Identification of novel HSP90α/ß isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as Huntington's disease.

A structure-based drug design strategy was used to optimize a novel benzolactam series of HSP90α/ß inhibitors to achieve >1000-fold selectivity versus the HSP90 endoplasmic reticulum and mitochondrial isoforms (GRP94 and TRAP1, respectively). Selective HSP90α/ß inhibitors were found to be equipotent to pan-HSP90 inhibitors in promoting the clearance of mutant huntingtin protein (mHtt) in vitro, however with less cellular toxicity. Improved tolerability profiles may enable the use of HSP90α/ß selective inhibitors in treating chronic neurodegenerative indications such as Huntington's disease (HD). A potent, selective, orally available HSP90α/ß inhibitor was identified (compound 31) that crosses the blood-brain barrier. Compound 31 demonstrated proof of concept by successfully reducing brain Htt levels following oral dosing in rats.

A simple experimentally based model using proprioceptive regulation of motor primitives captures adjusted trajectory formation in spinal frogs.

Spinal circuits may organize trajectories using pattern generators and synergies. In frogs, prior work supports fixed-duration pulses of fixed composition synergies, forming primitives. In wiping behaviors, spinal frogs adjust their motor activity according to the starting limb position and generate fairly straight and accurate isochronous trajectories across the workspace. To test whether a compact description using primitives modulated by proprioceptive feedback could reproduce such trajectory formation, we built a biomechanical model based on physiological data. We recorded from hindlimb muscle spindles to evaluate possible proprioceptive input. As movement was initiated, early skeletofusimotor activity enhanced many muscle spindles firing rates. Before movement began, a rapid estimate of the limb position from simple combinations of spindle rates was possible. Three primitives were used in the model with muscle compositions based on those observed in frogs. Our simulations showed that simple gain and phase shifts of primitives based on published feedback mechanisms could generate accurate isochronous trajectories and motor patterns that matched those observed. Although on-line feedback effects were omitted from the model after movement onset, our primitive-based model reproduced the wiping behavior across a range of starting positions. Without modifications from proprioceptive feedback, the model behaviors missed the target in a manner similar to that in deafferented frogs. These data show how early proprioception might be used to make a simple estimate initial limb state and to implicitly plan a movement using observed spinal motor primitives. Simulations showed that choice of synergy composition played a role in this simplicity. To generate froglike trajectories, a hip flexor synergy without sartorius required motor patterns with more proprioceptive knee flexor control than did patterns built with a more natural synergy including sartorius. Such synergy choices and control strategies may simplify the circuitry required for reflex trajectory construction and adaptation.

N-[6-amino-2-(heteroaryl)pyrimidin-4-yl]acetamides as A2A receptor antagonists with improved drug like properties and in vivo efficacy.

In the present article, we report on a strategy to improve the physical properties of a series of small molecule human adenosine 2A (hA2A) antagonists. One of the aromatic rings typical of this series of antagonists is replaced with a series of aliphatic groups, with the aim of disrupting crystal packing of the molecule to lower the melting point and in turn to improve the solubility. Herein, we describe the SAR of a new series of water-soluble 2,4,6-trisubstituted pyrimidines where R1 is an aromatic heterocycle, R2 is a short-chain alkyl amide, and the typical R3 aromatic heterocyclic substituent is replaced with an aliphatic amino substituent. This approach significantly enhanced aqueous solubility and lowered the log P of the system to provide molecules without significant hERG or CYP liabilities and robust in vivo efficacy.

Lead optimization of 4-acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6-pyridylpyrimidines as A2A adenosine receptor antagonists for the treatment of Parkinson's disease.

4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinson's disease. The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of l-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.

Trunk sensorimotor cortex is essential for autonomous weight-supported locomotion in adult rats spinalized as P1/P2 neonates.

Unlike adult spinalized rats, approximately 20% of rats spinalized as postnatal day 1 or 2 (P1/P2) neonates achieve autonomous hindlimb weight support. Cortical representations of mid/low trunk occur only in such rats with high weight support. However, the importance of hindlimb/trunk motor cortex in function of spinalized rats remains unclear. We tested the importance of trunk sensorimotor cortex in their locomotion using lesions guided by cortical microstimulation in P1/P2 weight-supporting neonatal spinalized rats and controls. In four intact control rats, lesions of hindlimb/trunk cortex caused no treadmill deficits. All spinalized rats lesioned in trunk cortex (n = 16: 4 transplant, 6 transect, 6 transect + fibrin glue) lost an average of about 40% of their weight support. Intact trunk cortex was essential to their level of function. Lesion of trunk cortex substantially increased roll of the hindquarters, which correlated to diminished weight support, but other kinematic stepping parameters showed little change. Embryonic day 14 (E14) transplants support development of the trunk motor representations in their normal location. We tested the role of novel relay circuits arising from the grafts in such cortical representations in E14 transplants using the rats that received (noncellular) fibrin glue grafting at P1/P2 (8 allografts and 32 xenografts). Fibrin-repaired rats with autonomous weight support also had trunk cortical representations similar to those of E14 transplant rats. Thus acellular repair and intrinsic plasticity were sufficient to support the observed features. Our data show that effective cortical mechanisms for trunk control are essential for autonomous weight support in P1/P2 spinalized rats and these can be achieved by intrinsic plasticity.

