Máté Gyimesi

Ádám I Horváth

Demeter Túrós

Sharad Kumar Suthar

Máté Pénzes

Csilla Kurdi

Louise Canon

Carlos Kikuti

Kathleen M Ruppel

Darshan V Trivedi

James A Spudich

István Lőrincz

Anna Á Rauscher

Mihály Kovács

Endre Pál

Sámuel Komoly

Anne Houdusse

András Málnási-Csizmadia

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

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Spasticity -characterized by involuntary increased tone of skeletal muscles -is a common sensorimotor disorder in patients with brain and spinal cord injuries after stroke, trauma, cerebral palsy, or in multiple sclerosis and several myopathies of different etiologies [START_REF] Li | Spasticity, Motor Recovery, and Neural Plasticity after Stroke[END_REF]Trompetto et al., 2014). Spasticity-related physical and mental conditions of patients often permanently disable self-supporting life management and ability to work [START_REF] Martin | Epidemiological, humanistic, and economic burden of illness of lower limb spasticity in adults: a systematic review[END_REF][START_REF] Rychlik | Quality of life and costs of spasticity treatment in German stroke patients[END_REF]. Economic burden for post-stroke patients with spasticity are 4-fold higher than those for patients without spasticity [START_REF] Zorowitz | Poststroke spasticity: sequelae and burden on stroke survivors and caregivers[END_REF], which includes direct costs of medications and hospitalization and indirect costs of caregiver assignments and independent daily life facilitating instruments.

Spasticity develops as a consequence of complex rearrangements of supraspinal inputs, which transforms the synaptic regulation of motoneurons that drives muscle contraction. The resulting exaggerated stretch reflex is accompanied with disrupted balance in regulation from the premotor cortex and reticular formation of the brainstem, which finally leads to aberrantly increased potentiation of motoneurons and hypercontraction of muscles [START_REF] Enslin | Management of Spasticity After Traumatic Brain Injury in Children[END_REF][START_REF] Mukherjee | Spasticity mechanisms -for the clinician[END_REF]Trompetto et al., 2014).

Current muscle relaxants in medical practice target the central nervous system (e.g. baclofen (GABA B agonist) and tizanidine (α2 agonist)), the neuromuscular junction (botulinum toxins) or the sarcoplasmic reticulum in muscle cells (dantrolene) (Fig. 1A). Due to their aspecific mechanism of action centrally acting muscle relaxants have a wide range of neurological and cardiovascular side effects (drowsiness, dizziness, depression, low blood pressure) [START_REF] Meleger | Muscle relaxants and antispasticity agents[END_REF][START_REF] Smit | Heart conduction problems in a tetraplegic patient caused by a single therapeutic dosage of Baclofen[END_REF], and low levels of efficacy [START_REF] Orsnes | Effect of baclofen on gait in spastic MS patients[END_REF], which often necessitates the application of an intrathecal pump surgically implanted inside the body [START_REF] Medical Advisory | Intrathecal baclofen pump for spasticity: an evidence-based analysis[END_REF]. Systemic treatment with botulinum toxins is impossible due to their lethal effect on respiratory and cardiac functions, and local treatments are painful, costly and often ineffective [START_REF] Careta | Report of Allergic Reaction After Application of Botulinum Toxin[END_REF][START_REF] Pavone | Botulinum Toxin Treatment for Limb Spasticity in Childhood Cerebral Palsy[END_REF]. Dantrolene has been associated with severe hepatotoxicity and genotoxicity [START_REF] Nih | ))[END_REF], therefore its clinical use is now restricted to lifethreatening malignant hyperthermia. Surgical treatment of severe spasticity is limited to selective dorsal rhizotomy, when the sensory roots that transfer abnormal information from the muscles to the spinal cord are transected. However, the advantage of dorsal rhizotomy over oral medications is only marginal, because the treatment often leads to paralysis, chronic back pain, sensory dysfunction, inflammation, constipation and cyst formation in the spinal column (cf. NICE interventional guidance 373).

Besides nervous system injury related spasticity, permanent muscle spasms emerge as the leading source of prolonged pain in non-specific low back pain and chronic musculoskeletal pain disorder, fibromyalgia, altogether affecting more than 10% of global population [START_REF] Marques | Prevalence of fibromyalgia: literature review update[END_REF][START_REF] Vos | Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study[END_REF]. Although treatments of muscle spasm in these syndromes with systemic muscle relaxants are statistically effective, recommendations for their use in clinics underline precautions due to the reported adverse side effects resulting from non-specific targeting of the nervous system [START_REF] Tofferi | Treatment of fibromyalgia with cyclobenzaprine: A meta-analysis[END_REF]van Tulder et al., 2003).

These considerations emphasize the unmet medical need for the development of a newgeneration anti-spastic drug, which directly targets the effector protein of muscle contraction while avoiding neurological and cardiovascular side effects. Nervous system independent inhibition of muscle contraction in spastic patients could also overcome the need for targeting the otherwise disrupted synaptic balance of neuronal regulation.

Selective inhibition of skeletal muscles, however, raises the challenge of developing a small molecule that distinguishes between the structurally and sequentially highly similar myosin-2 isoforms. The ideal candidate inhibits only fast skeletal muscle fast isoforms (MyHC IIa,b,x/d), because the slow isoform in skeletal muscles (myosin heavy chain, MyHC I) expressed from the MYH7 gene is identical to β-cardiac myosin, the most abundant motor in the ventricles of the heart [START_REF] Coluccio | Myosins and Disease[END_REF]

Results

In order to relax muscle fibers, we aimed to arrest the actomyosin force generation cycle in the actin-detached weak actin-binding state [START_REF] Takacs | Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke[END_REF], when myosin heads are decoupled from actin filaments (Malnasi-Csizmadia and Kovacs, 2010;[START_REF] Sweeney | Muscle Contraction[END_REF]) (Fig. 1C). Using rational pharmacological design in a model MyHC IIa myosin structure we developed and optimized a small-molecule inhibitor (MPH-220) into the myosin-2 class specific blebbistatin-binding pocket (Allingham et al., 2005;[START_REF] Kovacs | Mechanism of blebbistatin inhibition of myosin II[END_REF][START_REF] Limouze | Specificity of blebbistatin, an inhibitor of myosin II[END_REF][START_REF] Straight | Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor[END_REF] (Fig. 1D, Fig. S2). MPH-220 enabled selective inhibition of fast skeletal myosin-2 isoforms (further referred to as skeletal myosins, Fig. S1) isolated from rabbit psoas muscle, while -even at extremely high concentrations -it did not inhibit the ATPase activity of slow skeletal/β-cardiac myosin (further referred to as cardiac myosin) isolated from left ventricle of porcine heart nor smooth muscle myosin-2 (Fig. 1E, Table S1), nor any non-muscle myosin-2 (NM2) isoforms (Fig. 1F, Table S1). Sequence analysis of the inhibitor binding site revealed an important difference between the cardiac and skeletal myosins near the morpholine ring of MPH-220 at a location playing a key role in the myosin force generation pathway (Malnasi-Csizmadia and Kovacs, 2010). The drug-interacting amino acid is uniquely and invariably leucine in fast skeletal myosin heavy chains (Leu 476 in human MyHC IIa), while this position is occupied by phenylalanine in all other myosin-2 isoforms (Fig. 1B, Fig. S1). We hypothesized that this structural difference can be the basis for the extreme selectivity of MPH-220 toward skeletal myosins. Therefore we assessed the role of a Phe/Leu exchange through mutational analysis of human non-muscle myosin-2C (NM2C), which is not inhibited by MPH-220 in the wild-type form possibly due to the presence of Phe 490 in the corresponding position. According to our hypothesis the expressed NM2C F490L variant became sensitive to MPH-220 (Fig. 1F, Fig. S3), corroborating that the single amino acid change determines the observed selectivity.

Considering the selective inhibitory nature of MPH-220 on myosin samples (Fig. 1E-F), and its potential to become a new-generation anti-spastic drug candidate we performed ATPase inhibition assays on human myosin samples. Expressed human β-cardiac myosin was essentially unaffected by MPH-220, while the non-selective myosin-2 inhibitor, blebbistatin, completely blocked its ATPase activity (Fig. 1G, Table S1). We further tested MPH-220 on skeletal and cardiac myosin samples isolated from patient-derived biopsies (Fig. 1H, Table S1). In line with their myosin-2 isoform composition, skeletal myosin samples from m.vastus lateralis and m.soleus -containing 56% and 28% fast isoforms in human muscles (Fig. S4) -were inhibited by 70% and 29%, respectively, confirming the hypothesis that only the fast isoform fraction is targeted by MPH-220. More importantly, β-cardiac myosin isolated from human heart left ventricle was unaffected by MPH-220 (Fig. 1H, Table S1). By contrast, blebbistatin reduced the activity of m.vastus lateralis and cardiac myosin samples by more than 90%, confirming that MPH-220 inhibition is proportional to the ratio of fast myosin isoform in different tissues (Fig. 1H, Table S1). These results provide the basis for future safe systemic administration of MPH-220 to human patients. Left ventricle human biopsy contains cca. 10% α-cardiac myosin isoform (expressed form the MYH6 gene (Fig. S1)) [START_REF] Miyata | Myosin heavy chain isoform expression in the failing and nonfailing human heart[END_REF], which also contains Phe in the proximal part of HP-helix Phe 474 ). The lack of inhibition of myosin samples from the byopsies confirms that α-cardiac myosin could not be inhibited by MPH-220. We also note that gene products of MYH1, MYH2 and MYH4 encoding fast skeletal muscle myosin heavy chains IIx/d, IIa and IIb, respectively, are not expressed in adult human heart in significant amount ( [START_REF] England | Heavy and light roles: myosin in the morphogenesis of the heart[END_REF], and NCBI Gene Expression database).

