Medicinal Chemistry and Use of Myosin II Inhibitor ... - ACS Publications

Perspective Cite This: J. Med. Chem. 2018, 61, 9410−9428

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Medicinal Chemistry and Use of Myosin II Inhibitor (S)‑Blebbistatin and Its Derivatives Bart I. Roman,*,†,‡,§ Sigrid Verhasselt,†,§ and Christian V. Stevens*,†,‡ †

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Research Group SynBioC, Department of Green Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Campus Coupure, Coupure Links 653 bl. B, 9000 Gent, Belgium ‡ Cancer Research Institute Ghent, De Pintelaan 185, 9000 Gent, Belgium ABSTRACT: (S)-Blebbistatin, a chiral tetrahydropyrroloquinolinone, is a widely used and well-characterized ATPase inhibitor selective for myosin II. The central role of myosin II in many normal and pathological biological processes has been revealed with the aid of this small molecule. The first part of this manuscript provides a summary of myosin II and (S)-blebbistatin literature from a medicinal chemist’s perspective. The second part of this perspective deals with the physicochemical deficiencies that trouble the use of (S)blebbistatin in advanced biological settings: low potency and solubility, fluorescence interference, (photo)toxicity, and stability issues. A large toolbox of analogues has been developed in which particular shortcomings have been addressed. This perspective provides a necessary overview of these developments and presents guidelines for selecting the best available analogue for a given application. As the unmet need for high-potency analogues remains, we also propose starting points for medicinal chemists in search of nanomolar myosin II inhibitors.

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INTRODUCTION

This work provides an overview of the medicinal chemistry of the blebbistatin series. It covers the discovery of (S)blebbistatin ((S)-1) as an inhibitor of myosin II ATPase activity, its synthesis, its mechanism of action, and its molecular targets. Furthermore, attention is given to its strengths and deficiencies as a pharmaceutical tool and research tool. The efforts made to mend these deficiencies are covered in an overview of the structure−activity and structure−property relationships (SAR and SPR, respectively) of (S)-blebbistatin and its analogues. An overview of all (S)blebbistatin derivatives developed up to now is presented, together with instructions for their optimal use as research tools. Finally, our perspectives on the development of superior myosin II inhibitors are provided.

The central role of myosin II in many normal and aberrant biological processes has in large part been revealed with the aid of (S)-blebbistatin ((S)-1, Scheme 1). This micromolar inhibitor of myosin II ATPase activity combines rapid and reversible inhibitory reactions with good cell-membrane permeability. These properties have made (S)-blebbistatin an essential tool for research on processes and diseases involving myosin II function and especially for dissecting precise myosin II driven cellular events. Since its discovery in 2001,1 (S)blebbistatin ((S)-1) has triggered a surge in myosin II related research (Figure 1). The affinity of (S)-blebbistatin ((S)-1) is not limited to particular myosin II isoforms: it covers cardiac-, skeletal-, and smooth-muscle myosin II and nonmuscle myosin II.2−10 This lack of selectivity and its low potency (IC50 values in the micromolar range) render (S)-blebbistatin ((S)-1) unusable as a starting point for the development of targeted therapeutic tools. The molecule also has several physicochemical liabilities that hamper its application as a research tool. For example, it displays poor water solubility and its (fluorescent) precipitates interfere in (fluorescence) read-outs.6−8,11−14 Additionally, it has cytotoxic side effects in certain contexts and is lightsensitive and phototoxic.4,6,15−20 Because of these deficiencies, the development of (S)blebbistatin derivatives as superior inhibitors of myosin II isoforms is an active research challenge. The aim of these endeavors is to obtain highly valuable research tools for cellbiology studies or starting points for the development of targeted therapeutic tools. © 2018 American Chemical Society

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MYOSINS Myosins are cellular proteins that act as motors, consuming fuel in the form of adenosine triphosphate (ATP). They are ATP-hydrolyzing enzymes that convert the chemical energy stored in ATP into mechanical force and movement. They use actin as a transport track or to produce tension. Myosins participate in many biological processes, of which musclebased movement is the most striking one.21−23 The myosin superfamily is divided into different classes, on the basis of sequence homology. The class II myosins were the first to be discovered and form the largest subfamily. They are generally referred to as conventional myosins. In addition, at least 34 classes of unconventional myosins are known. These Received: March 30, 2018 Published: June 7, 2018 9410

DOI: 10.1021/acs.jmedchem.8b00503 J. Med. Chem. 2018, 61, 9410−9428

Journal of Medicinal Chemistry

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Scheme 1. Topics Covered in This Perspective

regulatory light chains are encoded by three distinct genes.21,27−32 Each heavy chain is further built up of three different domains (Figure 2A): (i) the N-terminal head domain, which is also referred to as the motor or catalytic domain because it contains the actin- and ATP-binding sites required for motor activity (see ATPase Cycle); (ii) the neck domain or lever arm, which binds the essential and regulatory light chains and amplifies structural changes in the motor domain; and (iii) the tail domain, which mediates heavy-chain homodimerization via an α-helical coiled coil and ends with a C-terminal nonhelical tailpiece. The tail domain determines the cellular localization and is responsible for filament assembly (Figure 2B). The antiparallel association into filaments allows myosin II to crosslink actin filaments and contract them during the ATPase cycle (vide infra).21,27−31,33−37 The head domain functionally consists of four subdomains linked by flexible connectors (helixes, switches, and loops; Figure 3): an N-terminal subdomain, an upper 50 kDa

Figure 1. Evolution of the number of publications in the SciFinder database containing the terms “myosin II” or “blebbistatin” from 1995 to 2015.

were assigned a class number in the order of discovery, with the exception of class I myosins, which were discovered well after class II myosins. Not all myosin classes are present in all phyla. For instance, no class II myosins have been identified in plants, while several myosin classes are exclusive to plants (VIII, XI, and XIII). To date, 12 myosin classes have been discovered in humans (I−III, V−VII, IX, X, XV, XVI, XVIII, and XIX), of which myosin II is the most abundant.21−27 Myosin II. Structural Properties. All class II myosins are composed of six noncovalently associated polypeptides: they exist as a homodimer of two heavy chains, which each bind an essential light chain and a regulatory light chain (Figure 2A). The essential light chains stabilize the heavy chains. Depending on the isoform, the regulatory light chains have different functions, some of which have not yet been fully elucidated. The myosin II heavy chains, essential light chains and

Figure 3. Simplified schematic diagram of the myosin II head domain, which ends in the lever arm (ATP- and actin-binding sites marked with *). Adapted with permission of Annual Reviews, from Sweeney, H. L.; Houdusse, A. Annu. Rev. Biophys. 2010, 39.23 Permission conveyed through Copyright Clearance Center, Inc.

subdomain, a lower 50 kDa subdomain, and a converter subdomain. The upper and lower 50 kDa subdomains are separated by a deep 50 kDa cleft. The ATP-binding site is situated close to the apex of the 50 kDa cleft, at the boundary of the N-terminal and upper 50 kDa subdomains, and consists of the P-loop and switch I. The actin-binding site spans the upper and lower 50 kDa subdomains. Coupling between the actin-binding interface and the ATP-binding site is mediated via the 50 kDa cleft through switch II. A seven-stranded βsheet core and associated loops form a transducer region.21,23,27,33,35,36,38−44

Figure 2. (A) Structure of the myosin II complex, consisting of two heavy chains, two essential light chains, and two regulatory light chains. (B) Association of myosin II into filaments and cross-linking of actin filaments. 9411



