HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Just-in-Time Compound Pooling Increases Primary Screening Capacity without Compromising Screening Quality.

Compound pooling, or multiplexing more than one compound per well during primary high-throughput screening (HTS), is a controversial approach with a long history of limited success. Many issues with this approach likely arise from long-term storage of library plates containing complex mixtures of compounds at high concentrations. Due to the historical difficulties with using multiplexed library plates, primary HTS often uses a one-compound-one-well approach. However, as compound collections grow, innovative strategies are required to increase the capacity of primary screening campaigns. Toward this goal, we have developed a novel compound pooling method that increases screening capacity without compromising data quality. This method circumvents issues related to the long-term storage of complex compound mixtures by using acoustic dispensing to enable "just-in-time" compound pooling directly in the assay well immediately prior to assay. Using this method, we can pool two compounds per well, effectively doubling the capacity of a primary screen. Here, we present data from pilot studies using just-in-time pooling, as well as data from a large >2-million-compound screen using this approach. These data suggest that, for many targets, this method can be used to vastly increase screening capacity without significant reduction in the ability to detect screening hits.

Development of industrially feasible concentrated 30% and 40% nanoemulsions for intravenous drug delivery.

Emulsions for parenteral nutrition loaded with drugs are used for optimized drug delivery, but in case of poorly oil soluble drugs, the injection volume can be too large when using commercial 10-20% oil emulsions. To reduce the injection volume, the feasibility of producing injectable, physically stable concentrated emulsions up to 40% oil content was investigated. Emulsions were made from fish oil, stabilized with egg lecithin, using high-pressure homogenization. Emulsions with oil contents of 10%-40% were investigated to assess basic correlations between increasing oil content, applied production pressures, homogenization cycles and resulting bulk droplet size, content of larger particles, zeta potential, viscosity and short-term stability. The observed correlations showed that in high-pressure homogenization, the contribution of the dispersive effect dominated the coalescence effect at low and Optimum production conditions for 30% and 40% nanoemulsions, i.e. 800 bar and 2 -3 homogenization cycles, were established on lab scale. These production conditions are industrially feasible. The obtained droplet sizes (about 200 nm) and the content of larger droplets were comparable to 10% commercial emulsions of parenteral nutrition, being important for in vivo tolerability and organ distribution. Despite the high oil concentration, the viscosity of the nanoemulsions was sufficiently low for injection. The short-term storage study showed physical stability for 1 month. A concentrated nanoemulsion base formulation from regulatory accepted excipients is now available, ready for loading with drugs.

Depletion of dopamine in the prefrontal cortex decreases the basal electrophysiological activity of mesolimbic dopamine neurons.

One hypothesis regarding the etiology of schizophrenia proposes that disruption of the dopaminergic innervation of the prefrontal cortex leads to an increase in dopamine (DA) transmission in subcortical regions. In the present study, we examined the effect of 6-hydroxydopamine lesions of the medial prefrontal cortex (mPFC) dopamine innervation on the spontaneous electrophysiological activity of ventral tegmental DA neurons recorded in vivo. DA cell activity was assessed along three dimensions: (1) the relative proportion of DA neurons exhibiting spontaneous activity, (2) their basal firing rate, and (3) the mean percentage of spikes fired in bursts. In lesioned rats, DA neurons in the ventral tegmental area (VTA) exhibited a significantly slower mean firing rate, as well as a significant reduction in the percentage of spikes fired in bursts relative to controls. In contrast, depletion of DA in the mPFC did not have a significant effect on the relative proportion of VTA DA neurons exhibiting spontaneous activity. We suggest that by reducing the basal electrophysiological activity of VTA DA neurons, mPFC DA depletion may lead to an increase in the level of responsivity of the system to excitatory stimuli. Thus, the magnitude of increase in action potential-dependent DA release that occurs in response to a challenge may be augmented in lesioned rats.

Activation of dopamine cell firing by repeated L-DOPA administration to dopamine-depleted rats: its potential role in mediating the therapeutic response to L-DOPA treatment.

