Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness.

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 µm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 µM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Phase separation by ssDNA binding protein controlled via protein-protein and protein-DNA interactions.

Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential.

Blebbistatin is a widely used inhibitor of myosin 2 that enables the study of a broad range of cytoskeleton-related processes. However, blebbistatin has several limitations hindering its applicability: it is fluorescent, poorly water soluble, cytotoxic, and prone to (photo)degradation. Despite these adverse effects, being the only available myosin 2-specific inhibitor, blebbistatin is rather a choice of necessity. Blebbistatin has been modified to improve its properties and some of the new compounds have proven to be useful replacements of the original molecule. This review summarizes recent results on blebbistatin development. We also discuss the pharmacological perspectives of these efforts, as myosins are becoming promising drug target candidates for a variety of conditions ranging from neurodegeneration to muscle disease, wound healing, and cancer metastasis.

Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

Structural model of weak binding actomyosin in the prepowerstroke state.

We present the first in silico model of the weak binding actomyosin in the initial powerstroke state, representing the actin binding-induced major structural changes in myosin. First, we docked an actin trimer to prepowerstroke myosin then relaxed the complex by a 100-ns long unrestrained molecular dynamics. In the first few nanoseconds, actin binding induced an extra primed myosin state, i.e. the further priming of the myosin lever by 18° coupled to a further closure of switch 2 loop. We demonstrated that actin induces the extra primed state of myosin specifically through the actin N terminus-activation loop interaction. The applied in silico methodology was validated by forming rigor structures that perfectly fitted into an experimentally determined EM map of the rigor actomyosin. Our results unveiled the role of actin in the powerstroke by presenting that actin moves the myosin lever to the extra primed state that leads to the effective lever swing.

Magnesium modulates actin binding and ADP release in myosin motors.

We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, ß-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg(2+)-dependent manner (0.3-9.0 mm free Mg(2+)) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg(2+) in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg(2+) in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg(2+) coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg(2+) concentrations, demonstrating that the ADP release rate constant is slowed by Mg(2+) in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg(2+) reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg(2+) inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg(2+)-dependent alterations in actin binding. Overall, our results suggest that Mg(2+) reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.

Azidoblebbistatin, a photoreactive myosin inhibitor.

Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC(50) ≥ 50 µM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC(50) = 5 µM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.

Emerging complex pathways of the actomyosin powerstroke.

Actomyosin powers muscle contraction and various cellular activities, including cell division, differentiation, intracellular transport and sensory functions. Despite their crucial roles, key aspects of force generation have remained elusive. To perform efficient force generation, the powerstroke must occur while myosin is bound to actin. Paradoxically, this process must be initiated when myosin is in a very low actin-affinity state. Recent results shed light on a kinetic pathway selection mechanism whereby the actin-induced activation of the swing of myosin's lever enables efficient mechanical functioning. Structural elements and biochemical principles involved in this mechanism are conserved among various NTPase-effector (e.g. kinesin-microtubule, G protein exchange factor and kinase-scaffold protein) systems that perform chemomechanical or signal transduction.

Viscosity dependence of passage through a fluctuating bottleneck.

We generalize the model of a rate process involving the passage of an object through a fluctuating bottleneck. The rate of passage is considered to be proportional to a power function of the radius of the bottleneck with exponent α > 0. The fluctuations of the bottleneck are coupled to the motion of the surrounding medium and governed by Langevin dynamics. We show numerically and also explain analytically that for slow bottleneck fluctuations the long time decay rate of the process has a fractional power law dependence on the solvent viscosity with exponent α/(α + 2). The results are consistent with the experimental data on ligand binding to myoglobin, and might also be relevant to other reactions for which exponents between 0 and 1 were reported.

para-Nitroblebbistatin, the non-cytotoxic and photostable myosin†II inhibitor.

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.

Internal friction in enzyme reactions.

The empirical concept of internal friction was introduced 20 years ago. This review summarizes the results of experimental and theoretical studies that help to uncover the nature of internal friction. After the history of the concept, we describe the experimental challenges in measuring and interpreting internal friction based on the viscosity dependence of enzyme reactions. We also present speculations about the structural background of this viscosity dependence. Finally, some models about the relationship between the energy landscape and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions are also discussed.