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ATPase Cycle. Movement of myosin along the actin filament requires energy, which is provided by ATP hydrolysis in the head domain during an ATPase cycle (Scheme 2). This cycle

from lower eukaryotic species such as Acanthamoeba and Dictyostelium, and (iv) the myosins from fungi.21 In humans, the myosin II subfamily only comprises skeletal-, cardiac-, and smooth-muscle myosins, as well as nonmuscle myosin II (NM II). Skeletal-muscle myosin II is the motor of skeletal-muscle contraction and is responsible for posture and all voluntary movements, whereas cardiac-muscle myosin II drives contraction of the heart.21 Smooth-muscle myosin II is responsible for involuntary contractions of hollow organs (except the heart), such as the stomach, intestine, uterus, and blood vessels.51 NM II is a collective term covering three distinct paralogues: NM IIA, NM IIB, and NM IIC. All animal cells express at least one NM II isoform.21,28,36,51−53 NM II plays an important role in embryonic development and in the normal functioning of the adult organism; relevant events include cytokinesis, cell−cell adhesion, and cell migration.28,29,54,55 Overactivity of myosin II ATPase is, however, associated with a range of human diseases, such as viral infections,56−60 liver fibrosis and portal hypertension,61 arthrofibrosis,62 glaucoma,63 methamphetamine-use relapse,64,65 cancer metastasis,66−71 cardiovascular diseases,72 and chronic respiratory diseases.73 Inhibitors of the ATPase activity of particular myosin II isoforms would therefore constitute valuable tools for dissecting the exact role of the protein in physiological functions or for developing targeted treatments against the aforementioned diseases and disorders.

Scheme 2. Schematic Representation of the ATPase Cyclea

a

Adapted from Winkelmann et al. Nat. Commun. 2015, 6,47 which is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

starts with myosin tightly bound to actin (A·M). Next, a sequence of conformational changes is triggered in the myosin head domain by contact between the γ-phosphate group of an ATP molecule and the P-loop of the ATP-binding site. Both the P-loop and switch I move to enclose the ATP molecule through a network of hydrogen bonds. Additionally, these movements induce twisting of the seven-stranded β-sheet, which causes cleft opening in the actin-binding site. This results in a largely decreased actin affinity, and hence actin dissociation occurs (Scheme 2, step a, M·ATP).23,27,33,38,43−47 In order for ATP hydrolysis to occur, switch II closes to form the enzymatically active site (for more details, consult Kiani and Fischer).48,49 This movement is coupled to bending of the relay helix, rotation of the converter subdomain by 60°, and priming of the lever arm (Scheme 2, step b, recovery stroke, orange arrows). ATP is then hydrolyzed into adenosine diphosphate (ADP) and Pi (Scheme 2, step c). At this stage, the hydrolysis products remain bound to the myosin head, forming a myosin·ADP·Pi complex that still has weak actin affinity.27,33,38,41,45,46,50 Next, cleft closure enables tight myosin-head rebinding at a new position on the actin filament. This rebinding is accompanied by Pi release (Scheme 2, step d, formation of A·M·ADP). The temporal ordering and structural basis of these events is still under debate. The converter subdomain transmits the involved, relatively small conformational changes of the head domain to the lever arm, resulting in an amplification and a 60° swing. In this so-called power stroke (green bent arrow), the myosin head pulls the actin filament over a distance of approximately 10 nm (Scheme 2, step e, green upward arrow). At the end of the power stroke, ADP is released (Scheme 2, step f), which allows the start of a new cycle.27,33,38,41,45,46,50 Myosin II Isoforms and Physiological Functions. Class II myosins can be subdivided into four groups on the basis of sequence analysis of the head domain: (i) the sarcomeric myosins from striated muscle, (ii) the vertebrate smoothmuscle myosins and nonmuscle myosins, (iii) the myosins

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(S)-BLEBBISTATIN: CHEMICAL AND BIOLOGICAL CHARACTERIZATION Discovery. Blebbistatin was discovered by Cheung et al. during a high-throughput screen (HTS) for inhibitors of NM IIA ATPase activity in 2001.1,74 From the 16 300 compounds in the evaluated small-molecule library (DIVERSet E, Chembridge Inc.), only two HTS hits also inhibited NM IIA dependent processes in cells. These molecules were presumed to be structures 2 and 3 (Scheme 3). Retesting of a freshly Scheme 3. Discovery of (±)-Blebbistatin ((±)-1) as an Oxidation Product of Compound 274,75

dissolved dimethyl sulfoxide (DMSO) sample of compound 2, however, showed no influence on NM IIA ATPase activity. Curiously, over the next 2 days, the latter DMSO solution of 2 had converted from colorless to bright yellow while standing in air, with a concomitant gain in NM IIA ATPase inhibition. This suggested degradation of molecule 2 in the original library as well, as a result of repeated freeze−thaw cycles. Further investigation led to the discovery of (±)-blebbistatin ((±)-1), 9412



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Scheme 4. Lucas-Lopez Synthesis4,77,a

a Reagents and conditions: (i) (1) 1.1 equiv amide 4, 1 equiv POCl3, dry CH2Cl2, rt, 3 h; (2) 1 equiv amine 5, dry CH2Cl2, reflux, 16 h. (ii) 3 equiv LiHMDS, dry THF, −78 to 0 °C, 3 h. (iii) (1) 1.2 equiv LiHMDS, dry THF, −78 °C, 30 min; (2) 2.4 equiv oxaziridine 9, dry THF, −10 °C, 16 h. b Determination of enantiomeric excess via chiral-HPLC analysis. cUpon recrystallization from CH3CN, with [α]26 D = −464°.

Scheme 5. Verhasselt Synthesis79−81,a

Reagents and conditions: (i) (1) 2 equiv POCl3, dry CH2Cl2, rt, 24 h; (2) 1.05 equiv amine 5, dry CH2Cl2, 35 °C, 24 h. (ii) (1) 2.1 equiv LiHMDS, dry THF, 0 °C, 1 h; (2) 2.4 equiv oxaziridine 9, dry THF, −15 °C, 16 h. bDetermination of enantiomeric excess via chiral-HPLC analysis. cUpon recrystallization from CH3CN. a

cyclization of amidine 6 using excess LiHMDS resulted in quinolinone 8 (Scheme 4, step ii, 90%). This intermediate proved stable for extended periods of time in the absence of air and light, but as expected, decomposed to (±)-blebbistatin ((±)-1) when dissolved in DMSO and exposed to direct sunlight in the presence of air for a prolonged period of time or when adsorbed on silica and irradiated with 365 nm light. Asymmetric α-hydroxylation of quinolinone 8 using Davis’ oxaziridine methodology (Scheme 4, step iii, oxaziridine 9),78 yielded (S)-blebbistatin ((S)-1) in both high yield and high enantiomeric excess (82%, ee 86%). A single recrystallization further increased the ee of (S)-blebbistatin ((S)-1) to >99%. The overall yield starting from pyrrolidinone 4 was 30%. The above synthesis was later optimized and shortened by Verhasselt et al.79−81 Limitations in amidine formation were resolved by intensifying the conditions and by using automated reversed-phase flash chromatography for compound isolation, leading to an increase in the yield of 6 from 41% to 78% yield (Scheme 5). Subsequent intramolecular cyclization and asymmetric α-hydroxylation were merged into a high-yielding enantioselective one-pot procedure. The resulting two-step

an oxidation product of component 2, as the active compound in the degraded solution. The inhibitor was named after its ability to block the cell blebbing that often accompanies cytokinesis, a process which requires NM IIA ATPase activity.75 Separation via chiral HPLC and testing of both enantiomers revealed (−)-blebbistatin ((−)-1) as the active species. (+)-Blebbistatin ((+)-1) was inactive.1,2,74−77 At this stage, determination of the absolute configuration of the active isomer using X-ray crystallographic analysis of the enantiomers failed. The active isomer was later proven to bear the (S)configuration (vide infra). Syntheses of (S)-Blebbistatin. The first and enantioselective synthesis of (S)-blebbistatin ((S)-1) was developed by Lucas-Lopez et al. (Scheme 4).4,77 In their three-step sequence, rings A, C, and D (see Scheme 1 for numbering) of the scaffold were connected via condensation of amine 5 with amide 4 upon amide activation with POCl3 (Scheme 4, step i). Amidine formation was the limiting step in the sequence, with a moderate yield of 6 (41%) and formation of amide dimer 7. Other activating agents (i.e., SOCl2, MeOTf, and PCl5) did not give superior outcomes. Intramolecular 9413