In order to further analyze the molecular background of selectivity we crystalized fast skeletal muscle myosin (isolated from high-quality rabbit m.psoas) in the presence of MPH-220 (Fig. 2A, Table S2). The crystal structure confirmed that MPH-220 binds to the blebbistatin-binding cleft and its morpholine ring is in close proximity to Leu 476 . This residue is at the N-terminus of the so-called HP-helix of the L50 subdomain [START_REF] Cope | Conservation within the myosin motor domain: implications for structure and function[END_REF], which is preceded by the switch-2 loop and followed by the relay region of the motor. Comparison of the MPH-220-bound skeletal and blebbistatin-bound Dictyostelium myosin-2 structures confirms that while the tricyclic cores of the inhibitors including the chiral OH are positioned similarly, Phe 466 of the HP-helix in Dictyostelium myosin-2 comes into steric hindrance with MPH-220 (Fig. 2B). Likewise, the position of this Phe in cardiac myosin structure also causes steric hindrance with MPH-220 (Planelles-Herrero et al., 2017) (Fig. 2C). This steric hindrance based on crystal structures together with the biochemical results strongly support that the unique Leu in the HP-helix of fast skeletal muscle myosin isoforms is responsible for the extreme selectivity of MPH-220. This mechanism for selective inhibition among myosin-2 isoforms is analogous to how blebbistatin is selective for myosin-2s due to the presence of the switch-2 Ser/Ala residue in myosin-2s (Allingham et al., 2005), while this position is occupied by Tyr/Phe in myosins from other classes that are uninhibited by blebbistatin. The resolved crystal structure also confirmed that MPH-220 selects and stabilizes the pre-powerstroke state. Overall, the structure validated the in silico approach to design molecules for selective inhibitor development (Fig. S5).

We further studied the effect of MPH-220 on muscle force generation in living animals (Fig. 3A). In agreement with the ATPase inhibitory properties (Fig. S6), the S(-) enantiomer of MPH-220 was drastically more effective than the R(+) enantiomer when animals were treated orally with MPH-220 (Fig. S7). Consequently, the force-relaxing effect of the racemic mixture was significantly lower than that of the S(-) enantiomer. While the racemic mixture caused maximum 40% reduction, the S(-) enantiomer could decrease hindleg force by 70% in a concentrationdependent manner, (Fig. 3B-C). The 30% residual isometric force level for S(-)-MPH-220 is in line with the presence of uninhibitable slow skeletal muscle isoforms in rat hindleg muscles. This attribute is of high importance because unlike centrally acting muscle relaxants MPH-220 would never cause overdose related patient immobilization due to the remaining slow myosin-mediated muscle tone. Importantly, the decreased force persisted for more than 10 hours (Fig. 3C), which indicates optimal properties for pharmacological development. The force-relaxing effect after oral administration was only marginally slower than that after intraperitoneal injection (Fig. S7), which indicated optimal absorption properties of MPH-220. High endothelial permeability was further confirmed by Caco-2 assay, which showed that MPH-220 is highly permeable with significantly faster transfer rate in the absorptive direction over the excretive direction (Fig. S7).

While skeletal muscle relaxation could be efficiently achieved by MPH-220, cardiac function was not perturbed in the concentration range that was effective in skeletal muscle force relaxation experiments (Fig. 3F, Fig. S8). More importantly, neither cardiac nor respiratory functions differed significantly from the excipient control during the 10-hour experiments suggesting a reasonable safety range between the effective and toxic doses of MPH-220 (Fig. 3E-F). The maximum 25% decrease in heart rate and blood flow parameters is most probably due to the 10-hour-long anesthesia without food and drink after an overnight starvation period, and not the effect of myosin inhibition (Fig. S8). Moreover, the identical direction of the change in heart rate and blood flow parameters solely reflects slowing down the heart during anesthesia, because cardiac myosin inhibition preceding heart failure increases heart rate while drastically lowering blood flow as shown with the non-selective inhibitor, AmBleb (Fig. S8).

We also examined MPH-220 distribution in rat tissues in a time-dependent manner and observed a dose-dependent, few-fold accumulation of MPH-220 in skeletal muscle samples over other tissues including brain, heart, kidney and blood (Fig. 3J-L). Accumulation in skeletal muscle persists after 3 hours when MPH-220 is completely eliminated from blood and other tissues, while its concentration in target muscles is still in the effective concentration range (10-fold higher than the determined IC 50 ). MPH-220 concentration was only temporarily elevated in the liver, which metabolizes MPH-220. We note that MPH-220 concentration in the diaphragm was much lower compared to other striated muscle samples, which explains the lack of respiratory dysfunctions after MPH-220 treatments. This feature is expected to prove even more favorable in human patients due to the substantially higher ratio of MyHC I slow isoforms in human diaphragm (Fig. S4). The pronounced accumulation of MPH-220 in skeletal muscles might be the result of high concentration of fast skeletal myosin isoforms, which bind the inhibitor with high affinity. This hypothesis is supported by the significantly lower accumulation of the R(+) enantiomer (Fig. S9), which -besides its weaker inhibitory efficacy (Fig. S6) -further explains the fourfold weaker force relaxation by the R(+) enantiomer (cf. Fig. 3B). The species-specific MyHC distribution (Fig. S4) also emphasizes the need for testing these pharmacodynamic properties of MPH-220 in larger animals (dogs, mini pigs), which -unlike rodents -have higher slow isoform proportions, more similar to human tissues.

Direct and efficient targeting of skeletal muscle contraction by MPH-220 raises the possibility of the development of a new generation nervous system-independent anti-spastic drug. Therefore, we investigated the efficiency of MPH-220 to improve spasticity-related gait disorders in rats with brain injury. We applied the recently developed spastic cerebral palsy animal model to provoke paralysis-coupled spasticity in rats (Fig. 4A) [START_REF] Yu | Establishing a rat model of spastic cerebral palsy by targeted ethanol injection[END_REF], and gait disorders were analyzed by non-invasive methods focusing on the motoric functions of rats combined with neural network based deep learning movement analysis (Fig. 4B, Movie S1). Similarly to human patients with stroke or brain trauma, rats showed individually variable gait patterns with different degrees of palsy in the lower limbs. Thus, we analyzed the effect of MPH-220 treatment individually on each animal and focused on treatment-induced changes in spasticity-related gait components.

Rat-A could not use the right forelimb and the left hindleg due to paralysis-coupled spasticity, which resulted in an almost complete one-sided circling behaviour in the open-field box two days after the surgery (Movie S2). 40 minutes after MPH-220 treatment the ratio of left turns increased from 15% to 38%, which further improved to 51% three hours after the treatment (Fig. 4C). Even though Rat-A walked significantly less after treatment, it was obvious that a major improvement was achieved: after relaxing spasticity in its left hindleg Rat-A was able to use this leg and make quasi-normal steps (Movie S3). Rat-B and C also had paralysis-coupled spasticity in the hindlegs, although, to a lower degree than Rat-A. The most obvious spasticity-related feature in the open field test was the random falling (Movie S4) and the spontaneous cramping of the right hindleg of Rat-C (Movie S5). Importantly, the relative frequency of both symptoms decreased drastically after MPH-220 treatment (Fig. 4D). Besides these serious gait abnormalities, Rat-B and C could not stand up in the cylinder assay before MPH-220 treatment. In agreement with the kinetics observed in the open field assay, three and five hours after oral MPH-220 treatment both rats could stand up in the cylinder touching the wall with both forepaws with the same number of events as their healthy littermates (Fig. 4D, Fig. S10). Next morning, the positive effects on falling and cramping frequencies, and on the ability to stand up in the cylinder disappeared as MPH-220 was excreted from their bodies. Rat-D showed spastic gait disorders only in the right hindleg without signs of severe palsy, while all other legs remained unaffected. This animal could not use the right hindleg normally due to stiff muscles, which resulted in abnormal positioning of all four paws (Fig. 4E, Fig. S11). The spastic leg was offset from the body axis, which hindered normal progression of the limb during walking, and resulted in lopsided gait patterns with tilted body axis (Fig. 4F, Fig. S11, Movie S6). After MPH-220 treatment the gait of this animal improved significantly as it started to use both hindlegs normally. The improved movement was the consequence of relaxed spasticity in the hindlegs, which enabled the animal to pull its leg into the body-axis thereby making normal walking steps (Fig. 4E-F, Fig. S11, Movie S6). The enhanced gait functions remained persistent for five hours, when paw positions and body symmetry was close to those of the healthy animals. Similarly to the other experiments, the effect relapsed to the pretreatment state after 21 hours. These effects could be reproduced five and twelve days after the first treatment corroborating the specific anti-spastic effect of MPH-220 treatment.

