been shown to possess immunomodulatory as well as psychoactive properties. We previously have shown that interleukin (IL)-1beta-stimulated human astrocytes, but not microglia, produce NO. In this study, we investigated the effects of the synthetic cannabinoid WIN55,212-2 on the production of several key inflammatory mediators by human fetal astrocytes activated by IL-1beta. Expression of the cannabinoid receptors CB1 and CB2 was detected on human astrocytes. WIN55,212-2 (10(-5) M) potently inhibited inducible NO synthase (iNOS) and corresponding NO production by IL-1beta-stimulated astrocytes. The CB1 and CB2 receptor-specific antagonists SR141716A and SR144528, respectively, partially blocked this suppressive effect. In addition, treatment of astrocytes with WIN55,212-2 downregulated in a concentration-dependent manner IL-1beta-induced tumor necrosis factor (TNF)-alpha release. Treatment with WIN55,212-2 also inhibited production of the chemokines CXCL10, CCL2 and CCL5 by IL-1beta-activated astrocytes. These findings indicate that WIN55,212-2 inhibits the production of inflammatory mediators by IL-1beta-stimulated human astrocytes and suggest that comparable agents may have therapeutic potential for the management of brain inflammation.

Astrocytes↗
PubMed2003

Tolerance to the memory disruptive effects of cannabinoids involves adaptation by hippocampal neurons.

The effects of chronic exposure to cannabinoids on short-term memory in rats were assessed during repeated daily injections of an initially debilitating dose (3.75 mg/kg) of the potent CB1 cannabinoid receptor ligand, WIN 55,212-2. Delayed nonmatch to sample (DNMS) performance was assessed over a 35-day exposure period in which performance was initially disrupted during the first 21 days of exposure but recovered by day 30 and was stable at pre-drug levels for 5 days thereafter. Withdrawal was precipitated by injections of the CB1 receptor antagonist SR141716A and transiently reduced performance for 2 days but was restabilized to pre-drug levels within 3-4 days. Concomitant recording from identified CA1 and CA3 hippocampal neurons demonstrated a marked correspondence in the time course of suppression of peak firing in the sample and delay phases of the task to the drug-induced performance deficits over the same days of exposure. Hippocampal encoding of task-relevant events and performance levels "tracked" each other on a daily basis throughout the chronic cannabinoid treatment and withdrawal regimen. However, hippocampal neuronal activity in the nonmatch phase of the task was unaffected by the chronic cannabinoid treatment or withdrawal, suggesting that only a select population of hippocampal neurons and synapses are involved in cannabinoid-sensitive short-term memory processes.

Action Potentials↗
PubMed2006

Developmental switch from LTD to LTP in low frequency-induced plasticity.

The stimulation of the Schaffer collateral/commissural fibers at low frequency (1 Hz) for 3-5 min can trigger a slow-onset form of low-frequency stimulation (LFS)-long-term potentiation (LTP) (LFS-LTP) in the CA1 area of the adult rat hippocampus. Here we have examined the developmental profile of this plasticity. In 9-15 day-old rats, the application of 1 Hz for 5 min induced long-term depression (LFS-LTD). In 17-21 day-old rats, 1 Hz stimulation had no effect when applied for 5 min but mediated LTD when stimulus duration was increased to 15 min. Over 25 day-old, 1 Hz stimulation mediated LFS-LTP. LFS-LTD was dependent on both N-methyl-D-aspartate (NMDA) and mGlu5 receptor activation. Antagonists of mGlu1alpha and cannabinoid type 1 receptor were ineffective to block LTD induction. LFS-LTD was not associated with a change in paired-pulse facilitation ratio, suggesting a postsynaptic locus of expression of this plasticity. Next, we examined whether LFS-LTD was related to 'chemical' LTDs obtained by the direct stimulation of mGlu5 and NMDA receptors. The saturation of LFS-LTD completely occluded NMDA- and (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG)-induced LTD. CHPG-LTD and NMDA-LTD occluded each other. In addition, we observed that NMDA-LTD was dependent on mGlu5 receptor activation in 9-12 day old rats while it was not in animals older than 15 day-old. Therefore we postulate that during LFS application, NMDA and mGlu5 receptor could interact to trigger LTD. Low-frequency-mediated synaptic plasticity is subject to a developmental switch from NMDA- and mGlu5 receptor-dependent LTD to mGlu5 receptor-dependent LTP with a transient period (17-21 day-old) during which LFS is ineffective.