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Scheme 6. Synthesis of Racemic Precursor (±)-1782,a

a

Reagents and conditions: (i) (1) 1.2 equiv amide 10, 1.2 equiv POCl3, dry CH2Cl2, rt, 3 h; (2) 1 equiv amine 5, dry CH2Cl2, reflux, 18 h. (ii) 2.5 equiv LiHMDS, dry THF, −78 to 0 °C, 3 h. (iii) 0.5 equiv sodium dichloroisocyanurate (13), THF/H2O (1:1), rt, 4 h. (iv) 1.85 equiv NaOH, H2O/THF (9:4), rt, 18 h. (v) 4 equiv DIPEA, 3 equiv TIPSOTf, dry CH2Cl2, reflux, 6 h. (vi) 4.5 equiv CAN, CH3CN/H2O (1:1), 0 °C, 8 h.

Table 1. Introduction of (Hetero)aromatic D-rings in Precursor (±)-17, Followed by TIPS Deprotection82,a,b

a Reagents and conditions: (i) 3 equiv Cs2CO3, 1.2 equiv ArI, 0.05−0.1 equiv CuI, 0.1−0.2 equiv (±)-18, molecular sieves (4 Å), dry toluene, 120 °C, 24 h. (ii) 3 equiv TBAF, THF, rt, 2 h. bThe yields are the combined yield of steps i and ii; the intermediates were not purified.

preparation in 78% overall yield (Scheme 5) is presently the most efficient synthesis of (S)-blebbistatin ((S)-1). A disadvantage of the syntheses of Lucas-Lopez and Verhasselt is that scaffold construction starts from a D-ringcontaining building block. This approach does not allow for a divergent synthesis of analogues with a varying decoration pattern on ring D, which would enable tuning of the physicochemical properties (vide infra). Lawson et al. investigated an alternative synthetic route for (±)-blebbistatin analogues comprising the late-stage introduction of (hetero)-

aromatic D-rings onto racemic ABC-tricyclic system (±)-17 (Scheme 6).82 The latter building block was prepared from pyrrolidinone 10 in six steps using a para-methoxyphenyl (PMP) protective group on the pyrrolidine nitrogen. Key steps were the racemic α-chlorination of quinolinone 12 with sodium dichloroisocyanurate, 13, and the subsequent nucleophilic substitution of chloride for hydroxide in alkaline aqueous medium. The authors did not comment on this twostep hydroxylation sequence (which contrasts the direct introduction in the previous routes). CuI-catalyzed N-arylation 9414



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Scheme 7. Failed Attempts to Determine the Absolute Stereochemical Configuration of (S)-14,a

a

Reagents and conditions: Strategy A: 1 equiv NBS, dry DMF, rt, 24 h. Strategy B: (1) 1.1 equiv amide 4, 1 equiv POCl3, dry CH2Cl2, rt; (2) 1 equiv amine 32, dry CH2Cl2, reflux. Strategy C: 0.5 equiv DMAP, 4 equiv dry pyridine, 10 equiv 3-bromobenzoyl chloride (36) or 4-bromobenzoyl chloride (37), dry CH2Cl2, rt, 24 h. bTime and yield were not reported. cThe enantiomeric excess was not reported, but [α]26 D values were −568 and −607 after recrystallization of (−)-38 and (−)-39, respectively, from EtOAc/hexane.

Scheme 8. Synthesis of Enantiopure (−)-4′-Bromoblebbistatin ((−)-31) and (−)-Blebbistatin ((−)-1) and Confirmation of the (S)-Configuration of (−)-4′-Bromoblebbistatin ((−)-31) by X-ray-Crystallographic Analysis4,a

a

Reagents and conditions: (i) 1 equiv NBS, dry DMF, rt, 2 days. (ii) (1) 1 equiv POCl3, dry CH2Cl2, rt, 3 h; (2) 1.1 equiv amine 5, dry CH2Cl2, reflux, 16 h. (iii) 3 equiv LiHMDS, dry THF, −78 to 0 °C, 3 h. (iv) (1) 1.2 equiv LiHMDS, dry THF, −78 °C, 30 min; (2) 2.4 equiv oxaziridine 9, dry THF, −10 °C, 16 h. (v) 3.7 equiv Et3N, 1% (m/m) Pd/C, H2, MeOH/DMF (1:1), rt, 24 h. bDetermination of enantiomeric excess via chirald 83 from Cambridge HPLC analysis. cUpon recrystallization from CH3CN, [α]26 D = −526. X-ray-structure image was rendered in Mercury 3.9 84 4 Structural Database entry CCDC-238392, originally uploaded by Lucas-Lopez et al.

of (±)-17 with iodobenzene using diamine 18 as a ligand and direct desilylation with tetra-n-butylammonium fluoride

(TBAF) yielded (±)-blebbistatin derivatives (±)-16 and (±)-25−30 in low to moderately high yields (Table 1). 9415