Beside the individual patterns of spastic behavior that could be improved by MPH-220 treatment, deep learning analysis was used to assess the over-ground locomotion of all animals during 15 minutes of free movement in the open field test. We used 28 parameters characterizing the 3D position of the limbs and body axis in three movement speed regimes (Table S3) to quantify the locomotor features based on pose-estimating deep learning algorithms and exposed these to a principal components analysis (PCA). In the global PCA space -including all three speedregimes -the major gait component (PC1) was significantly different from the healthy range, indicating that pyramidal tract damage-induced spastic walk asymmetry is reflected in the major principal gait component (Fig. 5A). Most importantly, MPH-220 treatment significantly decreased PC1 scores, the effect of which was maximal at 3 hours and no longer detected after 21 hours. Importantly, while PC1 scores were significantly different from the untreated level, the treated values did not differ significantly from the healthy range, which confirms that MPH-220 ameliorated gait pattern of the treated animals to that of the healthy rats. However, when we analyzed the three speed-regimes separately, we found that pyramid tract damage mostly affected PC1 in the slow and medium-speed regimes, while PC2 and PC3 were significantly affected in the fast regime (Fig. 5B, Fig. S11). More importantly, PC1 could be significantly improved by MPH-220 treatment in the slow and medium-speed regimes, while PC3 was significantly improved in the fast regime. We identified the major parameters that predominantly contributed to PC1 and PC3 in the slow, medium and fast regimes, and found a marked difference in the parameter sets defining PC1 and PC3 (Fig. 5C-D, Fig. S11, Table S3). Analysis of these components revealed that in the slow and medium-speed regimes MPH-220 treatment has the largest effect on the volumes of the ellipsoids representing paw positions and on the distances between their centers, both suggesting a much ordered stepping pattern and straightened body axis (Fig. 5E). On the other hand, in the fast regime, MPH-220 has the most pronounced effect on the angles of the ellipsoids, which indicates that the treated rats could keep their legs closer to the body axis similarly to the healthy running pattern. These results further solidify that MPH-220 is capable of treating the spastic component of movement abnormalities after brain injury, exhibiting a promising potential to address the unmet medical need for effective anti-spastic therapies.

Discussion

Our biochemical and in vivo results demonstrate that MPH-220 selectively inhibits fast skeletal myosin-2 isoforms, which enables effective improvements of spastic gait disorders after brain damage as a consequence of efficient reduction of muscle force. These findings emphasize that nervous system injury-derived muscle spasticity can be treated by directly targeting the effector protein of muscle contraction, thereby avoiding adverse neurological and cardiovascular side effects that are characteristic of currently used muscle relaxants. Moreover, as MPH-220 proved a promising candidate for further anti-spastic drug development, we tested its pharmacological properties by investigating its off-target effects on human enzymes including kinase panels, Gprotein coupled and nuclear hormone receptors (Fig. S12). We also demonstrate that MPH-220 is not mutagenic and not cytotoxic (Fig. S12-13) which, together with its high-absorption properties, confirm that MPH-220 is safe and possesses optimal attributes for further drug development as a new-generation anti-spastic drug candidate. Although we acknowledge that MPH-220 may cause general muscle weakness as observed in our animal disease model. We believe that this effect will be significantly less pronounced in human patients due to the higher ratio of MyHC I slow skeletal muscle myosin isoform. In human skeletal muscles the presence of 40-60% MyHC I isoform, which is not inhibited by MPH-220, highlights another important aspect of this type of muscle relaxation as opposed to nervous system targeting medications. Although general weakness of aspecific muscle groups may appear by using both types of muscle relaxants, the MPH-220 based anti-spastic effect will never cause complete immobilization of patients due to the residual muscle tone from the uninhibited slow isoform fraction. Another concern may arise regarding extraocular muscles, which express MYH13 gene product containing Leu required for MPH-220 inhibition (Fig. S1). Nevertheless, in adult human extraocular muscles significant amount of α-cardiac myosin heavy chain is expressed [START_REF] Kjellgren | Myosin heavy chain isoforms in human extraocular muscles[END_REF], therefore, these muscles will also not be completely immobilized by MPH-220. However, these effects might be investigated during future clinical trials.

Moreover, based on the complex pathophysiology of spasticity development (Trompetto et al., 2014) we suppose that early treatment with an effective anti-spastic drug will significantly delay the onset and markedly reduce the severity of spastic symptoms after nervous system injuries.

Beside traumatic brain and spinal cord injuries in stroke and cerebral palsy, spasticity is also a common symptom in several other diseases of broad etiology (Table 1). Spasticity is a major component of upper motor neuron lesion syndromes including multiple sclerosis, amyotrophic lateral sclerosis, spastic paraplegia, multiple system atrophy and atypical Parkinsonism, and also a characteristic symptom in several skeletal muscle myopathies such as myotonia congenita, myotonia fluctuans, or certain neurodegenerative diseases. Altogether, spasticity in these syndromes affect nearly 1% of the human population (cca. 55 million spastic patients) and more than 10% of people suffers from often-painful muscle stiffness in low back pain and fibromyalgia [START_REF] Marques | Prevalence of fibromyalgia: literature review update[END_REF][START_REF] Vos | Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study[END_REF]. Consequently, a nervous systemindependent, safe and effective treatment is a significant unmet need from both clinical and socio-economical perspectives. Therefore MPH-220 is a promising anti-spastic drug candidate to improve quality of life and may contribute to extended life expectancy [START_REF] Blair | Survival and mortality in cerebral palsy: observations to the sixth decade from a data linkage study of a total population register and National Death Index[END_REF][START_REF] Gillard | The negative impact of spasticity on the health-related quality of life of stroke survivors: a longitudinal cohort study[END_REF][START_REF] Slaman | Can a lifestyle intervention improve physical fitness in adolescents and young adults with spastic cerebral palsy? A randomized controlled trial[END_REF][START_REF] Yi | Emerging Issues in Cerebral Palsy Associated With Aging: A Physiatrist Perspective[END_REF] of these patients and would provide new therapeutic protocols for clinicians in these diverse indications.

References:

Allingham, J.S., Smith, R., and Rayment, I. (2005). The structural basis of blebbistatin inhibition and specificity for myosin II. Nat Struct Mol Biol 12, 378-379. Berman, H., Henrick, K., and Nakamura, H. (2003). Announcing the worldwide Protein Data Bank. Nat Struct Biol 10, 980.

Tong, C.W., Stelzer, J.E., Greaser, M.L., Powers, P.A., and Moss, R.L. (2008). Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function. Circulation research 103, 974-982. Trompetto, C., Marinelli, L., Mori, L., Pelosin, E., Curra, A., Molfetta, L., and Abbruzzese, G. (2014). Pathophysiology of spasticity: implications for neurorehabilitation. BioMed research international 2014, 354906.

Trybus, K.M. (2000). Biochemical studies of myosin. Methods 22, 327-335.

Urban, P.P., Wolf, T., Uebele, M., Marx, J.J., Vogt, T., Stoeter, P., Bauermann, T., Weibrich, C., Vucurevic, G.D., Schneider, A., et al. (2010). Occurence and clinical predictors of spasticity after ischemic stroke. Stroke 41, 2016[START_REF] Coluccio | Myosins and Disease[END_REF]. Vagin, A., and Teplyakov, A. (2010). Molecular replacement with MOLREP. Acta crystallographica Section D, Biological crystallography 66, 22-25.

Van Naarden Braun, K., Doernberg, N., Schieve, L., Christensen, D., Goodman, A., and Yeargin-Allsopp, M. ( 2016 analysis, and validation, SKS: investigation, formal analysis, MP: investigation, methodology development, CK: investigation, formal analysis, LC: investigation, methodology development, crystallization and structure determination, formal analysis, CK: investigation, methodology development, structure refinement and formal analysis, KMR: investigation, preparation of human cardiac myosin sample, DVT: investigation, preparation of human cardiac myosin sample, JAS: funding acquisition and supervision for human cardiac myosin sample, IL: investigation, methodology development, formal analysis, AÁR: funding acquisition, writingreview & editing, MK: funding acquisition, supervision, writing -review & editing, EP: investigation, SK: conceptualization, funding acquisition, AH: methodology development, formal analysis, funding acquisition for crystallization and structure determination, AMC: conceptualization, formal analysis, funding acquisition, investigation, methodology development, supervision, validation, visualization, writing

Declaration of Interests:

The authors declare the following competing interests: Employment: AMC, MG are owners of Optopharma Ltd., AÁR and MG are part-time employed by Optopharma Ltd. Related patents: PCT/EP2017/051829, WO/2017/129782, HU1800129A2; PCT/HU2019/050017, WO/2019/202346A2, WO/2019/202346A3. JAS is a cofounder and member of the scientific advisory boards of Cytokinetics and MyoKardia, biotechnology companies developing small molecules that target the sarcomere for the treatment of various muscle diseases. KMR is on the scientific advisory board at MyoKardia. JAS, DVT and KMR are cofounders of Kainomyx Inc., a biotechnology company focused on developing small molecules to target tropical diseases.

Data availability:

The atomic model is available in the PDB (Berman et al., 2003) under PDB ID 6YSY. S3). (D) Heatmap of major parameters contributing to PC1 (blue-orange) and PC3 (purple-orange) (cf. Table S3). (D) Schematic representation of MPH-220 effects on paw positions in slow and fast regimes.

Method details

Materials

All chemicals were purchased from Sigma-Aldrich (Germany) if otherwise not stated. Sulphobutyl-ether-β-cyclodextrin (Dexolve TM (SBECD)) was obtained from Cyclolab (Hungary). HPLC-grade acetonitrile, chloroform and water were purchased from VWR (PA, USA). Blebbistatin was purchased from Sellekchem (TX, USA), AmBleb and NBleb are products of Optopharma Ltd., and isoflurane was purchased from Rotacher-Medical GmbH (Germany). Ames Microplate Format Mutagenicity Assay kit was purchased from Xenometrix (Switzerland).