Virtual affinity fingerprints for target fishing: a new application of Drug Profile Matching.

We recently introduced Drug Profile Matching (DPM), a novel virtual affinity fingerprinting bioactivity prediction method. DPM is based on the docking profiles of ca. 1200 FDA-approved small-molecule drugs against a set of nontarget proteins and creates bioactivity predictions based on this pattern. The effectiveness of this approach was previously demonstrated for therapeutic effect prediction of drug molecules. In the current work, we investigated the applicability of DPM for target fishing, i.e. for the prediction of biological targets for compounds. Predictions were made for 77 targets, and their accuracy was measured by Receiver Operating Characteristic (ROC) analysis. Robustness was tested by a rigorous 10-fold cross-validation procedure. This procedure identified targets (N = 45) with high reliability based on DPM performance. These 45 categories were used in a subsequent study which aimed at predicting the off-target profiles of currently approved FDA drugs. In this data set, 79% of the known drug-target interactions were correctly predicted by DPM, and additionally 1074 new drug-target interactions were suggested. We focused our further investigation on the suggested interactions of antipsychotic molecules and confirmed several interactions by a review of the literature.

Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke.

The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.

Post-reperfusion acute MR diffusion in stroke is a potential predictor for clinical outcome in rats.

Middle cerebral artery occlusion (MCAO) models show substantial variability in outcome, introducing uncertainties in the evaluation of treatment effects. Early outcome predictors would be essential for prognostic purposes and variability control. We aimed to compare apparent diffusion coefficient (ADC) MRI data obtained during MCAO and shortly after reperfusion for their potentials in acute-phase outcome prediction. Fifty-nine male rats underwent a 45-min MCAO. Outcome was defined in three ways: 21-day survival; 24 h midline-shift and neurological scores. Animals were divided into two groups: rats surviving 21 days after MCAO (survival group, n = 46) and rats dying prematurely (non-survival/NS group, n = 13). At reperfusion, NS group showed considerably larger lesion volume and lower mean ADC of the initial lesion site (p < 0.0001), while during occlusion there were no significant group differences. At reperfusion, each survival animal showed decreased lesion volume and increased mean ADC of the initial lesion site compared to those during occlusion (p < 10-6), while NS group showed a mixed pattern. At reperfusion, lesion volume and mean ADC of the initial lesion site were significantly associated with 24 h midline-shift and neurological scores. Diffusion MRI performed soon after reperfusion has a great impact in early-phase outcome prediction, and it works better than the measurement during occlusion.

Temperature dependence of internal friction in enzyme reactions.

Our aim was to elucidate the physical background of internal friction of enzyme reactions by investigating the temperature dependence of internal viscosity. By rapid transient kinetic methods, we directly measured the rate constant of trypsin 4 activation, which is an interdomain conformational rearrangement, as a function of temperature and solvent viscosity. We found that the apparent internal viscosity shows an Arrhenius-like temperature dependence, which can be characterized by the activation energy of internal friction. Glycine and alanine mutations were introduced at a single position of the hinge of the interdomain region to evaluate how the flexibility of the hinge affects internal friction. We found that the apparent activation energies of the conformational change and the internal friction are interconvertible parameters depending on the protein flexibility. The more flexible a protein was, the greater proportion of the total activation energy of the reaction was observed as the apparent activation energy of internal friction. Based on the coupling of the internal and external movements of the protein during its conformational change, we constructed a model that quantitatively relates activation energy, internal friction, and protein flexibility.

Contribution of 2D and 3D structural features of drug molecules in the prediction of Drug Profile Matching.

Drug Profile Matching (DPM), a novel virtual affinity fingerprinting method capable of predicting the medical effect profiles of small molecules, was introduced by our group recently. The method exploits the information content of interaction patterns generated by flexible docking to a series of rigidly kept nontarget protein active sites. We presented the ability of DPM to classify molecules excellently, and the question arose, what the contribution of 2D and 3D structural features of the small molecules is to the intriguingly high prediction power of DPM. The present study compared the prediction powers for effect profiles of 1163 FDA-approved drug compounds determined by DPM and ChemAxon 2D and 3D similarity fingerprinting approaches. We found that DPM outperformed the 2D and 3D approaches in almost all therapeutic categories for drug classification except for mechanically rigid structural categories where high accuracy was obtained by all three methods. Moreover, we also tested the predictive power of DPM on external data by reducing the parent data set and demonstrated that DPM can overcome the common screening problems of 2D and 3D similarity methods arising from the presence of structurally diverse molecules in certain effect categories.