The administration of L-dihydroxyphenylalanine (L-DOPA) to patients with Parkinson's disease is known to produce acute effects that include the reduction of rigidity as well as delayed therapeutic actions involving the resumption of complex motor behavior. In order to examine the potential role of dopamine (DA) cell activity in mediating these responses, the effects of acute and repeated L-DOPA administration on the electrophysiological activity of the residual dopamine (DA) neurons were examined in rats that had received partial 6-hydroxydopamine (6-OHDA)-induced DA lesions. DA cell activity was assessed along three dimensions: (1) the relative proportion of DA neurons exhibiting spontaneous spike firing, (2) their basal firing rate, and (3) their firing pattern. Following 6-OHDA-induced DA depletion, rats were treated for 1 month with saline or L-DOPA. In addition, rats from each group received either an acute injection of L-DOPA or saline on the day of recording. In rats receiving repeated saline treatment, the DA neurons recorded following acute L-DOPA administration were firing at significantly slower basal firing rates and exhibited less burst firing when compared to saline-pretreated rats given acute saline. In contrast, DA cells recorded from rats that had received repeated L-DOPA administration for 4 weeks followed by an acute saline injection did not exhibit any significant differences from DA cells of intact control rats with respect to basal firing rate or firing pattern; however, there was a substantial increase in the proportion of DA neurons exhibiting spontaneous spike firing after correcting for 6-OHDA-induced cell loss. In addition, in rats receiving repeated L-DOPA treatment, the DA cells recorded following acute administration of L-DOPA showed significantly less of a reduction in firing rate when compared to the cells recorded following acute L-DOPA in the saline treatment group. These results show that: (1) acute L-DOPA administration appears to exert its actions by DA autoreceptor stimulation, whereas (2) repeated L-DOPA administration increases the proportion of spontaneously active DA neurons in partially lesioned rats. As a result, repeated L-DOPA administration would be expected to cause an increase in spike-dependent DA release as a consequence of the greater proportion of DA cells showing spontaneous activity. This may be the major factor underlying the delayed therapeutic benefits of L-DOPA therapy in the treatment of Parkinson's disease.

Structure-activity relationship studies of CNS agents. Part 14: Structural requirements for the 5-HT1A and 5-HT2A receptor selectivity of simple 1-(2-pyrimidinyl)piperazine derivatives.

The 5-HT1A and 5-HT2A receptor affinity of model 1-(2-pyrimidinyl)-piperazine derivatives 15-21 and 23-32 has been determined. 2-(N-Methylpiperazino)-4,6-di(2-thienyl)pyrimidine 26 is a new, highly active and selective 5-HT2A receptor ligand. The topography of a molecule and the stereoelectronic effects of the thiophene rings are the major factors responsible for the high affinity and selectivity of 26 towards 5-HT2A sites.

Effects of amygdala lesions on reflex facilitation and conditioned response acquisition during nictitating membrane response conditioning in rabbit.

The present study demonstrated that large lesions of the amygdala disrupt the maintenance of reflex facilitation of the unconditioned nictitating membrane (NM) response and slow the acquisition of conditioned NM responses in rabbit. Before behavioral training, the central nucleus of the amygdala and adjacent areas were lesioned electrolytically. In the 1st experiment, the lesioned animals exhibited no reflex facilitation of the unconditioned NM response at conditioned stimulus (CS)-unconditioned stimulus (US) intervals of 125-8,000 ms. In the 2nd and 3rd experiments in which one CS-US interval (500 ms) was used, the lesions disrupted the maintenance of reflex facilitation but did not alter the facilitation exhibited in the 1st block of training. The lesions retarded the acquisition of conditioned NM responses when the 1000-Hz tone CS intensity was 65 dB but not when the intensity was 85 dB.

Quantitative structure-activity relationship analysis of cation-substituted polyaromatic compounds as potentiators (amplifiers) of bleomycin-mediated degradation of DNA.

A set of 21 polyheteroaromatic compounds substituted with flexible cationic groups and of similar molecular size has been analyzed for binding with DNA and for effects of the bleomycin-mediated degradation of the DNA double helix. Increases in apparent rates of the DNA digestion were observed in all cases under the experimental conditions of noncompetitive binding of these compounds and bleomycin to DNA. Surprisingly, the quantitative structure-activity relationship analysis revealed two distinct correlations despite close structural similarities for the set of bleomycin amplifiers. These unusual results are explained in terms of the formation of two stereochemically different ternary complexes of activated bleomycin-DNA-amplifier. The relevance of this finding for the design of new bleomycin amplifiers is discussed.