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Because of the numerous steps required for the preparation of precursor (±)-17, its racemic nature, and the low overall yield, late-stage incorporation of (hetero)aromatic D-rings via the Lawson route is not advantageous except for the preparation of large libraries. There is still room for a high yielding, enantioselective, and divergent preparation of D-ring modified (S)-blebbistatin analogues. Determination of the Absolute Stereochemistry of (−)-Blebbistatin ((−)-1). Originally, X-ray crystallographic analysis of the active enantiomer (−)-blebbistatin ((−)-1) did not provide sufficiently high-quality data for an absolute stereochemical determination. In analogy to literature asymmetric α-hydroxylations of cyclic enolates by oxaziridine 9,78 the (−)-enantiomer was believed to possess the (S)configuration. Lucas-Lopez et al. therefore attempted to prepare heavyatom bromine-containing analogues in order to obtain betterquality crystals (Scheme 7).4 The first strategy, involving the preparation of (−)-4′-bromoblebbistatin ((−)-31) via direct bromination of (−)-blebbistatin ((−)-1) with N-bromosuccinimide (NBS), failed, as purification of the resulting products proved too difficult (Scheme 7, Strategy A). The same problem was encountered with bromination of the intermediates en route to (−)-blebbistatin ((−)-1). The synthesis of (−)-6bromo-blebbistatin ((−)-35) from brominated aniline 32 was also unsuccessful, because of the low yield of amidine 33 (Scheme 7, Strategy B). The authors successfully prepared (−)-O-(3-bromobenzoyl)blebbistatin ((−)-38) and (−)-O-(4bromobenzoyl)blebbistatin ((−)-39) through acylation of the chiral alcohol in (−)-blebbistatin ((−)-1) with bromobenzoyl chlorides 36 and 37, respectively (Scheme 7, Strategy C). Unfortunately, recrystallization did not yield sufficiently highquality crystals for X-ray analysis. Ultimately, (−)-4′-bromoblebbistatin ((−)-31) was synthesized from amide 40 (Scheme 8).4 An enantiopure sample of (−)-4′-bromoblebbistatin ((−)-31) was obtained after recrystallization from acetonitrile, for which the absolute stereochemistry was determined as S. Further, enantiopure (S)-4′bromoblebbistatin ((S)-31) was reduced to (S)-blebbistatin ((S)-1; Scheme 8, step e, 99%, ee >99%). Lastly, chiral-HPLC, 1 H NMR, and MS analyses proved that this sample of (S)blebbistatin ((S)-1) was identical to (−)-blebbistatin ((−)-1), thereby finally confirming its absolute stereochemistry. Mechanism and Structural Basis of (Nonmuscle) Myosin II ATPase Inhibition. As discussed above (Scheme 2), the ATPase cycle can be divided into six different events (a−f). (S)-Blebbistatin does not affect ATP binding or ATP hydrolysis (Scheme 2, steps a−c). Structural studies on Dictyostelium discoideum myosin II and kinetic analysis have shown that (S)-blebbistatin binds to the myosin·ADP·Pi complex in the 50 kDa cleft between the ATP-binding site and actin-binding interface (Scheme 9 and Figure 4). This leads to a stabilized, long-lived (S)-blebbistatin·myosin·ADP·Pi complex, in which the 50 kDa cleft is kept partially open. As a result, (S)-blebbistatin ((S)-1) traps myosin in a state with low actin affinity and inhibits the release of Pi after ATP hydrolysis (Scheme 9, step d). In this way, it prevents force generation (i.e., the power stroke, Scheme 9, step e) from occurring.16,85,86 The cocrystal structure of (S)-blebbistatin ((S)-1) bound to the Dictyostelium discoideum myosin II·ADP·vanadate complex (PDB: 1YV3)86 also provides a rationale for the selective activity of (S)-blebbistatin ((S)-1) over its (R)-enantiomer:

Scheme 9. Nonmuscle Myosin II ATPase Inhibition Mechanism of (S)-Blebbistatin ((S)-1)a

a

During the ATPase cycle, (S)-blebbistatin ((S)-1) binds to the myosin·ADP·Pi complex and forms a long-lived, stable (S)blebbistatin·myosin·ADP·Pi complex with weak actin affinity. This prevents Pi release and force generation (i.e., the power stroke) from occurring.

Figure 4. Cocrystal structure of (S)-blebbistatin ((S)-1) bound to the head domain of the Dictyostelium discoideum myosin II·ADP·vanadate complex (PDB: 1YV3).86 Heteroatoms are colored as follows: nitrogen: dark blue, oxygen: red, phosphorus: green, vanadium: purple.

hydrogen-bond formation between the chiral hydroxyl group of (S)-blebbistatin ((S)-1) and the amide hydrogen of Gly240 and carboxylate oxygen of Leu262 (Figure 5). These bonds ensure a correct orientation of the molecule in the binding pocket. However, most of the binding strength originates from hydrophobic interactions. For example, rings A and B are surrounded by Tyr261, Thr474, Tyr634, Gln637, and Leu641; ring C interacts with Ile455, Ser456, and Ile471, whereas the side chains of Leu262, Phe466, Glu467, Cys470, and Val630 enclose ring D.86 Molecular Targets of (S)-Blebbistatin. Multiple groups have examined the ATPase-inhibitory potency of blebbistatin (1, in racemic form or as the (S)-enantiomer) against diverse members of the myosin superfamily through in vitro ATPase assays (Table 2).2−11,87−89 In such an assay, ATP consumption, Pi production, or ADP generation is quantified as a measure of ATPase activity. These studies have shown that (S)-blebbistatin ((S)-1) inhibits the ATPase activity of most myosin II class members, with half-maximum inhibitory concentrations (IC50) in the micromolar range. An exception is Drosophila melanogaster NM II, which is uniquely insensitive 9416



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from binding.86 This has recently been confirmed by a mutagenesis analysis in NM IIA, B, and C and smooth muscle myosin II, where an A456F mutation installed resistance to blebbistatin.89 This alteration did not significantly influence the motor activity or phosphorylation-dependent regulation. Further, in silico modeling and kinetic studies indicate that the presence of a Met residue in position 466 of Drosophila melanogaster NM II (rather than an Ile residue in the corresponding position 455 of human NM IIA) causes sufficient steric bulk to prevent binding of (S)-blebbistatin ((S)-1), thereby abolishing the inhibition of NM II ATPase activity.88

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STRENGTHS, DEFICIENCIES, AND IMPROVED ANALOGUES Since its discovery as a selective myosin II inhibitor, (S)blebbistatin ((S)-1) has rapidly been embraced as a research tool to investigate myosin II dependent processes in different species and cell types. Its main advantages are its cellmembrane permeability and its rapid and reversible effect (i.e., within minutes). As such, it has played a significant role in the understanding of the function of myosin II in cellular events, including cytokinesis, cell−cell adhesion, and cell migration.2,28,29,54 In cardiac-muscle research, (S)-blebbistatin ((S)-1) serves as a valuable tool for excitation−contraction uncoupling in electrophysiological studies.91,92 Finally, it has provided insight into the cellular and molecular events that drive a range of myosin II related diseases, such as viral infections,56−60 liver fibrosis and portal hypertension,61 arthrofibrosis, 62 glaucoma, 63 methamphetamine-use relapse,64,65 cancer metastasis,66−71 cardiovascular diseases,72 and chronic respiratory diseases.73 Unfortunately, (S)-blebbistatin ((S)-1) bears deficiencies that encumber or prohibit its use as a therapeutic tool or research tool. These include low potency, lack of selectivity

Figure 5. Detail of the (S)-blebbistatin ((S)-1) binding site in Dictyostelium discoideum myosin II (PDB: 1YV3).86 Carbon in amino acid residues: gray, nitrogen: blue, oxygen: red, sulfur: yellow, carbon of (S)-blebbistatin ((S)-1): orange, hydrogen bonds: red dashed lines.

toward (±)-blebbistatin ((±)-1) at concentrations up to 200 μM. Interestingly, (±)-blebbistatin ((±)-1) does not inhibit the ATPase activity of unconventional myosins from classes I, V, X, and XV, even at 150 μM. These data indicate that (±)-blebbistatin ((±)-1), and hence (S)-blebbistatin ((S)-1), is selective for myosin II but possesses low selectivity within this class. A rationale for these observations stems from comparison of the residues interacting with (S)-blebbistatin in the Dictyostelium discoideum myosin II cocrystal structure (PDB: 1YV3)86 to the homologous residues among the different myosins. All myosins listed in Table 2 possess the Gly240 and Leu262 residues required for orientation control. The insensitivity of myosins I, V, X, and XV can be explained by the substitution of residue Ser456 with residues bearing large, aromatic side chains (i.e., Tyr or Phe) that prevent (S)-blebbistatin ((S)-1)