Age Factors↗
PubMed1998

A phase I double-blind, placebo-controlled, single rising dose study to determine the safety, tolerability, and pharmacokinetics of oral YM934 in healthy male volunteers.

In this phase I study a novel potassium channel opener (YM934) was administered to young healthy male volunteers to identify a dose with minimal cardiovascular effects. The study was carried out using a double-blind, placebo-controlled rising dose design with oral YM934 in single doses of 20 micrograms, 60 micrograms, and 180 micrograms. During each study day frequent blood samples were obtained for drug assay, measurements of cardiovascular parameters, and parasympathetic activity. The drug was well tolerated, with headache being the only significant adverse event. No relationship could be detected between dose or plasma concentration of YM934 and headache. No significant changes in routine laboratory and pharmacodynamic parameters occurred between placebo and active drug treatment. The pharmacokinetics of YM934 were characterized by a rapid oral absorption, an elimination half-life of 30 hours, and an oral clearance of 1.7 L/h. The obtained data allow estimation of a YM934 dose regimen with minimal cardiovascular (side) effects to be used for future efficacy studies.

Administration, Oral↗
PubMed2002

Genotype resistance profiles in patients failing an NNRTI-containing regimen, and modifications after stopping NNRTI therapy.

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) develops quickly and independently if they are used in combination with NRTIs or protease inhibitors (PIs) as rescue therapy, mainly due to the low genetic barrier of this class of drugs. In this study we examined clinical, therapeutic, and virologic characteristics in 88 patients with mutations conferring resistance to NNRTIs, and in 11 patients 1 year after stopping NNRTI therapy. Between patients administered Nevirapine (NVP) and those taking Efavirenz (EFV), no statistical differences were found in CD4 cell count, HIV viral load, time on NNRTI therapy, or number of PIs administered previously. A slow decline in the detectability of mutations encoding NNRTI resistance was found.

Alkynes↗
PubMed2000

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis.

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.

Animals↗
PubMed2001

Cannabinoid receptor agonist WIN 55,212-2 inhibits rat cortical dialysate gamma-aminobutyric acid levels.

The effects of the cannabinoid receptor agonist WIN 55,212-2 (0.1-5 mg/kg i.p.) on endogenous extracellular gamma-aminobutyric acid (GABA) levels in the cerebral cortex of the awake rat was investigated by using microdialysis. WIN 55,212-2 (1 and 5 mg/kg i.p.) was associated with a concentration-dependent decrease in dialysate GABA levels (-16% +/- 4% and -26% +/- 4% of basal values, respectively). The WIN 55,212-2 (5 mg/kg i.p.) induced-inhibition was counteracted by a dose (0.1 mg/kg i.p.) of the CB(1) receptor antagonist SR141716A, which by itself was without effect on cortical GABA levels. These findings suggest that cannabinoids decrease cortical GABA levels in vivo, an action that might underlie some of the cognitive and behavioral effects of acute exposure to marijuana.

Animals↗
PubMed2006

Expression of functional CB1 cannabinoid receptors in retinoic acid-differentiated P19 embryonal carcinoma cells.

Although primary neuronal cell cultures, usually obtained from embryonic or early postnatal rodents, have been used in vitro to study the neural cannabinoid signalling system, development of cell lines with neural properties exhibiting native expression of cannabinoid receptors is desirable. This study was undertaken to investigate the expression of CB1 and CB2 cannabinoid receptors in neurons that develop from retinoic acid (RA)-primed mouse P19 embryonal carcinoma cells. Both undifferentiated P19 cells and RA-treated P19 neurons were positive, by using reverse transcription-polymerase chain reaction (RT-PCR), for CB1 (but not CB2) mRNA. Neuronal differentiation increased the CB1 mRNA expression, and Western blotting with a CB1 receptor antibody showed a strong immunoreactive band at approximately 62 kDa in membranes from P19-derived neurons. The cannabinoid receptor agonists CP 55,940 and HU-210 produced concentration-dependent inhibition of forskolin-induced (3 microM) cyclic AMP production in the P19-derived neurons (29% at 1 microM CP 55,940 and 34% at 1 microM HU-210), which could be blocked by the CB1-selective receptor antagonist AM251, but not by the CB2-selective antagonist AM630. Furthermore, glutamate (100 microM) induced a sustained increase in [Ca2+]i in P19-derived neurons that could be concentration-dependently blocked by the cannabinoid receptor agonists WIN 55,212-2. Thus, the protocol used provides an in vitro model system expressing CB1 cannabinoid receptors at the level of mRNA, protein, and AM251-sensitive agonist-induced inhibition of intracellular cyclic AMP accumulation, which may be useful to investigate the developmental regulation, expression and function of neuronal cannabinoid receptors.