Molecular basis for potentiation of bleomycin-mediated degradation of DNA by polyamines. Experimental and molecular mechanical studies.

The bleomycin-mediated degradation of DNA is stimulated (amplified) by certain DNA binding compounds, such as polyamines, that distort the double helix. Computer modelling studies suggest that putrescine (1), spermidine (2), and spermine (3) bind preferentially on the floor of the major groove of (dGdC)5.(dGdC)5. This interaction results in a bend of the oligomer helix toward the major groove and enlargement of the minor groove, both effects being in the order 1 less than 2 less than 3. These polyamine-induced distortions, as obtained from theoretical studies, parallel the experimental values of the amplification activities of 1-3 in the bleomycin-mediated degradation of poly(dGdC).poly(dGdC). The amplification mechanism of non-competitive binding of amplifier molecules in the major groove, and bleomycin in the minor groove, is proposed. It is suggested that the amplifier-induced conformational changes of the DNA helix increase affinity of the activated bleomycin complex toward the DNA minor groove and, consequently, result in an increased efficiency of the bleomycin-mediated degradation of the helix.

Interaction of unfused tricyclic aromatic cations with DNA: a new class of intercalators.

Unfused tricyclic aromatic ring systems 1-6 with one or two cationic side chains have been synthesized and their interactions with DNA and synthetic polymers probed with a variety of techniques. Molecular mechanics calculations indicate that the torsional angle between ring planes in the minimum energy conformation of the tricyclic molecules can range from 0 degree to as high as 50 degrees depending on the type of rings and substituents. Viscometric titrations with linear and supercoiled DNA, linear dichroism, and NMR studies indicated that all compounds with torsional angles of approximately 20 degrees or less bind to DNA by intercalation. The more highly twisted intercalators caused significant perturbation of DNA structure. Unfused intercalators with twist angles of approximately 20 degrees have reduced binding constants, suggesting that they could not form an optimum interaction with the DNA base pairs. Unfused intercalators with twist less than 20 degrees formed strong complexes with DNA. The structures of these unfused intercalators are more analogous to typical groove-binding molecules, and an analysis of their interaction with DNA provides a better understanding of the subtle differences between intercalation and groove-binding modes for aromatic cations. The results indicate that intercalation and groove-binding modes should be viewed as two potential wells on a continuous energy surface. The results also suggest design strategies for intercalators that can optimally complement DNA base pair propeller twist or that can induce bends in DNA at the intercalation site.

Molecular basis for bleomycin amplification: conformational and stereoelectronic effects in unfused amplifiers.

Sixteen unfused heterobiaromatic and biphenyl compounds substituted with an amino side chain (protonated in water) have been tested for (i) binding with DNA and (ii) their effect on the digestion of the DNA double helix by a bleomycin-iron complex. Only the DNA intercalating molecules amplify the digestion of DNA. One 2,2'-bipyridine derivative tested is an inhibitor of the bleomycin reaction because it removes ferrous ion from the bleomycin complex. Polarity of the intercalating unfused biaromatic system is of primary importance for effective binding of the molecule with native DNA and, at the same time, for its amplification activity. The molecules that have the biaromatic system polarized extensively in the direction of the side cationic chain, so that the intercalating sites constitutes a positive part of the dipole, show strong binding with DNA and good amplification activity. For strong intercalative forces that determine the amplification activity, it is important that both the heteroaromatic subsystems of the molecule have positive ends of their dipoles positioned away from the side chain. This work provides general guidelines for synthesis of new highly effective bleomycin amplifiers.

Assessing the driving potential of the handicapped.

Proper and early assessment of the handicapped person's potential for driving a vehicle depends upon an understanding of the factors involved in a driver evaluation. The driver evaluation factors discussed in this paper are: driving aids and their application; the handicapped driver's physical, perceptive, and cognitive capabilities; and the psychosocial characteristics that influence a client's ability and need for driving.