Table 2. Half-Maximum Inhibitory Concentrations (IC50) of (±)-Blebbistatin ((±)-1) for the ATPase Activity of Diverse Myosins and Sequence Comparison with Selected (S)-Blebbistatin-Contact Residues in Dictyostelium discoideum Myosin II species D. discoideum Acanthamoeba H. sapiens H. sapiens G. gallus M. musculus G. gallus A. irradians O. cuniculus S. scrofa D. melanogaster R. norvegigus Acanthamoeba M. musculus B. taurus H. sapiens

myosin type II II nonmuscle IIA nonmuscle IIB nonmuscle IIB nonmuscle IIC smooth-muscle IIc striated-muscle II skeletal-muscle II β-cardiac-muscle II nonmuscle II Ib Ic Va X XV

IC50a (μM) a

4.9−13 83 4−14a 4.6a 1.8 3.2a 5.5−23.5 2.3 0.22−4.32a 1.2 >200d >150d >150d >150d >150d >100d

selected (S)-blebbistatin-contact residuesb Gly240 Gly Gly Gly Gly Glu Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

Leu262 Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu

Ser456 Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Tyr Tyr Tyr Phe Tyr

references Ile455 Ile Ile Ile Ile Ile Ile Ile Ile Ile Met Ile Ile Ile Ile Ile

3, 6−8, 11, and 87 3 2−4 and 89 89 3 89 10 and 89 51 3, 5−8, and 79−81 3 88 3 3 3 and 89 3 3

a

Data obtained with (S)-blebbistatin ((S)-1) were recalculated to mixtures containing 50% (S)-blebbistatin ((S)-1) and 50% (R)-blebbistatin (R)1, assuming that (R)-blebbistatin (R)-1 would not contribute to inhibition. bSequence alignments were performed on the UniProt Web site (accession numbers are P08799, P05659, P35579, P35580, Q789A4, Q6URW6, P10587, P24733, Q28641, P79293, Q99323, Q05096, P10569, Q99104, P79114, and Q9UKN7, respectively).90 cThe IC50 value for M. gallopavo3 and B. taurus9 smooth-muscle myosin II ATPase inhibition are 80 and 4.3−11 μM, respectively; these data are not included in the table, because no sequence information is available for these myosins. dNo inhibition was observed at the highest concentration evaluated. 9417



Perspective

Table 3. A-Ring Modification of (S)-Blebbistatin ((S)-1): Synthesis and ATPase-Inhibitory Activitya

ATPase-inhibitory activity

product and references (S)-14,77,81,94 (S)-4393 (S)-4493 (S)-4593 (S)-4693 (S)-474,77 (S)-4881,94 (S)-4981,94 (S)-5081,94 (S)-5181,94

b

R 6-Me 5-Me 7-Me 8-Me H 7-NO2

D. discoideummyosin II

human NM IIA

yield (%)

eec (%)

% inhibition at 50 μM

IC50 (μM)

30−78 20 19 31 19 3 49 15 95 96

86/>99 64/99 90/>99 86/>99 86/>99 76 72/>99 92/96 98 98e

92 88 90 35 88 ND ND ND ND ND

7.1 ± 0.4 NDd ND ND ND 28 ± 3 ND ND ND ND

rabbit skeletal-muscle myosin II % inhibition at 50 μM 97 95 94 72 95 ND ND ND ND ND

± ± ± ± ±

2 1 1 2 1

% inhibition at 5 μM 93 86 86 31 84 ND ND ND ND ND

± ± ± ± ±

1 1 1 9 1

IC50 (μM) 1.02 ND ND ND ND ND 7.97 8.46 14.5 >20f

± 0.05

± 0.02 ± 1.22 ± 2.2

a Reagents and conditions: (i) 6 equiv N,N′-dimethylbarbituric acid, 4 × 0.1 equiv Pd(PPh3)4, dry CH2Cl2, reflux, 4 × 1.5 h. (ii) 5 × 10 equiv MnO2, DMF, rt, 5 × 5 min. bFor compounds (S)-1 and (S)-43−49: overall yield from relevant N-aryl pyrrolidinone and 2-aminobenzoate; for compounds (S)-50 and (S)-51: yield of steps i and ii, respectively. cDetermination of ee via chiral-HPLC analysis; higher values are upon recrystallization from CH3CN. dND: not determined. eBased on the result for (S)-50. fConcentrations exceeding 20 μM resulted in compound precipitation in the assay buffer.

across myosin II isoforms, fluorescence, poor water solubility, possible cytotoxic side effects, light sensitivity, and phototoxicity. A large body of research has been devoted to mending each of these disadvantages, of which an overview is presented in the following paragraphs. Synthesis of Analogues and ATPase SAR. (S)Blebbistatin ((S)-1) inhibits the ATPase activity of several myosin II isoforms at micromolar concentrations. The low potency and poor selectivity within the myosin II subfamily are important deficiencies that prevent its widespread use as a pharmacological tool with in vivo model systems. A considerable body of research has targeted the development of (S)-blebbistatin analogues with improved potency and, to a lesser extent, selectivity. SARs have accordingly been established. Synthetic aspects in the following paragraphs are limited to those modifications that were not covered in the earlier section on the syntheses of (S)-blebbistatin. A-Ring-Modified Analogues. The number of A-ring modified analogues thus far prepared is limited to nine compounds. A series of analogues with simple modifications was prepared by Lucas-Lopez et al. The position of the methyl was varied in derivatives (S)-43−46,93 whereas the influence of a nitro group was evaluated in (S)-7-nitro-6-norblebbistatin ((S)-47, Table 3).4,77 These molecules were prepared using the above-described Lucas-Lopez route (Scheme 4). Noteworthy was a decreased asymmetric induction in the synthesis of (S)-43 (ee 64%), as steric clash with the methyl substituent disfavors coordination of the lithium cation with the enolate

oxygen (7Li NMR observations). Also, ring closure en route to (S)-45 proceeded sluggishly, because of steric clash between the methyl substituent and the nitrogen-coordinated lithium cation. A series of analogues with more extensive A-ring modifications was prepared by Verhasselt et al. (Table 3).81,94 In search of higher ATPase-inhibitory potency through additional hydrophobic interactions and π−π stacking with Tyr261, Tyr634, and Leu641 of the myosin II binding pocket (Figure 5), extension of the ring system at positions C6 and C7 was explored. (S)-Benzo[h]blebbistatin ((S)-48, Table 3), (S)(1H)-pyrrolo[3,2-h]blebbistatin ((S)-51), the corresponding indoline ((S)-50), and the N-allyl-protected synthetic intermediate ((S)-49) were synthesized and evaluated. The syntheses followed the earlier-described Verhasselt route (see Scheme 5). Analytical amounts of the (R)-enantiomers were prepared for chiral-HPLC analysis. Table 3 provides a comparison between the ATPase IC50 values of the A-ring-modified analogues and parent compound (S)-blebbistatin ((S)-1) for a number of myosin II isoforms. Removal of the methyl substituent or displacement to position C5 or C7 has little effect on activity. Methyl displacement to C8 markedly reduces activity, which was rationalized as being due to steric hindrance between the 8-methyl substituent and residue Tyr634 in a cocrystallization experiment with Dictyostelium discoideum myosin II. Nitro substitution at C7 and extension of the tricyclic core also have a negative impact on ATPase-inhibitory potency. Precipitation in the assay buffer 9418