Individual premotor drive pulses, not time-varying synergies, are the units of adjustment for limb trajectories constructed in spinal cord.

Complex actions may arise by combining simple motor primitives. Our studies support individual premotor drive pulses or bursts as execution primitives in spinal cord. Alternatively, the fundamental execution primitives at the segmental level could be time-varying synergies. To distinguish these hypotheses, we examined sensory feedback effects during targeted wiping organized in spinal cord. This behavior comprises three bursts. We tested (1) whether feedback altered the structure of individual premotor drive bursts or primitives, and (2) whether feedback differentially modulated different drive bursts or pulses in the three burst sequence. At least two of the three bursts would need to always be comodulated to support a time-varying synergy. We used selective muscle vibration to control spindle feedback from a single muscle (biceps/iliofibularis). The structures of premotor drive bursts were conserved. However, biceps vibration (1) scaled the amplitudes of two bursts coactivated during the initial phase of wiping independently of one another without altering their phase, and (2) independently phase regulated the third burst but preserved its amplitude. Thus, all three bursts were regulated separately. Durations were unaffected. The independent effects depended on (1) time of vibration during wiping, (2) frequency of vibration, and (3) limb configuration. Because each of the three bursts was independently modulated, these data strongly support execution using individual premotor bursts rather than time-varying synergies at the spinal level of motor organization. Our data show that both sensory feedback and central systems of the spinal cord act in concert to adjust the individual premotor bursts in support of the straight and unimodal wiping trajectory.

Dopamine signaling and the distal reward problem.

Actions and their associated consequences, such as reward attainment, are often temporally distant. Animals nevertheless learn such associations thereby solving the 'distal reward' problem. We sought to determine whether dopamine signaling plays a role in such learning. Wild-type and dopamine type I receptor knockout mice executed three left/right choices leading to one of eight differentially rewarded goal sites. Compared with wild-type mice, knockouts exhibited selective impairments in decision making at choice points distal, but not proximal, to goal sites. We conclude that dopamine's role in reinforcement learning depends on the temporal relationship of actions to reward and that dopamine signaling through D1 receptors constitutes a component of those brain mechanisms responsible for solving the distal reward problem.

Adaptation of prefrontal cortical firing patterns and their fidelity to changes in action-reward contingencies.

Animals adapt action-selection policies when the relationship between possible actions and associated outcomes changes. Prefrontal cortical neurons vary their discharge patterns depending on action choice and rewards received and undoubtedly play a pivotal role in maintaining and adapting action policies. Here, we recorded neurons from the medial precentral subregion of mouse prefrontal cortex to examine neural substrates of goal-directed behavior. Discharge patterns were recorded after animals developed stable action-selection policies, wherein four possible action sequences were invariably related to different reward magnitudes and during adaptation to changes in the action-reward contingencies. During the adaptation period, when the same action sequence resulted in different reward magnitudes, many neurons (38%) exhibited significantly different discharge patterns for identical action sequences, well before reaching the reward site. In addition, trial-to-trial reliability of ensemble pattern production leading up to reward was found to vary both positively and negatively with increases and decreases in reward magnitude, respectively. Pairwise analyses of simultaneously recorded neurons revealed that decreased reliability in part reflected fluctuations between different ensemble activity patterns as opposed to within-pattern variability. Increases in reliability were related to an increased probability of both selecting highly rewarding actions and completing such actions without pause or reversal, whereas decreases in reliability were associated with the opposite pattern. Thus, we suggest that both the spatiotemporal pattern and fidelity of prefrontal cortical discharge are impacted by action-outcome relationships and that each of these features serve to adapt action choices and maintain behaviors leading to reward.

Improvements in the signal-to-noise ratio of motor cortex cells distinguish early versus late phases of motor skill learning.

There are numerous experience-driven changes in cortical circuitry that correlate with improved performance. Improved motor performance on a reach-to-grasp task in rodents is associated with changes in long-term potentiation (LTP), synaptogenesis, and movement representations in primary motor cortex (M1) by training days 3, 7, and 10, respectively. We recorded single-cell activity patterns in M1 during reach-to-grasp training to test how neural-spiking properties change with respect to LTP, synaptogenesis, and motor map changes. We also tested how neural-spiking changes relate directly to improved performance by monitoring muscle activity patterns. We found that signal-to-noise ratios (SNRs) of M1 spiking were significantly improved with practice but only after 7-12 d. Three sources of noise were assessed: signal-dependent noise exemplified by the slope of the relationship between mean spike count and count variance per burst, signal-independent noise exemplified by the offset of this relationship, and background firing rates before and after bursts. Signal-independent noise and pre-burst firing rates were reduced with practice. Early performance gains (days 1-6) were dissociated from SNR improvements, whereas later performance gains (day 7-12) were related directly to the magnitude of improvement in both muscle recruitment reliability and success rates. With training, an increased number of cells exhibited firing rates that were correlated with muscle recruitment patterns, with lags suggesting a primary direction of influence from M1 to muscles. These results suggest a functional linkage from local synaptogenesis in M1 to improved spiking reliability of M1 cells to more reliable recruitment of muscles and finally to improved behavioral performance.