Proteins

Rabbit actin was prepared as described previously from rabbit muscle acetone powder (Pel-Freez Biologicals Llc., AR, USA) [START_REF] Spudich | The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin[END_REF]. Skeletal muscle myosin subfragment-1 (S1) was prepared from rabbit m.psoas according to an earlier published protocol [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF]) by α-chymotryptic digestion (0.25 mg/ml α-chymotrypsin, 10 min, 25 o C; reaction was stopped with 3 mM PMSF). Psoas S1 prep was further purified on MonoS 5/50 GL column to produce high-quality prep for crystallization. Cardiac muscle myosin S1 was isolated from left ventricle of porcine heart following cardiac myofibril preparation protocol (Tong et al., 2008) by α-chymotryptic digestion (0.05 mg/ml α-chymotrypsin, 10 min, 25 o C; reaction was stopped with 3 mM PMSF). Human cardiac muscle myosin subfragment-1 (S1) was prepared from adult human left ventricular tissue samples obtained from ethically consented donors (AnaBios Corporation) according to earlier published protocol [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF] by α-chymotryptic digestion (0.25 mg/ml α-chymotrypsin, 9 min, 25 o C; reaction was stopped with 3 mM PMSF). Smooth muscle myosin S1 was prepared from chicken gizzard (Trybus, 2000) by activated papain digestion [START_REF] Seidel | Fragmentation of gizzard myosin by alpha-chymotrypsin and papain, the effects on ATPase activity, and the interaction with actin[END_REF]) (0.2 mg/ml papain, 12 min, 25 o C; reaction was stopped with 5 mM sodium iodoacetate). Human skeletal muscle myosin was prepared similarly to rabbit skeletal muscle myosin [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF]. Briefly, biopsies were washed in Rigor Buffer (20 mM MOPS pH 7.4, 40 mM KCl, 2 mM MgCl 2 ) and myosin was extracted in Myosin Extraction Buffer (20 mM MOPS pH 7.4, 500 mM KCl). Extracted myosin was precipitated in 21-volume ultrapure water with 1 mM EDTA. Myosin was collected in tabletop centrifuge, dissolved in Myosin Extraction Buffer and kept on ice until the measurement. The Sf9 codon optimized sequences for NM2 motor domains (Met 1 -Arg 775 for NM2A, Met 1 -Arg 782 for NM2B (B0) and Met 1 -Arg 799 for NM2C (C0)) fused to an Ala-Ser linker and an artificial α-actinin lever arm (as described earlier for NM2C [START_REF] Heissler | Comparative kinetic and functional characterization of the motor domains of human nonmuscle myosin-2C isoforms[END_REF]) with C-terminal FLAG-tag sequences were synthesized and cloned into pFastBac1 expression vector. The NM2C-F490L construct was also synthesized by Biomatik Co in pFastBac1 expression vector. The pFastBac1/NM2 constructs were overexpressed in Sf9 cells after bacmid preparation from DH10Bac cells. To reduce proteolytic digestion during expression, Pepstatin-A (0.1 mg/l) was added to Sf9 cells [START_REF] Gotoh | Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors[END_REF]. Expressed human β-cardiac myosin S1 [START_REF] Kawana | Biophysical properties of human beta-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy[END_REF] was prepared as earlier described [START_REF] Sommese | Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human beta-cardiac myosin motor function[END_REF].

ATPase measurement

Steady-state ATPase measurements were carried out in 50 µl volume in a flat bottom 384-well plate (Nunc-Thermo Fischer) using NADH-PK/LDH coupled assay described previously [START_REF] Gyimesi | The mechanism of the reverse recovery step, phosphate release, and actin activation of Dictyostelium myosin II[END_REF]

Molecular dynamic simulation

Simulations were carried out using the AMBER16 program suite. The ff14SB force field [START_REF] Maier | ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB[END_REF] was used in all subsequent simulations to model protein interactions. The initial structure of the Fast Human Skeletal myosin-2 motor domain was modeled using structural homology with the SWISS-MODEL server [START_REF] Waterhouse | SWISS-MODEL: homology modelling of protein structures and complexes[END_REF] using the Human Cardiac myosin-2 motor domain (PDB ID 5n69). ADP.VO 4 was substituted to ADP.PO 4 . Coordinates and partials charges for MPH-220 were refined using antechamber of the AMBERTOOLS package with the AM1 semi-empirical Hamiltonian. Force filed parameters for the MPH-220 atoms were adopted from GAFF force field [START_REF] Wang | Development and testing of a general amber force field[END_REF] based on structural similarity following the suggestions of the parmchk2 program. In order to determine the initial position of MPH-220 we RMSD least-square fitting the model's structure with the blebbistatin-Dyctiostelium motor domain (PDB ID 3bz9) binding pocket. The initial position was then determined by RMSD least-square fitting the tricyclic ring of MPH-220 to blebbistatin's tricyclic ring. The water molecules resolved by crystallography were retained and the model was expanded in an 8-Angstrom clearance dodecahedron box with TIP3P water molecules for explicit solvation and counterions were added to neutralize the system's net charge. The complex was minimized with 2000 steps steepest descent followed by 4000 steps of conjugate gradient method while applying 10 kcal/(mol Å 2 ) to all atoms except water molecules. The minimization is continued with the same protocol while removing the restraint from all H atoms. The same restraints were applied for the following steps. The complex was heated in 3 100-K 20-ps steps to 300 K under NVT conditions, then subsequently equilibrated to 1 bar in 20 ps under NPT conditions. NPT conditions were applied in all further steps. The system was cooled back to 10 K in the next 20 ps. Restraints from this point were only applied to the protein backbone. The system was heated back to 300 K in 10 ps and the simulation was continued for 1ns. The restraint force was then gradually reduced in 1 ns simulations steps to 5, 1, 0.5 and 0 kcal/(mol Å 2 ). The simulation was then allowed to continue and equilibrate under NPT conditions for a further 200 ns to obtain the final structures. The trajectories were sampled every 50 ps during the simulation.

Crystallisation and data processing

MyCH IIb-MPH-220 crystals were obtained by vapor diffusion using a protein solution at 10 mg/ml containing 10 mM MOPS pH 7.3; 50 mM NaCl; 3 mM MgCl 2 ; 0.2 mM ATP; 3 mM NaN 3 ; 1 mM DTT; 0.1 mM EGTA; 0.1 mM PMSF; 1 µg/ml leupeptin, 2mM MgADP, 2mM VO 4 , 0.5 mM MPH-220 against a reservoir containing 35% PEG600, 20mM DTT; 100mM HEPES pH 7.0; 5% DMSO at 291K. Optimization of crystallization conditions was performed with trypsin in situ proteolysis. Crystals were frozen then shot in the Proxima2 beamline (synchrotron Soleil). X-ray diffraction images were processed with AutoProc [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF]. The crystal belongs to the P2 1 2 1 2 1 space group with one molecule per asymmetric unit.

The data collection and refinement statistics are presented in (Table S2).

Structure determination and refinement

Structure of the motor domain was initially solved by molecular replacement using 1QVI [START_REF] Gourinath | Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head[END_REF] as a search model in Molrep(Vagin and Teplyakov, 2010), and completed with a second search for the lever arm and its essential light chain from 5N69 (Planelles-Herrero et al., 2017) with Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF]. Refinement constraints for the ligand were automatically generated with Grade [START_REF] Bricogne | BUSTER version 2.10[END_REF]. The final model was obtained after several cycles of building in Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and refinement with the latest 2019 release of BUSTER-TNT [START_REF] Bricogne | BUSTER version 2.10[END_REF].

Isometric force measurement

Isometric force of rat left hindleg was measured by a force meter (Supertech Ltd.) after the hindleg was fixed and stimulated according to earlier published protocol [START_REF] Pratt | A stepwise procedure to test contractility and susceptibility to injury for the rodent quadriceps muscle[END_REF]. A non-elastic wire was attached to the ankle of the rat and pulled until pre-strained position. Spike (R) software controlled stimulator (BioStim, Supertech Ltd.) was used to trigger the n. femoralis through metal electrodes with 100 Hz, 1 sec, 4V, 2.5 minute intervals. Data detection was performed with CED Micro 1401 data acquisition interface. Inhibitors were either dissolved in 200 µl DMSO for intraperitoneal injection or in 1ml 30% SBECD solution (dissolved in Gibco TM Dulbeccos's Phosphate Buffered Saline (D-PBS)) for oral administration.

Non-invasive measurement of cardiovascular and respiratory functions

Cardiovascular and respiratory functions were measured using a non-invasive pulse oximeter (MouseOx Plus, Starr Life Sciences Corp., PA, USA). Blood flow parameters shown on figures corresponds to the measured pulse distention, which is the measure of change in blood vessel volumes in the light path of the sensor, which reflects local blood pressure changes. Rats were anesthetized and the neck region was trimmed using a small animal trimmer to facilitate accurate measurements by size specific collar sensor.

Concentration measurement from tissue samples

Animals were treated i.p. or per os with MPH-220, over-anesthetized and tissue samples were collected and frozen at -80°C. Tissue samples were thawed and homogenized in D-PBS, and chloroform was added to the homogenate. Samples were thoroughly shaken, vortexed and sonicated. After sonication, samples were centrifuged and the chloroform layer was collected.

Chloroform extraction was repeated two more times. Chloroform was drying by evaporation in a fume-hood overnight (48 hours). After chloroform drying, water-acetonitrile solution (50%/50%) was added to the samples, which were again vortexed and sonicated again. Samples were centrifuged and supernatants were collected and transferred into ultra-centrifuge vials and ultracentrifuged at 60,000 rpm, 10°C for 60 minutes before HPLC-MS injection. Peak area of compound in HPLC was determined at 400 nm and concentration of compound was determined from the calibration curve. HPLC-MS conditions were the following: Merck Purosphere STAR RP-18 endcapped 250 mm X 4.6 mm column, mobile phase: isocratic 50% H 2 O (0.1% TFA) -50% ACN (0.09% TFA), injection volume: 20 µl HPLC, flow rate: 0.5 ml/min, run time: 25 min, detection at 260, 280, 300, 400 and 450 nm, capillary voltage: 3.5 kV, cone voltage: 30 V.