Drug effect prediction by polypharmacology-based interaction profiling.

Most drugs exert their effects via multitarget interactions, as hypothesized by polypharmacology. While these multitarget interactions are responsible for the clinical effect profiles of drugs, current methods have failed to uncover the complex relationships between them. Here, we introduce an approach which is able to relate complex drug-protein interaction profiles with effect profiles. Structural data and registered effect profiles of all small-molecule drugs were collected, and interactions to a series of nontarget protein binding sites of each drug were calculated. Statistical analyses confirmed a close relationship between the studied 177 major effect categories and interaction profiles of ca. 1200 FDA-approved small-molecule drugs. On the basis of this relationship, the effect profiles of drugs were revealed in their entirety, and hitherto uncovered effects could be predicted in a systematic manner. Our results show that the prediction power is independent of the composition of the protein set used for interaction profile generation.

Subcellular Dissection of a Simple Neural Circuit: Functional Domains of the Mauthner-Cell During Habituation.

Sensorimotor integration is a pivotal feature of the nervous system for ensuring a coordinated motor response to external stimuli. In essence, such neural circuits can optimize behavioral performance based on the saliency of environmental cues. In zebrafish, habituation of the acoustic startle response (ASR) is a simple behavior integrated into the startle command neurons, called the Mauthner cells. Whereas the essential neuronal components that regulate the startle response have been identified, the principles of how this regulation is integrated at the subcellular regions of the Mauthner cell, which in turn modulate the performance of the behavior, is still not well understood. Here, we reveal mechanistically distinct dynamics of excitatory inputs converging onto the lateral dendrite (LD) and axon initial segment (AIS) of the Mauthner cell by in vivo imaging glutamate release using iGluSnFR, an ultrafast glutamate sensing fluorescent reporter. We find that modulation of glutamate release is dependent on NMDA receptor activity exclusively at the AIS, which is responsible for setting the sensitivity of the startle reflex and inducing a depression of synaptic activity during habituation. In contrast, glutamate-release at the LD is not regulated by NMDA receptors and serves as a baseline component of Mauthner cell activation. Finally, using in vivo calcium imaging at the feed-forward interneuron population component of the startle circuit, we reveal that these cells indeed play pivotal roles in both setting the startle threshold and habituation by modulating the AIS of the Mauthner cell. These results indicate that a command neuron may have several functionally distinct regions to regulate complex aspects of behavior.

Chiral HPLC separation of enantiomeric blebbistatin derivatives and racemization analysis in vertebrate tissues.

Simple and consistent chiral HPLC methods for the efficient separation of enantiomeric blebbistatin derivatives, namely parent compound blebbistatin and derivatives 4-nitroblebbistatin, 4-aminoblebbistatin, 4-dimethylaminoblebbistatin, and 4-t-butylblebbistatin were developed using cellulose tris(3,5-dimethylphenylcarbamate) as a stationary phase (Lux cellulose-1 column). Blebbistatin, 4-aminoblebbistatin, and 4-dimethylaminoblebbistatin racemates were well-separated in normal-phase HPLC conditions while 4-nitroblebbistatin and 4-t-butylblebbistatin were effectively separated in both normal- and reversed-phase HPLC conditions. Furthermore, the order of elution of enantiopure compounds was found to be independent of mobile phase compositions and conditions used, and solely depends on the interaction between the enantiomer and the chiral stationary phase. We found that despite the chiral center being present far from the D-ring in the blebbistatin structure, the D-ring substitutions prominently affect the chiral separation. Ex vivo racemization studies of the most popular blebbistatin derivative (S)-(-)-4-aminoblebbistatin in rat blood and brain tissues revealed that the compound does not convert into the inactive enantiomer. This confirms that (S)-(-)-4-aminoblebbistatin is a useful tool compound in cellular and molecular biology studies without the risks of racemization and degradation effects.