Perspective

Scheme 10. Preparation of 4′-Substituted (S)-Blebbistatin Analoguesa

Reagents and conditions: (i) 1.5 equiv NIS, BF3·2H2O/MeOH (7:3), 50 °C, microwave, 30 min (small scale). (ii) NaN3, CuI, sodium ascorbate, DMEDA, DMSO/H2O (5:1), rt, 30 min.7,11 (iii) 2.5 equiv NCS, BF3·2H2O/MeOH (7:2), 100 °C, microwave, 30 min.6,7 (iv) 10 equiv HCOONH4, Pd/C, MeOH, rt, 15 min.80 (v) 0.05 equiv Pd(PPh3)4, 6 equiv K2CO3, dry MeOH, 50 °C, 1.5 h. (vi) 0.1 equiv NaI, 1.05 equiv Cs2CO3, 1.05 equiv benzyl bromide, dry CH3CN, 50 °C, 1 h.80. bThe yield was not reported. cThe ee was not reported. dReagent stoichiometry, yield, and ee were not reported. eOverall yield from relevant N-aryl pyrrolidinone and 2-aminobenzoate via the Lucas-Lopez or Verhasselt sequence. fThe reaction mixture initially consisted of 50 mol % (S)-54 and 50 mol % (S)-55. gThe ee was derived from the ee of (S)-58. hThe reaction mixture initially consisted of 94 mol % (S)-57 and 6 mol % dibenzylated product. a

Scheme 11. Synthesis of 3′-Modified (S)-Blebbistatin Analogues79−81,a

Reagents and conditions: (i) 0.05 equiv Pd (PPh3)4, 6 equiv K2CO3, dry MeOH, 50 °C, 1 h. (ii) 6 equiv N,N′-dimethylbarbituric acid, 0.1 equiv Pd(PPh3)4, dry CH2Cl2, reflux, 1 h. (iii) 2 × 4 equiv Et2NOH, dry CH2Cl2, reflux, 2 × 24 h. (iv) 30 equiv H2SO4, H2O, 70 °C, 14 h. (v) 1.05 equiv Cs2CO3, 0.2 equiv DMAP, 1.3 equiv acryloyl chloride (for (S)-66) or 1.4 equiv propionyl chloride (for (S)-67), dry CH3CN, rt, 30 min. (vi) 0.15 equiv guanidine hydrochloride, 1.01 equiv acrylic anhydride (for (S)-68) or 1.01 equiv propionic anhydride (for (S)-69), EtOH, 40 °C, 30 min. All ee values were obtained via chiral-HPLC analysis. bOverall yield from relevant N-aryl pyrrolidinone and 2-aminobenzoate via the Lucas-Lopez or Verhasselt sequence. cDetermination of ee via the chiral-HPLC analysis of the corresponding methyl ester. a

was moreover noted above at 10, 20, and 40 μM for compounds (S)-48, (S)-49, and (S)-50, respectively.

In conclusion, small lipophilic substituents can be tolerated on C5, C6, or C7 but not on C8, and they are synthetically best 9419



Perspective

Scheme 12. Synthesis of (S)-2,3-dihydro-1H-pyrrolo[2,3-c′]blebbistatin ((S)-74) and (S)-1H-pyrrolo[2,3-c′]blebbistatin ((S)75)80,81,a

a Reagents and conditions: (i) Verhasselt route. (ii) 6 equiv N,N′-dimethylbarbituric acid, 0.1 equiv Pd(PPh3)4, dry CH2Cl2, reflux, 1 h. (iii) 10 equiv MnO2, CHCl3, rt, 3 h. All ee values were obtained via chiral-HPLC analysis. bOverall yield from 71. cAfter recrystallization from CH3CN.

accessible on C6 and C7. No beneficial binding interactions were noted on linear extension of the tricyclic-core scaffold. Overall, the works of Lucas-Lopez4,77 and Verhasselt81,94 indicate that there is little potential for ATPase-potency enhancement via decoration of the A-ring of the (S)blebbistatin scaffold. D-Ring-Modified Analogues. Modification of the D-ring of (S)-blebbistatin ((S)-1, for numbering see Scheme 1) has mainly focused on the 3′- and 4′-positions, as the cocrystal structure with Dictyostelium discoideum myosin II (PDB: 1YV3) indicates that space for modifications is predominantly available in that region of the scaffold. The displacement of the phenyl ring for other aromatic moieties has also been investigated. Képiró et al. synthesized a first series of D-ring-modified analogues. Small amounts of (S)-4′-iodoblebbistatin ((S)-52) and (S)-4′-azidoblebbistatin ((S)-53) were obtained from (S)blebbistatin ((S)-1) by iodination and halogen-azide exchange, whereas larger amounts were prepared from 1-(4-iodophenyl)pyrrolidin-2-one via the Lucas-Lopez sequence and again azidation (Scheme 10).7,11 Yield and enantiopurity were not reported. The same researchers prepared (S)-4′-chloroblebbistatin ((S)-26, ee not provided) by direct chlorination of (S)blebbistatin ((S)-1, Scheme 10), a sample of (S)-4′-nitroblebbistatin ((S)-54) using the Lucas-Lopez route and preparative chiral HPLC (ee >99%),6,7 and small amounts of (S)-4′-aminoblebbistatin ((S)-55, in unknown yield and ee) by reduction of (S)-4′-nitroblebbistatin ((S)-54).8 Improved syntheses of (S)-4′-nitroblebbistatin ((S)-54) and (S)-4′aminoblebbistatin ((S)-55) with detailed compound characterization were later reported by Verhasselt et al. (Scheme 10).80,81 The latter group also synthesized analogues with substituents in the 4′-position of a more widely varying size: (S)-4′-allyloxyblebbistatin ((S)-56) was prepared in high enantiopurity from 1-(4-(allyloxy)phenyl)pyrrolidin-2-one using the Verhasselt route,80,81 and further conversion afforded (S)-4′-hydroxyblebbistatin ((S)-57) and (S) 4′-benzyloxyblebbistatin ((S)-58, Scheme 10). Finally, as described above

(Scheme 8), Lucas-Lopez et al. synthesized enantiopure (S)4′-bromoblebbistatin ((S)-31).4 Substitution at the 3′-position was explored by Verhasselt et al. in a series of analogues bearing groups of varying polarity and size (Scheme 11).79−81 (S)-3′-(Allyloxy)blebbistatin ((S)59), (S)-3′-(diallylamino)blebbistatin ((S)-60), and (S)-3′cyanoblebbistatin ((S)-61) were obtained via an adapted Lucas-Lopez route and subsequent modification. A key point was the choice of an allyl protecting group or cyano function. Compounds (S)-59−61 were converted into polar analogues (S)-3′-hydroxyblebbistatin ((S)-62), (S)-3′-aminoblebbistatin ((S)-63), (S)-3′-carbamoylblebbistatin ((S)-64), and (S)-3′carboxyblebbistatin ((S)-65). Selective esterification or monoamidation afforded (S)-3′-acryloxyblebbistatin ((S)-66, 61%, ee >99%), (S)-3′-propionyloxyblebbistatin ((S)-67, 99%, ee 99%), (S)-3′-acrylamidoblebbistatin ((S)-68, 73%, ee 99%), and (S)-3′-propionylamidoblebbistatin ((S)-69, 91%, ee >99%). The corresponding (R)-enantiomers were prepared in high enantiopurity, and the absolute configuration of (S)-62 and (R)-62 was determined by X-ray analysis. More extensive D-ring modifications were also reported by Verhasselt et al.: ring fusion at the 3′,4′-position with a lipophilic naphthyl group incorporated in (S)-benzo[c′]blebbistatin ((S)-70), an indoline as a conformationally restrained 3′-amino-substituted analogue in (S)-2,3-dihydro1H-pyrrolo[2,3-c′]blebbistatin ((S)-74), and a more polar indole moiety in (S)-1H-pyrrolo[2,3-c′]blebbistatin ((S)75).80,81 Compound (S)-70 was obtained in excellent enantiopurity via the Verhasselt sequence. Preparation of (S)-74 and (S)-75 started from pyrrolidinone 71 and followed the same route. Amidine synthesis proved difficult because of the electron-withdrawing protonated amino group (under the reaction conditions) present on the aryl amide. An enantiopure mixture of allylated intermediate (S)-72 and its oxidation product (S)-73 was ultimately obtained. The desired indoline (S)-74 (Scheme 12, step ii, 73%, ee >99%) and indole (S)-75 were subsequently obtained in high ee (Scheme 12, step iii, 70%, ee >99%). 9420