Spasticity animal model

Targeted brain damage was performed to establish a rat model of spastic cerebral palsy. Briefly, animals were pre-anesthetized with 4% isoflurane, which was reduced to 2% after the fixation of the animal on the stereotactic apparatus. First, a 2 cm-long longitudinally incision between ears was carried out, which was followed by the cleaning of the surface of the skull by hydrogen peroxide to visualize the Bregma point. Injection coordinates were determined based on The Rat Brain in Stereotaxic Coordinates, 6th Edition [START_REF] Paxinos | The rat brain in stereotaxic coordinates, 6th edn[END_REF]: ML -0.8 mm, AP -10 mm and DV 9.7 mm from the dura. A 25 µl Neuros Syringe (Hamilton, NV, USA) with needle size 22G was used to gently inject 15 µl ethanol for left pyramidal tract lesion. After the injection the syringe was left in place for 10 minutes, followed by its removal from the brain and the sealing of the wound.

Neural network based deep-learning analysis of movements in open-field tests

The over-ground locomotion of the animals was recorded in an 80 by 80 cm wide plastic openfield box using an 8 mm thick transparent Plexiglas as the bottom panel over the course of 15 minutes. The arena was placed on a custom-made wooden frame in order to raise it 1 meter above the ground level, while keeping the transparent bottom unobstructed. A Microsoft Kinect V2 for XBOX ONE (2013) RGB-D sensor was secured approx. 80 cm below the Plexiglas facing upwards to capture depth information from inside the open-field arena (Fig. 4B). A Resnet-50 deep convolutional neural network was trained to predict distinct body parts (nose, neck, center, right forelimb, left forelimb, right hindlimb, left hindlimb, base of the tail) of the animals on the 8-Bit depth recordings using the DeepLabCut toolbox [START_REF] Mathis | DeepLabCut: markerless pose estimation of user-defined body parts with deep learning[END_REF] in the Google Colaboratory python environment. The network training parameters were identical as described in [START_REF] Nath | Using DeepLabCut for 3D markerless pose estimation across species and behaviors[END_REF] using ~1000 frames total for the final training and validation sets (500.000 iteration of training achieved 1.75 mean pixel error for the training and 7.33 mean pixel error for the validation set). A custom MATLAB script was then used to process the previously modified depth archives (25.000 frames / recording) in conjunction with the error corrected numerical output (X and Y coordinates) of the deep learning algorithm of each labeled body part. The animals were first segmented based on the pixel intensity deviation relative to the background. Each frame was then translated and rotated based on the position and angle of the axis between the center of mass of the animal and the position of its nose to create egocentric recording. This transformation and rotation was then carried out on the body part labels as well.

The 3 dimensional position of each limb was calculated by combining the transformed output coordinates of the deep neural net with the depth information of the labeled body parts on each frame of the preprocessed and transformed depth recordings. The three dimensional movement maps were exported to further analyses.

In the final algorithm 95% CI least-squares elliptical fittings were computed on the 3D movement maps of each individual limb of the animals and the gait parameters were calculated (Table S3). Centroid displacement thresholds on moving average function of 100 frames were used to calculate distinct locomotion regimes (1 st Regime: <0.6 pixel deviation, 2 nd Regime: >0.6 and <1.2 pixel deviation, 3 rd Regime: >1.2 pixel deviation.). The gait parameters were scaled using the Z-score method and subjected to principal component analysis and the results were represented in the 3D PC space together with the 95% CI ellipsoids.

Cylinder assay

Motor functions of spastic rats were tested in an open-top, 21 cm diameter, clear plastic cylinder. Forelimb activity was calculated from number of touches of each forelimb against the wall of the arena. Forelimb touch was defined when the whole palm touched the sidewall of the cylinder. Number of sidewall touches was counted for 3 minutes.

Safety panel measurements

G-protein coupled receptor inhibition and activation was tested in the gpcrMAX panel of DiscoverX ® Profiling service (Eurofins). Caco-2 assays were carried out by Absorption and Permeability Services at Eurofins Discovery. Predictor TM hERG Fluroescence Polarization Assay, SelectScreen Kinase Profiling and SelectScreen Nuclear Receptor Profiling was carried out by Thermo Fisher Scientific. Ames MPF TM (Xenometrix, Switzerland) reverse mutagenicity assay was performed according to the manufacturer's guide on TA98 and TA100 bacterial strains in the absence and presence of phenobarbital/β-naphtoflavone-induced rat liver S9 fraction. Cytotoxicity measurements were performed with CyQUANT TM LDH Cytotoxicity assay (Thermo Fisher) according to the manufacturer's protocols with human adult dermal (Gibco, Thermo Fisher) and lung (Cell Applications Inc., CA, USA) fibroblast strains.

Supplementary Text

The broadest available human G-protein-coupled-receptor (GPCR) and kinase panels were used to test MPH-220 inhibition on 168 GPCRs and 484 wild-type and most frequent mutant kinases (Fig. S13A-B). Inhibition and activation levels did not approach the threshold used by regulatory agencies confirming the highly selective nature of MPH-220, which supports the safe use of MPH-220 in systemic treatments. We further tested the activation and inhibition of the most important hormone nuclear receptors and MPH-220 proved safe in this regard, too, because hormone receptors were neither activated (Fig. S13C) nor inhibited (Fig. S13D) due to the lack of receptor binding by MPH-220 (Fig. S13E). Cytotoxicity of MPH-220 was also tested on two human adult fibroblast cell lines for two and five days and both 20 µM and 50 µM MPH-220 proved non-cytotoxic in both strains (Fig. S13F). We also investigated the mutagenicity of MPH-220 by OECD/FDA approved Ames reverse mutagenicity assay up to the solubility limit of MPH-220. We used the two FDA-required bacterial strains sensitive for frameshift and basepair substitution mutations and neither strain showed any sign of mutagenicity of MPH-220 or any other derivatives containing a morpholine group (Fig. S14). These properties confirmed that MPH-220 is pharmacologically safe, which permits its development toward human clinical trials.