Analysis of Variance↗
PubMed2006

Cannabinoid agonist WIN55,212-2 induces apoptosis in cerebellar granule cells via activation of the CB1 receptor and downregulation of bcl-xL gene expression.

The endogenous cannabinoid system is involved in the regulation of a number of physiologic effects in both the central and peripheral nervous systems. Its role in the control of neuronal cell proliferation has attracted major attention because of its potential implications for new therapeutic strategies. In the present study, we demonstrated that treatment of cultured cerebellar granule cells with the synthetic cannabinoid WIN55,212-2, causes cell-body and nuclear shrinkage, which are hallmarks of neuronal apoptosis, as well as concentration-dependent decrease in cell viability. Staining with the fluorescent nuclear dye, Hoechst 33258, revealed apoptosis in 27.1% and 58.5% of cells exposed to 1 and 10 microM of WIN55,212-2, respectively (P < 0.01 and P < 0.001 vs. control respectively) after 36 hr. After 24 hr of exposure to WIN55,212-2, mRNA levels for the anti-apoptotic gene bcl-xL were reduced to 45.6% of those found in control (P < 0.01). These effects were completely reverted when cells were exposed to the synthetic cannabinoid in the presence of the specific CB1-receptor antagonist, SR141716A (1 microM). Moreover, the pro-apoptotic effect of 10 microM WIN55,212-2 could be reduced by the addition to the incubation medium of a cell-permeant inhibitor of caspase-1 (50 nM). Finally, WIN55,212-2 significantly increased caspase-1 activity after 24 hr. These findings show that the activation of CB1 receptors on cerebellar granule cells induces apoptotic cell death, which is associated with downregulation of the anti-apoptotic gene, bcl-xL, and at least in part, activation of caspase-1.

Analysis of Variance↗
PubMed2007

Acute, chronic and withdrawal effects of the cannabinoid receptor agonist WIN55212-2 on the sequential activation of MAPK/Raf-MEK-ERK signaling in the rat cerebral frontal cortex: short-term regulation by intrinsic and extrinsic pathways.

The cannabinoids (CB) modulate the extracellular signal-regulated kinase (ERK), leading to various forms of plasticity in the brain. Little is known, however, on the in vivo short- and long-term activation and regulation of the components of mitogen-activated protein kinase (MAPK)/ERK signaling by CB. The CB agonist WIN55212-2 (8 mg/kg) increased the immunodensities of phosphorylated c-Raf-1 (42%), MEK1/2 (63%), ERK1 (24%), and ERK2 (28%) in the rat cerebral frontal cortex. These effects were antagonized by SR141716A (rimonabant, 10 mg/kg), a selective CB(1) receptor antagonist. Repeated WIN55212-2 treatment (2-8 mg/kg for 5 days) resulted in tachyphylaxis to the acute activation of Raf-MEK-ERK signaling. Acute WIN55212-2 also induced a hypothermic effect in rats, which was reduced after repeated administration (tolerance). Treatment with SR141716A after chronic WIN55212-2 resulted in the expected cannabinoid withdrawal syndrome, without concomitant alterations in the phosphorylation state of c-Raf-1, MEK1/2, or ERK1/2. Pretreatment with SL327 (20 mg/kg, a MEK1/2 inhibitor) increased the basal phosphorylation of c-Raf-1 (40%) and MEK1/2 (74%; feedback regulation) and fully prevented the up-regulation of ERK1/2 (23-31%) induced by WIN55212-2. Pretreatment with MK801 (1 mg/kg, a NMDA receptor antagonist) effectively blocked the up-regulation c-Raf-1 (41%), MEK1/2 (57%) and ERK1/2 (25-30%) induced by the CB agonist. The main findings demonstrate that the acute stimulation of CB(1) receptors in the frontal cortex results in the sequential phosphorylation of Raf-MEK-ERK cascade, in which c-Raf-1 activation (rate-limiting process) plays a crucial role. Moreover, the in vivo stimulating effect of WIN55212-2 on Raf-MEK-ERK signaling is under the extrinsic regulation of an excitatory glutamatergic mechanism.