Perspective

Table 4. D-ring Modification of (S)-Blebbistatin ((S)-1): Known Analogues and ATPase-Inhibitory Activity

ATPase-inhibitory activity: IC50a b

product

R

ee (%)

D. discoideum myosin II (μM)

rabbit skeletal-muscle myosin II (μM)

references

(S)-1 (±)-25 (S)-26 (S)-31 (S)-52 (S)-53 (S)-54 (S)-57 (S)-55 (±)-15 (S)-56 (S)-58 (±)-29 (±)-27 (S)-62 (S)-63 (S)-61 (S)-65 (S)-64 (S)-59 (S)-66 (S)-67 (S)-60 (S)-68 (S)-69 (S)-70 (S)-74 (S)-75 (±)-28 (±)-30

H 4′-Me 4′-Cl 4′-Br 4′-I 4′-N3 4′-NO2 4′-OH 4′-NH2 4′-OMe 4′-OAllyl 4′-OBn 4′-Ph 3′-CF3 3′-OH 3′-NH2 3′-CN 3′-COOH 3′-CONH2 3′-OAllyl 3′-OAcryloyl 3′-OPropanoyl 3′-N(Allyl)2 3′-NHAcryloyl 3′-NHPropanoyl

>99 rac NDc,d >99 ND ND >99 >99 ND rac >99 >99 rac rac 99 >99 83 96 98 >99 >99 99 >99 99 >99 >99 >99 >99 rac rac

2.5−6.5 ND 1.9 ± 0.1 ND ND 5.2 ± 0.3 2.3 ± 0.1 ND 6.6 ± 2 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

0.11−2.16 ND ND ND ND ND 0.40−0.40 5.47 ± 1.32 1.0−5.4 ND 0.380 ± 0.003 >40e ND ND 19.3 ± 0.5 14.1 ± 0.1 48.5 ± 0.1 >100f >100f 9.41 ± 1.83 57.6 ± 7.8 23.5 ± 2.5 >10.8g >100f >100f >40e 7.70 ± 0.19 7.20 ± 0.59 ND ND

3, 5−8, 11, 79−81, and 87 82 6 and 7 4 7 and 11 7 and 11 6, 7, 80, and 81 79−81 8, 80, and 81 82 80 and 81 80 and 81 82 82 79−81 79−81 79−81 79−81 79−81 80 and 81 80 and 81 80 and 81 80 and 81 80 and 81 80 and 81 80 and 81 80 and 81 80 and 81 82 82

a Average ± SD for one literature source; range of averages for multiple sources. bThe ee of the final product used for biological testing; rac indicates a racemic mixture. cAlso prepared in racemic form by Lawson et al.82 dND: not determined. eHighest compound concentration used was 40 μM. fHighest compound concentration used was 100 μM. gHighest compound concentration used was 10.8 μM, as concentrations higher than 10.8 μM resulted in compound precipitation in the assay buffer.

The maximal extent of inhibition was the same for all active compounds, if solubility limits were not reached. Some SARs have nevertheless been deduced, which point toward a far larger influence of sterics than electronics.80 Small groups are equally well tolerated in the 4′-position as in the 3′-position, whereas for medium-sized groups, 4′-substitution is preferred. Large substituents are not tolerated at either position. The observations for larger groups could not be rationalized in terms of binding-pocket geometry. It has been suggested that the kinetics of the chemo-mechanical ATPase cycle also play an important role in ligand discrimination.

As described earlier, Lawson et al. also synthesized a range of D-ring-modified blebbistatin analogues, (±)-15 and (±)-25− 30, via their newly developed route (Scheme 6 and Tables 1 and 4).82 An overview of all hitherto reported analogues is presented in Table 4. A comprehensive overview of available myosin II ATPaseinhibitory-activity data of D-ring-modified blebbistatin analogues is presented in Table 4. Not all known analogues have been assessed, which is especially unfortunate for the Lawson series.82 2′-Substitution has not been evaluated so far. None of the D-ring modifications lead to an improvement in potency, whereas some alterations lead to a complete loss of activity. 9421



Perspective

Besides causing fluorescent precipitates, the application of (S)-blebbistatin ((S)-1) at concentrations exceeding its steadystate solubility also has other undesired consequences. Experiments carried out in oversaturated solutions create uncertainty about actual treatment concentrations, because these will depend on the time between addition and measurement. Furthermore, blebbistatin precipitates redissolve only slowly and have the tendency to attach to plastic or glass surfaces. This affects wash-out in commonly used experimental setups and compromises the reversibility of inhibition. The precipitates also perturb light-scattering-based measurements and may interrupt normal vascular flow in cardiac opticalmapping.8,14 Several groups have contributed to the development of blebbistatin analogues with a higher aqueous solubility while retaining good cell-membrane permeability. A first attempt was undertaken by Lucas-Lopez et al. by introducing an electronwithdrawing nitro group on the scaffold in (S)-7-nitro-6norblebbistatin ((S)-47).4,77 The authors claimed that fluorescence following 488 nm excitation was significantly reduced for (S)-7-nitro-6-norblebbistatin ((S)-47) as compared with that of (S)-blebbistatin ((S)-1), but both were later shown to be negligible.14,79 The use of this compound for resolving fluorescence interference and solubility-related issues is uncertain as solubility and permeability data were not provided. Its weaker potency as compared with that of (S)blebbistatin ((S)-1) also raises concerns (Table 3). Várkuti et al. later developed (S)-4′-nitroblebbistatin ((S)54) and (S)-4′-aminoblebbistatin ((S)-55, Table 5).6,8 The steady-state solubility of the nitro analogue was similar to that of (S)-blebbistatin ((S)-1). The protonated amino group (at physiological pH) in (S)-55, on the other hand, causes a steady-state aqueous solubility 30 times higher than that of (S)-blebbistatin ((S)-1). Concurrently, Verhasselt et al. developed (S)-3′-hydroxyblebbistatin ((S)-62) and (S)-3′aminoblebbistatin ((S)-63) as two myosin II inhibitors with a 30-fold higher water solubility than (S)-blebbistatin.79,81 The latter two molecules were further shown not to interfere in (fluorescence) readouts in cell-based systems. (S)-4′-Hydroxyblebbistatin ((S)-57) was significantly less soluble than the aforementioned polar analogues.80,81

Other Analogues. (±)-6-Bromo-4′-ethoxy-blebbistatin ((±)-76) was discovered by Cheung et al. in the same highthroughput screen as the one that found (±)-blebbistatin ((±)-1, Figure 6). It is also an oxidation product of a 4aminoquinoline (i.e., molecule 77).1,2,74

Figure 6. Discovery of (±)-6-bromo-4′-ethoxyblebbistatin ((±)-76) as an oxidation product of compound 77.1,2,74