Fig. S1

. Sequence analysis of myosin-2 isoforms from different species. Related to Figure 1 The tables show the amino acid residue at the starting point of the HP-helix in myosin-2 isoforms expressed from different MYH genes in eight vertebrate species and the muscle and non-muscle myosin-2 isoforms in D. melanogaster and C. elegans invertebrate species. The alignments below the tables represent the sequence environment of the relevant Leu (blue L) or Phe (black F) residue, including the switch-2 loop (red) and the N-terminal part of the HP-helix (light orange). These data clearly show that all fast skeletal myosin-2 isoforms in all species contain Leu in the morpholine-interacting position, while Phe is present in all slow type myosin-2s including both cardiac isoforms at this position. The characteristic difference is even more pronounced when comparing the neighboring residues in the HP-helix, which are practically invariant in all myosin isoforms and species. Relative ATPase activities are shown for different MPH-220 derivatives tested during the development of MPH-220. MPH-49 is the morpholine derivative of blebbistatin, which shows selective inhibition; however, its solubility remained blebbistatin-like (less than 50 µM in aqueous solutions). MPH-188 and MPH-248 are morpholine derivatives on different scaffolds of the patented MPH-family. MPH-188 showed drastic improvement in solubility (1 mM in aqeous solutions), but its IC 50 value for skeletal myosin was slightly worse than that of MPH-220. MPH-248 retained skeletal myosin specificity of the pyridine ring containing core (MPH-174), but its total inhibition and IC 50 values remained worse than those of MPH-220. We also tried to modify the structure of the morpholine ring by using homomorpholine (MPH-202), thiomorpholine (MPH-203) or piperazine (MPH-48) substituents, but neither molecule showed better properties than the morpholine counterpart (MPH-49). The IC 50 of MPH-203 was promising; however, its solubility was even lower than that of MPH-49, suggesting that the optimal ring structure for selective and efficient inhibition is the morpholine ring. We also tried to modify the ring structure of the tricyclic core, but neither modification showed improved properties, confirming that MPH-220 is the optimal structure. 1I) on wild type and F490L variant form of NM2C. All three derivatives showed insignificant inhibition on the wild type form, while all three inhibited the F490L form efficiently. Importantly, when non-selective blebbistatin derivatives with small modifications on the D-ring (blebbistatin -yellow, para-nitroblebbistatin -purple, para-aminoblebbistatin -pink) were used, both wild type and F490L NM2C forms were inhibited identically (D). These data confirm that the selectivity observed for MPH-220 is due to the specific combination of morpholine substitution on the inhibitor and the unique Leu in the HP-helix of fast skeletal muscle myosin isoforms (cf. Fig. 1B, Fig. S1). Literature-based analysis of myosin-2 isoform composition -determined by SDS-PAGE or immune/enzyme histochemistry -of different muscles in rat (A), dog (B), mini pig (C) and human (D) show significant differences in the proportion of the slow isoform (MyHC I, gray) between the species. While rat muscles contain predominantly fast isoforms (MyHC IIa, IIb, IIx/d, orange), the ratio of fast isoforms in human muscles is between 40-70%. Distributions in dogs and mini-pigs are in between the rat and human levels. These differences bear important consequences on the expected effect size in different species and may require unique consideration of dosage in non-clinical studies on different species (e.g. rats vs. dogs/pigs). The relatively high proportion of slow isoform in human muscles also has important positive consequence on the effect profile of an MPH-220 based anti-spastic drug, because overdosingrelated complete muscle tone loss is not expected to occur due to the residual non-inhibited slow myosin contractions. This latter condition confers a huge advantage over the currently used nervous system targeting muscle relaxants, where temporary but complete immobility can readily in case of slight overdose. The large difference in slow isoform proportion can also be seen in the diaphragm of different species (E), which indicates that MPH-220 will be even safer for respiratory functions in humans than in rats. (A) Skeletal muscle myosin from rabbit m.psoas (containing 95% fast MyHC isoforms) is efficiently inhibited by S(-) (red, IC 50,S(-) = 1.1 ± 0.1 µM, I total,S(-) = 94 ± 1%) and the racemic mixture of MPH-220 (orange, IC 50,R/S = 3.4 ± 0.2 µM, I total,R/S = 95 ± 1%). The R(+) enantiomer is not effective, because its IC 50 value is an order of magnitude higher than that of the S(-) enantiomer, and the total inhibition could not be achieved (yellow, IC 50,R(+) = 19 ± 3 µM, I total,R(+) = 60 ± 4%). Data are mean ± SD; n=3-6. (D-F) All three inhibitors were administered to rats in different concentration ranges and cardiovascular (heart rate, blood flow) and respiratory functions (breath rate, oxygen saturation) were monitored. 16 mg/kg AmBleb killed the animals within 15 minutes due to heart failure (D), while neither 13 mg/kg MPH-188 (E) nor 18 mg/kg MPH-220 (F) had any significant effect on cardiovascular and respiratory functions. Note that we measured MPH-220 effect on longer time scales (parallel with force measurements). (G) We repeated the experiments several times and found that MPH-220 does not affect cardiac functions to a greater extent than the solvent excipient during the long anesthetic period (cf. Fig. 2F-G). Data are mean ± SD, n=1-4. (H) hERG potassium channel inhibition measurement up to 5 µM MPH-220. We measured hERG inhibition up to 5 µM, because the concentrations of MPH-220 in cardiac tissue even upon treatment with high doses (up to 130 mg/kg) were in this concentration range (3-7 µM). At this concentration range we did not observe inhibition of hERG potassium channels and we did not observe any concentration dependence of inhibition on cardiomyocytes, which would otherwise potentiate a drug candidate for QT prolongation leading to life-threatening arrhythmias [START_REF] Pollard | An introduction to QT interval prolongation and non-clinical approaches to assessing and reducing risk[END_REF]. Six hours after oral treatment with 25 mg/kg S(-) and R(+) enantiomers of MPH-220, rat tissue samples were analyzed and remarkable difference in accumulation could be observed. 3-6-times more S(-) enantiomer was accumulated in skeletal muscle tissues than R(+) enantiomer. However, this difference was not due to incomplete absorption of the R(+) enantiomer as the concentration of both enantiomers was approximately the same in all other tissues. Moreover, R(+)-MPH-220 concentration was even higher in liver, spleen and blood. Cylinder assay was carried out on three healthy littermates of the pyramidal tract damaged rats. Individual differences could be observed both in their intention to stand up and in general activities. All three rats were tested two times with 6 hours difference between the two measurements. Importantly, the number of prancing was in the range in which the spastic rats showed improvement after MPH-220 treatment (cf. S3), 1, 3, 5, 9 and 21 hours after the first measurement (healthy control) or before and 1, 3, 5, 9 and 21 hours after 20 mg/kg oral MPH-220 treatment. (B) Averaged distances between the paws in each frame of the 3D recordings in healthy (gray band) and treated (colored dots) animals; green: left fore-and hindlimbs, pink: right fore-and hindlimbs, red: left and right hindlimbs, maroon: left and right forelimbs, purple: left fore-and right hindlimbs, blue: right fore-and left hindlimbs. Schematic representation of distances is shown above the plots. Statistical analysis show that left fore-and hindlimbs are significantly closer in the operated rats than in the healthy animals, however, 1-5 hours after MPH-220 treatment the difference becomes insignificant. The right fore-and hindlimbs are significantly farther in the operated rats before treatment, which becomes non-significant 1 hour after MPH-220 treatment. These together indicate that MPH-220 treatment resulted in a significantly straightened body axis. Data are mean ± SD; n=2. (C) Principal component (PC) analysis: PC1, PC2 and PC3 scores of healthy (gray) and operated (colored bars) rats in all, slow, medium-speed and fast movement regimes (upper left quartet). PC1 (blue, upper right quartet), PC2 (eggplant, lower left quartet) and PC3 (purple, lower right quartet) scores before (darker) and after (lighter) 20 mg/kg oral MPH-220 treatment in all, slow, medium-speed and fast movement regimes. Box plots with min-to-max whishkers, n=4. (D) Parameter contributions to PC1 (upper row), PC2 (middle row) and PC3 (lower row) in the slow (left column), mediumspeed (middle column) and fast (right column) movement regimes. Dotted lines represent the threshold for major contributions to PC (3.57%). Box plots with min-to-max whishkers, n=4. (E) Explained variance in all, slow, medium-speed and fast movement regimes; PC1, PC2 and PC3 are coloread according to panels C-D. (F) The computed gait parameters were scaled (Z-score) and averaged. The columns represent the individual gait parameters (1-28, cf. Table S3 copied below the heatmap) in the slow (S1-28), medium-speed (M1-28) and fast (F1-28) movement regimes. The separate row represents the average of healthy animals at zero minute, and the other six rows represent the parameter scores before and 1, 3, 5, 9 and 21 hours after MPH-220 treatment (n=4). PC analysis revealed that in the slow regime MPH-220 has the largest effect on X-Y axis length and the consequent volumes of the ellipsoids representing paw positions in 3D and certain distances between the centers of ellipsoids (blue brackets) contributing to PC1; in the fast regime MPH-220 has the largest effect on the angles of the ellipsoids (purple brackets) contributing to PC3. Table S1. Properties of the applied proteins. Related to Figure 1. *ATPase activity in the presence of 1% DMSO and 25 µM F-actin, mean ± SD is shown **Inhibitory parameters determined in measurements shown in Fig. 1, mean ± SD is shown (n= 3-12) ***ND: not-determined reliably due to low levels or lack of inhibition a ATPase activity of the α-actinin containing human NM2C-S1(C0) construct [START_REF] Heissler | Comparative kinetic and functional characterization of the motor domains of human nonmuscle myosin-2C isoforms[END_REF] was 3-10-times lower lower than that of the HMM and full-length variants [START_REF] Billington | Characterization of three full-length human nonmuscle myosin II paralogs[END_REF][START_REF] Zhang | The Myosin II Inhibitor, Blebbistatin, Ameliorates FeCl3-induced Arterial Thrombosis via the GSK3beta-NF-kappaB Pathway[END_REF], which was also reflected in the ATPase activites of our NM2A and NM2B constructs. ATPase activites of S1 variants of NM2A [START_REF] Kovacs | Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform[END_REF], NM2B [START_REF] Wang | Kinetic mechanism of non-muscle myosin IIB: functional adaptations for tension generation and maintenance[END_REF] and NM2C [START_REF] Heissler | Comparative kinetic and functional characterization of the motor domains of human nonmuscle myosin-2C isoforms[END_REF] are also 3-6 times higher than the α-actinin containing constructs used here. Table S3. Parameter description used in PCA calculations in Fig. 4 and Fig. S12. Related to Figure 4 and Figure 5. Video recordings were analyzed in three walking speed categories (slow (less than 0.036 m/s), medium (0.036-0.072 m/s) and high (more than 0.072 m/s)). The numbers encode different parameters of the ellipsoids fitted to data points from 3D position analysis. LF: left front limb, RF: right front limb, LH: left hind limb, RH: right hind limb.

L L L L F F L F F F L F rat L L F F F F F pig L L L F dog L L L F L F L chicken L L F F rabbit L F cow L L F F mouse L L L F F L F F F F gene MYSA/

  (Fig. 1B, Fig S1).

  ). Birth Prevalence of Cerebral Palsy: A Population-Based Study. Pediatrics 137.van Tulder, M.W., Touray, T.,Furlan, A.D., Solway, S., and Bouter, L.M. (2003). Muscle relaxants for non-specific low back pain. The Cochrane database of systematic reviews, CD004252.Verplancke, D., Snape, S., Salisbury, C.F.,Jones, P.W., and Ward, A.B. (2005). A randomized controlled trial of botulinum toxin on lower limb spasticity following acute acquired severe brain injury. Clinical rehabilitation 19, 117-125.

Figure 1 .