Animals↗
PubMed2003

Effects of ATP-sensitive K+ channel openers and tolterodine on involuntary bladder contractions in a pig model of partial bladder outlet obstruction.

AIMS: To compare in vivo the efficacy, potency, and bladder-vascular selectivity of ATP-sensitive potassium channel openers (KCOs), YM934 and (-)-cromakalim to a muscarinic antagonist, tolterodine in a novel partial outlet obstructed pig model. METHODS: Partially obstructed female Landrace pigs were implanted with telemetry transmitters to allow the continuous measurement of intravesical, abdominal and arterial pressures. A subcutaneous port catheter was used to adjust bladder volume. Bladder and arterial pressure were simultaneously monitored under isoflurane anesthesia before and after increasing i.v. doses of test compounds. RESULTS: Under anesthesia, voiding was completely inhibited, but spontaneous, nonvoiding bladder contractions were observed with mean amplitude of 16 +/- 1 cm H(2)O, duration of 35 +/- 2 seconds, and intercontraction interval of 43 +/- 4 seconds (n = 25). YM934 and (-)-cromakalim both caused dose-dependent decreases in bladder contraction area under the curve (AUC) with effective doses to inhibit AUC by 35% of 3.6 and 14.9 nmol/kg, i.v., respectively. However, concomitant reductions in mean arterial pressure of 12 and 13% were also observed. Tolterodine did not inhibit spontaneous bladder contractions at doses up to 100 nmol/kg, i.v. corresponding to plasma concentrations up to 41 ng/mL. CONCLUSIONS: The superior efficacy of KCOs to inhibit spontaneous bladder contractions relative to tolterodine support the hypothesis that KCOs may provide an alternate therapeutic mechanism to treat symptoms of overactive bladder if bladder-vascular selectivity can be sufficiently improved. The minimally invasive model described herein appears useful in the preclinical evaluation of potential therapeutics targeted to treat the overactive bladder.

Adenosine Triphosphate↗
PubMed2002

Inhibitor binding alters the directions of domain motions in HIV-1 reverse transcriptase.

Understanding the molecular mechanisms of HIV-1 reverse transcriptase (RT) action and drug inhibition is essential for designing effective antiretroviral therapies. Although comparisons of the different crystal forms of RT give insights into the flexibility of different domains, a direct computational assessment of the effect of inhibitor binding on the collective dynamics of RT is lacking. A structure-based approach is used here for exploring the dynamics of RT in unliganded and inhibitor-bound forms. Non-nucleoside RT inhibitors (NNRTI) are shown to interfere directly with the global hinge-bending mechanism that controls the cooperative motions of the p66 fingers and thumb subdomains. The net effect of nevirapine binding is to change the direction of domain movements rather than suppress their mobilities. The second generation NNRTI, efavirenz, on the other hand, shows the stronger effect of simultaneously reorienting domain motions and obstructing the p66 thumb fluctuations. A second hinge site controlling the global rotational reorientations of the RNase H domain is identified, which could serve as a target for potential inhibitors of RNase H activity.

Alkynes↗
PubMed2005

Quantum study of mutational effect in binding of efavirenz to HIV-1 RT.

Full quantum mechanical computational study has been carried out to study binding of efavirenz (EFZ), a second generation FDA approved nonnucleoside inhibitor, to HIV-1 reverse transcriptase (RT) and its K103N and Y181C mutants using the MFCC (molecular fractionation with conjugate caps) method. The binding interaction energies between EFZ and each protein fragment are calculated using a combination of HF/3-21G, B3LYP/6-31G* and MP2/6-31G* ab initio levels. The present computation shows that Efavirenz binds to HIV-1 RT predominantly through strong electrostatic interaction with the Lys101 residue. The small loss of binding to K103N mutant by Efavirenz can be attributed to a slightly weakened attractive interaction between the drug and Lys101 due to a conformational change of mutation. The small loss of binding to Y181C mutant by efavirenz can be attributed to the Glu698 residue moving closer to EFZ due to conformational change, which results in an increase of repulsive energy relative to the wild type (WT). The binding of efavirenz-derived DPC961 to HIV-1 RT is enhanced by an additional attractive interaction to residue Hid235 and reduced repulsion to Glu698, resulting in an increase of binding energy by about 4 kcal/mol.

Alkynes↗