Early skill learning is expressed through selection and tuning of cortically represented muscle synergies.

Skill learning may be based on integrating and adapting movement building blocks organized in the CNS. We examined at what level integration and adaptation occur during early skill learning, the level of individual muscles, muscle synergies or combinations of synergies through time, and whether these operations are expressed through the primary motor cortex (M1). Forelimb muscle and M1 cell activity were recorded over the first day of training on a reach-to-grasp task in rodents. Independent components analysis was used to assess how well muscle activation patterns could be described as time-varying combinations of synergies. In 3 of 11 animals, prereach M1 activity predicted the activation of different combinations of independent components (ICs) to perform the task. With training, animals increasingly adopted postures and prereach patterns of M1 activity that supported activation of the more successful combination. With training, animals also adjusted the activation magnitude (6 of 11 animals) and weights (11 of 11) of specific ICs that constituted the selected combination. Weights represent how IC activation patterns were distributed to forelimb muscles; this distribution pattern was adapted with training. M1 cells (37 of 100) had task-related firing rates that were significantly correlated with IC activation patterns. Changes in M1 firing rates were associated with corresponding changes in either the activation magnitude or weights of the correlated IC. Our data suggest that early skill learning is expressed through selection and tuning of M1 firing rates, which specify time-varying patterns of synergistic muscle contractions in the limb.

Jumping in frogs: assessing the design of the skeletal system by anatomically realistic modeling and forward dynamic simulation.

Comparative musculoskeletal modeling represents a tool to understand better how motor system parameters are fine-tuned for specific behaviors. Frog jumping is a behavior in which the physical properties of the body and musculotendon actuators may have evolved specifically to extend the limits of performance. Little is known about how the joints of the frog contribute to and limit jumping performance. To address these issues, we developed a skeletal model of the frog Rana pipiens that contained realistic bones, joints and body-segment properties. We performed forward dynamic simulations of jumping to determine the minimal number of joint degrees of freedom required to produce maximal-distance jumps and to produce jumps of varied take-off angles. The forward dynamics of the models was driven with joint torque patterns determined from inverse dynamic analysis of jumping in experimental frogs. When the joints were constrained to rotate in the extension-flexion plane, the simulations produced short jumps with a fixed angle of take-off. We found that, to produce maximal-distance jumping, the skeletal system of the frog must minimally include a gimbal joint at the hip (three rotational degrees of freedom), a universal Hooke's joint at the knee (two rotational degrees of freedom) and pin joints at the ankle, tarsometatarsal, metatarsophalangeal and iliosacral joints (one rotational degree of freedom). One of the knee degrees of freedom represented a unique kinematic mechanism (internal rotation about the long axis of the tibiofibula) and played a crucial role in bringing the feet under the body so that maximal jump distances could be attained. Finally, the out-of-plane degrees of freedom were found to be essential to enable the frog to alter the angle of take-off and thereby permit flexible neuromotor control. The results of this study form a foundation upon which additional model subsystems (e.g. musculotendon and neural) can be added to test the integrative action of the neuromusculoskeletal system during frog jumping.

Functional morphology of proximal hindlimb muscles in the frog Rana pipiens.

Musculoskeletal models have become important tools in understanding motor control issues ranging from how muscles power movement to how sensory feedback supports movements. In the present study, we developed the initial musculotendon subsystem of a realistic model of the frog Rana pipiens. We measured the anatomical properties of 13 proximal muscles in the frog hindlimb and incorporated these measurements into a set of musculotendon actuators. We examined whether the interaction between this musculotendon subsystem and a previously developed skeleton/joint subsystem captured the passive behavior of the real frog's musculoskeletal system. To do this, we compared the moment arms of musculotendon complexes measured experimentally with moment arms predicted by the model. We also compared sarcomere lengths measured experimentally at the starting and take-off positions of a jump with sarcomere lengths predicted by the model at these same limb positions. On the basis of the good fit of the experimental data, we used the model to describe the multi-joint mechanical effects produced by contraction of each hindlimb muscle and to predict muscle trajectories during a range of limb behaviors (wiping, defensive kicking, swimming and jumping). Through these analyses, we show that all hindlimb muscles have multiple functions with respect to accelerating the limb in its three-dimensional workspace and that the balance of functions depends greatly on limb configuration. In addition, we show that muscles have multiple, task-specific functions with respect to the type of contraction performed. The results of this study provide important data regarding the multifunctional role of hindlimb muscles in the frog and form a foundation upon which additional model subsystems (e.g. neural) and more sophisticated muscle models can be appended.