 1 Figure 1. MPH-220 inhibits skeletal muscle myosin with extreme selectivity independently of the nervous system. (A) Current muscle relaxants target the central nervous system or act peripherally, while MPH-220 directly inhibits myosin. (B) Human myosin-2 isoforms contain phenylalanine (black F) at the beginning of the HP-helix between the switch-2 loop (red) and the relay region, except for fast skeletal myosin isoforms, where this position is leucine (blue L) (cf. Fig. S1). (C) MPH-220 was designed to bind into the blebbistatin-binding cavity of the motor domain, to enable inhibition in the actin-detached state. (D) Close-up view of the communication center of the functional regions (actin-binding cleft, switch-2 loop of the active site and the HPrelay-converter region) in the homology model of MyHC IIa with bound MPH-220 (orange) reveals interaction between the morpholino group and Leu 476 in the HP-helix. (E) Actinactivated ATPase inhibition of six myosin-2 isoforms (cf. Table S1). (F) Actin-activated ATPase inhibition of NM2C and the NM2C F490L variant. (G) Actin-activated ATPase inhibition of expressed human β-cardiac myosin with MPH-220 and blebbistatin. (H) Actin-activated ATPase inhibition of human muscle myosin samples from biopsies of m. soleus, m. vastus lateralis and heart ventricle. In contrast to MPH-220, blebbistatin fully inhibited m. vastus lateralis sample and β-cardiac myosin samples. Mean ± SD is shown in all ATPase experiments, n=3-9.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Crystal structure of MPH-220-bound fast skeletal muscle myosin. (A) Cartoon representation of the heavy chain (gray) of rabbit skeletal muscle myosin-2 in Mg.ADP.VO 4 (spheres, blue) pre-power-stroke state with the essential light chain bound to the lever arm (Table S2). MPH-220 binds to the bottom of the actin-binding cleft close to the HP-helix, with its morpholine ring in close proximity of Leu 476 . Blue mesh: electron density corresponding to MPH-220 at sigma 1.0. (B) Comparison of the MPH-220 (orange) binding site in MyHC IIb (gray/beige) and the blebbistatin (yellow) binding site in Dictyostelium myosin-2 (dark gray/brown) (1YV3.pdb (Allingham et al., 2005)). The chiral OH groups of both inhibitors interact with the same residues Gly 248 and Leu 270 (Gly 240 and Leu 262 in Dictyostelium). Mesh: surfaces based on Van-der-Walls radii, calculated with PyMol. (Bottom) Residues involved in compound binding in Ligplot+ v2.2 (Laskowski and Swindells, 2011) representation; U50: upper 50-kDa subdomain and L50: lower 50-kDa subdomain (gray); sw-2: switch-2 loop (red); HP: HP-helix (beige). Hydrogen bonds (up to 3.2 Å) are shown as green lines. (C) MPH-220 bound MyHC IIb structure with superimposed cardiac HP-helix illustrates the steric clash between the morpholine ring of MPH-220 and the cardiac HP-helix Phe 473 (red) residue.

Fig. 5 .

 5 Fig. 5. Principal component analysis of spastic rats reveals details of improved gait components after MPH-220 treatment. Global (A) and speed-specific (B) principal component (PC) analysis of gait parameters of healthy (gray) and operated (colored) animals before (darker) and after (lighter) MPH-220 treatment. (C) Parameter contributions to PC1 in the slow and medium regimes and to PC3 in the fast regimes (cf. TableS3). (D) Heatmap of major parameters contributing to PC1 (blue-orange) and PC3 (purple-orange) (cf. TableS3). (D) Schematic representation of MPH-220 effects on paw positions in slow and fast regimes.

  Fig. S2. Inhibitory properties of different MPH-220 derivatives. Related to Figure 1.

  Fig. S3. Contrary to wild-type NM2C, the NM2C variant harbouring the F490L substitution is sensitive to inhibition with morpholine group containing MPH-220 derivatives. Related to Figure 1. ATPase activity was measured with MPH-49 (A), MPH-188 (B) and MPH-220 (C, same dataset as in Fig.1I) on wild type and F490L variant form of NM2C. All three derivatives showed insignificant inhibition on the wild type form, while all three inhibited the F490L form efficiently. Importantly, when non-selective blebbistatin derivatives with small modifications on the D-ring (blebbistatin -yellow, para-nitroblebbistatin -purple, para-aminoblebbistatin -pink) were used, both wild type and F490L NM2C forms were inhibited identically (D). These data confirm that the selectivity observed for MPH-220 is due to the specific combination of morpholine substitution on the inhibitor and the unique Leu in the HP-helix of fast skeletal muscle myosin isoforms (cf. Fig.1B, Fig.S1).

  Fig. S4. Myosin-2 isoform distribution within different muscle types in different species. Related to Figure 1.Literature-based analysis of myosin-2 isoform composition -determined by SDS-PAGE or immune/enzyme histochemistry -of different muscles in rat (A), dog (B), mini pig (C) and human (D) show significant differences in the proportion of the slow isoform (MyHC I, gray) between the species. While rat muscles contain predominantly fast isoforms (MyHC IIa, IIb, IIx/d, orange), the ratio of fast isoforms in human muscles is between 40-70%. Distributions in dogs and mini-pigs are in between the rat and human levels. These differences bear important

Fig. S5 .

 S5 Fig. S5. Crystal structure of MyHC IIb is similar to the modeled structure used for inhibitor development. Related to Figure 2. (A) Cartoon representation of the crystal structure of MyHC IIb (gray/beige), and the superimposed modelled structure of MyHC IIa from molecular dynamic simulations (yellow/maroon). MPH-220 binding site reveals that the position and orientation of MPH-220 in the crystal structure (orange) and in the modeled structure (green) is similar. Amino acid residue carbons in gray (crystal structure) and maroon (model), and carbons for MPH-220 in orange (crystal structure) and green (model) are shown. (B) Close-up of the MPH-220 binding site reveals that HP-helix position is well predicted; in particular, Leu 476 position is very similar. The alignment also reveals a moderate shift in MPH-220 position between the homology model and the crystal structure; in particular we see slightly different position and orientation of the morpholino group. Still, the homology model predicts well the sub-pocket explored by this cycle; residues Leu 476 , Glu 477 , Cys 480 , Asn 600 and Val 650 interact indeed with the morpholino group in both crystal structure and homology model. These results confirm that the modelled structure that we used for inhibitor development (cf. Fig. 1, Fig. S2) is reliable and the results from those experiments are trustworthy. (C) Close-up view of the crystal structure with bound MPH-220 colored according to B-factors from 21.1 Å 2 (dark blue) to 125.2 Å 2 (red). ADP, VO 4 and MPH-220 are represented by thicker lines, the sphere represents the Mg 2+ ion.

  Fig. S7. Isometric force reduction by MPH-220 after intraperitoneal and oral administration. Related to Figure 3.(A) Isometric force reduction in rat hindleg after oral MPH-220 treatment: ΔF S(-) = 58%, ΔF R(+) = 36%, ΔF R/S = 32%, τ S(-) = 88 min, τ R(+) = 121 min, τ R/S = 461 min; muscle fatigue in the control animals: ΔF contr = 8%, τ contr = 323 min. (B) 18 mg/kg MPH-220 was administered to anesthetized rats by intraperitoneal injection in 200 µl DMSO (pink circles) or orally through a gavage tube in 30% SBECD (orange circles). Isometric force decrease rates were very similar (τ i.p. = 34 min) except that oral administration resulted in a 17-minute leg phase -corresponding to absorption of MPH-220 -followed by force decrease phase (τ p.o. = 53 min). These observations support high gastrointestinal permeability of MPH-220, which was further confirmed by in vitro Caco-2 permeability assay (cf. Fig.3E). (C) Caco-2 permeability assay in the apical-to-basolateral (AB, orange) and basolateral-to-apical (BA, light orange) direction in the presence of two different transporter inhibitors.

  Fig. S9. S(-)-MPH-220 accumulates in muscle tissues with higher tendency than the R(+) enantiomer. Related to Figure 3.

  Fig. S10. Cylinder test analysis of healthy rats show large individual variance in the tendency to stand up. Related to Figure 4.

  Fig. S11. Gait analysis of MPH-220 treated rats. Related to Figure 4 and Figure 5 (A) Paw position distribution of healthy (upper three rows) and treated (lower three rows) animals in the three speed-regimes (cf. TableS3), 1, 3, 5, 9 and 21 hours after the first

Fig. S12 .

 S12 Fig. S12. Safety profiling of MPH-220 supports further drug development due to lack of mutagenicity and interaction with off-target enzymes. Related to STAR Methods. (A) We tested the effects of 5 µM MPH-220 on 186 different human G-protein-coupledreceptors (GPCRs) covering 60 different receptor families including adrenergic, dopamine, P2Y and serotonin receptor families (by DiscoverX-Eurofins). According to the highly selective nature of MPH-220, neither GPCR was inhibited or activated above 30%, which is a standard reference threshold for hit compounds. (B) Beside the gpcrMAX panel, inhibition of 484 kinases

  b Literature-based species dependent MyHC distribution is demonstrated in Fig. S4. c ND: not-determined due to very low amount of myosin content in the human biopsies -only relative ATPase activity was measured d Blebbistatin inhibition was not measured due to limited sample size

Table 1 .

 1 Prevalence and economic burden of diseases with broad etiologies in which muscle spasticity is one of the major characteristic symptoms a WHO Atlas multiple sclerosis resources in the world 2008 (who.int/mental_health/neurology) b NIH Amyotrophic Lateral Sclerosis (ALS) brochure (ninds.nih.gov/Disorders)

	Disease	Background	Symptom	Prevalence	Patient number	Ratio of
				(per 100,000)		spasticity
	Stroke	Blood flow disruption in the	Weakness, paralysis,	100-600 (Kim et	30 million	38-46%
		brain either by a blood clot	spasticity, coordination and	al., 2015; Yi et al.,		(Lundstrom et al.,
		or by hemorrhage	balance problems, memory	2020)		2008; Opheim et
			problems			al., 2014; Urban et
						al., 2010; Wissel
						et al., 2013)
						(11-14 million
						spastic patients)
	Multiple sclerosis	Autoimmune disease	Fatigue, gait difficulties,	300-400 in US	2.3 million	50% a
	(MS)	attacking the myelin sheaths	numbness, spasticity,	(Wallin et al.,		(1.5-2 million
		of nerve fibers	weakness, vision problems	2019)		spastic patients)
	Cerebral palsy (CP)	Brain development disorders	Muscle stiffness, spasticity,	150-300 (Kruse et	17 million	70-75% (Novak,
		or disruption, due to genetic	ataxia, tremors, gait disorders	al., 2009; Pulgar		2014; Pulgar et
		mutations, infections, trauma		et al., 2019; Van		al., 2019)
		or injury		Naarden Braun et		(12-13 million
				al., 2016)		spastic patients)
	Amyotrophic lateral	Degeneration of cortical	Muscle weakness, atrophy,	4-5 (Xu et al.,	14-15,000 (US) b	40% (Meyer et al.,
	sclerosis (ALS)	motor neurons, the motor	spasticity	2019)		2019)
		nuclei of the brainstem, and				(6,000 spastic
		the anterior horn cells of the				patients)
		spinal cord
	Traumatic brain	Blow or jolt to the head or a	Paralysis, sensory	1% (up to 15%)	1.7-2 million new	5-19% (Bose et
	injury	head injury that disrupts	impairments, headaches,	(Dewan et al.,	TBI/year g in US	al., 2015;
		normal functions	coordination, spasticity	2018),	(Enslin et al.,	Verplancke et al.,
				475,000	2020),	2005) (30-35
				children/year (US)	68-71 million	million spastic
				(Enslin et al.,	worldwide	patients)
				2020)	(Dewan et al.,
					2018)
	Multiple system	Progressive	Slow movement, muscle	2-6 (Bjornsdottir
	atrophy (MSA)	neurodegenerative disease	stiffness, spasticity,	et al., 2013)
		with alpha-synuclein	incoordination, tremors,
		accumulation in glia	bladder control problems
		(form of atypical
		Parkinsonism)
	Spastic paraplegia	Defects in transport of	Progressive gait disorder,	3-6
		proteins, lipids through long	spasticity
		nerve fibers (axons).
	Myotonia congenita	Abnormality of skeletal	Painless spasm, difficulty	1-2 (Romitti et al.,
	(Becker disease)	muscle membranes resulting	relaxing muscles (myotonia),	2015)
		in hyperexcitability	rigidity, abnormally enlarged
			muscles (hypertrophy)
	Spinal cord injury or	Nerve damage due to e.g.	Pain, increased reflexes in	2-8 (Witiw and
	tumors	thoracic spine herniated discs	legs that can cause spasticity,	Fehlings, 2015)
		damage the nerves	muscle weakness,
			numbness or tingling

  at 25 o C in the presence of 0.5 mM ATP and 25 µM F-actin in ATPase buffer (10 mM MOPS pH 7.0, 4 mM MgCl 2 , 2 mM β-mercaptoethanol) for 15 minutes. ATPase activity was calculated from linear regression of the time dependent absorbance data collected at 340 nm. Different concentrations of inhibitors were added to the reaction in 0.5 µl DMSO (1% of total volume). DMSO and actin-controls were measured for each measurement set. Animals 200-250 g male Wistar rats were obtained from Toxi-Coop (Hungary) or Animal Facility, Basic Medical Science Center at Semmelweis University (Hungary). Animals were maintained in standard housing conditions with 12-12-hour light-dark periods and were allowed free access to dry rat food and water. All procedures were conducted in accordance with the ARRIVE guidelines and the guidelines set by the European Communities Council Directive (86/609 EEC) and approved by the Animal Care and Use Committee of the Eötvös University (registration number: 48/1/2015) under the permission numbers: PE/EA/142-5/2020, PE/EA/143-7/2020.

  The Kinect sensor was connected to a Windows PC (i5-9500F, GeForce GTX 1050 2 GB, 16GB DDR4) placed in a separate room via the official adapter (Kinect Adapter for Windows PC) and a USB extension cable. The Kinect for Windows SDK V2.0.1410.19000 API-s were used to control the Kinect sensor in MATLAB v. R2019b with the Image Acquisition Toolbox Support Package for Kinect for Windows Sensor add-on.An in-house MATLAB program was used to calibrate the spatial position of the device by measuring the relative distance of the four corners of the arena to the sensor to ensure uniform depth information values in the whole arena during experiments. Another in-house MATLAB program was used for image acquisition by the infrared depth camera (512 x 424 / 30 FPS) and the RGB camera (1920 x 1080 / 30 FPS) simultaneously. Video recordings from the depth camera were stored in .mj2 archives using lossless 16-Bit Motion JPEG 2000 compression, while the RGB camera recordings were stored in MP4 formats to allow easy viewer access. The depth video recordings were subjected to a preprocessing MATLAB script to correct the erroneous depth estimates arising from the time-of-flight depth sensing, such as flying pixels and multipath interference. The modified archives were scaled to preserve intensity values of the region of interest (rat in the arena) and exported as 8-Bit uncompressed AVI videos for further use.

Table S2 . Data collection and refinement statistics (molecular replacement).

 S2 Related to Figure 2. Data from one crystal. *Values in parentheses are for highest-resolution shell.

		Length of X-Y major axis		Length of X-Z major axis		Angle of X-Y major axes	Angle of X-Z major axes		Volume				Distance			Body-axis	No. of frames
	LF	RF	LH	RH	LF	RF	LH	RH	LF	RF	LH	RH	LF	RF	LH	RH	LF	RF	LH	RH	LF-RF	LF-LH	LF-RH	RF-LH	LH-RH	RF-RH
	1	Pre-power-stroke 2 3 4 5 6	7	8	MyHC IIb + MPH-220 9 10 11 12 13 14 15	16	17	18	19	20	21	22	23	24	25	26	27	28
		(ADP.VO 4 ) state
		Data collection
		Space group				P 2 1 2 1 2 1
		Cell dimensions
		a, b, c (Å)				49.6 119.4 176.6
		α, β, γ (°)				90.0 90.0 90.0
		Resolution (Å)			88.3 -3.3 (3.4 -3.3)*
		R merge						0.003722 (0.2293)
		I / σI						10.46 (2.42)
		CC 1/2						0.999 (0.939)
		Completeness (%)		99.80 (99.88)
		Redundancy				2.0 (2.0)
		Refinement
		Resolution (Å)			3.3
		No. reflections			total: 34625 (3374)
									unique: 17313 (1687)
		R work						0.2260 (0.3450)
		R free							0.2464 (0.4555)
		Non-hydrogen atoms		7474
		Protein					7382
		Ligand/ion				60
		Water					32
		B-factors					100.49
		Protein					100.73
		Ligand/ion				91.05
		Water					62.66
		R.m.s. deviations
		Bond lengths (Å)		0.015
		Bond angles (°)		1.55

Acknowledgments:

We would like to thank Prof. Árpád Dobolyi for help in rat operations; and Máté Winternitz, Noémi Karnok, Kinga Oravecz and Henrietta Hóf for technical assistance; and beam line scientists of PX2A (SOLEIL synchrotron) for excellent support during data collection. We also thank Prof. James R. Sellers (NIH-NHLBI) for detailed discussions and helpful comments on the manuscript; and Prof. Matthias Preller (MHH) for helpful discussions regarding MD simulations.

Funding: This study has been supported by the Hungarian National Research, Development and Innovation Office (NVKP 16-1-2016-0051 to MG, AÁR and AMC) and the Hungarian Ministry of Finance (GINOP-2.1.7-15-2016-02580 to MG and AÁR). LC is the recipient of a PhD fellowship from Ligue contre le cancer IP/SC-16058. AH was supported by grants from CNRS, FRM DCM20181039553, ANR-17-CE11-0029-01, AFM 21805. The AH team is part of the Labex CelTisPhyBio:11-LBX-0038, which is part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL). DVT, KMR and JAS are supported by National Institutes of Health grants R01 GM033289 and R01 HL1171138. DVT is supported by an American Heart Association postdoctoral fellowship (17POST33411070).

Materials Availability

There are restrictions to the availability of MPH-inhibitors due to related patents: PCT/EP2017/051829, WO/2017/129782, HU1800129A2; PCT/HU2019/050017, WO/2019/202346A2, WO/2019/202346A3. Access to these materials should be required from Optopharma Ltd.

Data and Code Availability

The published article includes all datasets generated or analyzed during this study. The atomic model is available in the PDB under PDB ID 6YSY.

Author contributions: MG: conceptualization, formal analysis, funding acquisition, investigation, methodology development, supervision, validation, visualization and writing, ÁIH: investigation, formal analysis, and validation, DT: investigation, software development, formal Supplemental Information for "Single residue variation in skeletal muscle myosin enables selective targeting: a small-molecule inhibitor ameliorates spastic gait disorder" Máté Gyimesi, Ádám I. Horváth, Demeter Túrós, Sharad Kumar Suthar, Máté Pénzes, Csilla Kurdi, Louise Canon, Carlos Kikuti, Kathleen M. Ruppel, Darshan V. Trivedi, James A. Spudich, István Lőrincz, Anna Á. Rauscher, Mihály Kovács, Endre Pál, Sámuel Komoly, Anne Houdusse, András Málnási-Csizmadia*

STAR Methods

Resource availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, András Málnási-Csizmadia (malna@elte.hu) As one of the most important requirements for a lead compound, mutagenicity was measured by the OECD/FDA approved Ames reverse mutagenicity assay using the two most sensitive bacterial strains for testing frameshift (TA98, dark blue) and base-pair substitution (TA100, light blue) mutation both in the absence (open circles) and presence (closed circles) of activated rat liver S9 fraction. Relative mutagenicity did not show MPH-220 concentration dependency and, more importantly, experiments on neither strain resulted in any data points above the threshold (gray dotted line). Besides MPH-220 we also tested the mutagenicity of the other two morpholine derivatives of MPH-220 (cf. Fig. S2-3) to elucidate whether the lack of mutagenicity is associated with the morpholine group. Neither MPH-49 nor MPH-188 showed concentration dependent increase in mutagenicity levels and, more importantly, neither showed in data points above the OECD/FDA threshold. Lack of mutagenicity was observed for both TA98 and TA100 S.typhimurium strains both in the absence and presence of liver S9 fraction, suggesting that neither the inhibitors nor their major metabolites are mutagenic, which supports safe development of MPH-220 toward human clinical phases.