Fluorescence and Solubility. (S)-blebbistatin ((S)-1) can interfere with the acquisition of fluorescent signals, which limits its use as a research tool.6,12,13 Combined use with green-fluorescent-protein labeling (GFP; 420−490 nm excitation, 520−570 nm emission) 95 or fluorescent Ca2+ indicators (488−494 nm excitation, 502−526 nm emission) is, for example, potentially troublesome.91,92,96 It was originally assumed that the fluorescence of (S)-blebbistatin ((S)-1) in solution was significant enough to cause interference.4,6 Aqueous solutions nevertheless exhibit negligible fluorescence in the 500−600 nm emission range. Swift et al. and Verhasselt et al. have later shown that (S)-blebbistatin ((S)-1) precipitates were actually causing fluorescence hotspots and interference.14,79,81 The steady-state solubility of (S)-blebbistatin ((S)-1) in aqueous buffers at 25 °C is 7−11 μM at 0.1% (v/v) DMSO. It increases quasi-linearly with the DMSO concentration to 80 μM at 10% (v/v) DMSO.8,11 In cellular experiments, however, the molecule is often added in putative concentrations of 50− 100 μM with the intention of fully inhibiting myosin II driven processes.3 Researchers need to be aware that although (S)blebbistatin ((S)-1) concentrations can indeed initially reach 50 μM, the compound will thereupon precipitate toward equilibrium concentrations over a short time frame (±90 min).7,8

Table 5. Potency and Physicochemical Properties of Selected (S)-Blebbistatin Analogues79−81 Caco-permeability assaya Papp (10 compound (S)-1 (S)-54 (S)-55 (S)-56 (S)-57 (S)-59 (S)-62 (S)-63

b

potency relative to that of (S)-1 1.0 2.5 0.19 2.3 0.16 0.23 0.11 0.15

± ± ± ± ± ± ± ±

0.2 0.2 0.01 0.3 0.04 0.05 0.01 0.01

c

solubility (μm) 6.18 31.7 >200f 2.75 82.4 2.83 193 186

± 0.08 ± 1.6d ± ± ± ± ±

0.05 9.7 0.09 1 9

A−B 60.5 32.5 69.4 29.7 NDg ND 28.5 62.7

± ± ± ±

−6

cm s−1)

recovery (%)

B−A 1.8 1.0 3.0 0.6

± 0.3 ± 3.1

19.2 2.23 31.1 7.58 ND ND 17.2 26.2

± ± ± ±

0.3 0.02 3.5 0.05

± 0.1 ± 0.1

A−B 66 34 85 32 ND ND 49 79

± ± ± ±

photostability t1/2(min)

B−A 1 1 1 1

±1 ±1

66 72 82 65 ND ND 70 74

± ± ± ±

4 1 1 1

±1 ±4

14 >90e 44 8.8 4.7 15 11 13

Caco-2 A−B (pH 7.4/7.4) and B−A (pH 7.4/7.4) permeability at 20 μM. bBased on IC50 values of ATPase inhibition in rabbit skeletal-muscle myosin II. cSteady-state solubility in PBS pH 7.4 buffer (2%, v/v, DMSO). dThe saturation concentration for this compound was also determined by Várkuti et al. yet under different conditions: 3.3 ± 0.1 μM at 0.1% (v/v) DMSO and 3.6 ± 0.2 μM at 1% (v/v) DMSO.8 eSample collected after 90 min indicated 33% degradation. fHighest compound concentration used was 200 μM. The saturation concentration for this compound was also determined by Várkuti et al. yet under different conditions: 298 ± 2.5 μM at 0.1% (v/v) DMSO and 426 ± 1.7 μM at 1% (v/v) DMSO.8 gND: not determined. a

9422



Perspective

Table 6. Cytotoxicity of (S)-Blebbistatin ((S)-1) Dependent on Cell Type, Inhibitor Concentration, and Incubation Time entry

cell type

concentration (μM)

incubation time

cytotoxicity (%)

reference

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

U87 U87 Du145 Du145 LNCaP LNCaP F11-hTERT F11-hTERT FEMX-1 FEMX-1 HeLa D. discoideum MIAPaCa2 BxPC3 Capan2 PANC1 zebrafish embryo

200 200 200 200 200 200 200 200 200 200 20 20 400 400 400 400 10

24 h 3h 24 h 3h 24 h 3h 24 h 3h 24 h 3h 3 days 3 days 24 h 24 h 24 h 24 h 36 h

65 40 90 30 96 10 85 30 100 30 90 0 20 25 45 0 100

15 15 15 15 15 15 15 15 15 15 6 6 66 66 66 66 6

Table 7. Light Sensitivity of (S)-Blebbistatin ((S)-1) at Various Wavelengths entry

wavelength (nm)

observed effect on (S)-blebbistatin ((S)-1)

reference

1 2 3 4 5 6 7 8 9 10 11

295 351 365 390−470 425 436 + 510 450−490 458 480 488 543

loss of inhibitory action on ATPase activity loss of inhibitory action on actin-filament movement change in absorption spectrum change in absorption and emission spectra and area% (HPLC) loss of inhibitory action on Pi release change in absorption spectrum change in absorption and emission spectra loss of inhibitory action on actin-filament movement change in absorption spectrum loss of inhibitory action on actin-filament movement and cytokinetic-ring contraction none

16 19 18 15, 80, and 81 16 4 18 and 20 19 6 and 8 17 and 19 19

Verhasselt et al. performed a comprehensive follow-up study on the solubility and membrane permeability (Caco-2, A−B and B−A) of polar and apolar (S)-blebbistatin analogues (Table 5).80,81 Compounds (S)-55, (S)-62, and (S)-63 were shown to combine excellent aqueous solubility and a high cellmembrane permeability. Remarkably, all evaluated (S)blebbistatin analogues showed high A−B permeability, and none were substrates for efflux transporters. A low A−B recovery was noted for (S)-54, (S)-56, and (S)-62, likely due to retention in the cell monolayer. In conclusion, cell-based screenings at high inhibitor concentrations where precipitates would interfere with readouts are best conducted using (S)-3′-hydroxyblebbistatin ((S)62), (S)-3′-aminoblebbistatin ((S)-63), or (S)-4′-aminoblebbistatin ((S)-55). Of these molecules, (S)-3′-hydroxyblebbistatin ((S)-62) is synthetically most accessible. Cytotoxicity. A disadvantageous feature of (S)-blebbistatin ((S)-1) in long-term experiments is its potential cytotoxicity (Table 6). Mikulich et al. showed that 200 μM (S)-blebbistatin ((S)-1) induces 65−100% cell death in various human cell lines (entries 1, 3, 5, 7, and 9) after prolonged incubation (24 h). The effect is much less pronounced in short-term experiments (3 h, entries 2, 4, 6, 8, and 10).15 Képiró et al. found that (S)-blebbistatin ((S)-1) causes 90% cell death in HeLa cells at 20 μM after 3 days of incubation (entry 11), whereas no cytotoxic effects were observed on Dictyostelium discoideum cells under the same conditions (entry 12).6

Pancreatic-adenocarcinoma-cell viability was only impaired at very high concentrations of (S)-blebbistatin ((S)-1). For instance, cytotoxicity for MIAPaCa2 (entry 13), BxPC3 (entry 14), and Capan2 (entry 15) cells exposed to 400 μM (S)blebbistatin ((S)-1) for 24 h was 20−45%, whereas no cytotoxic effects on PANC1 cells were observed at this concentration (entry 16).66 Finally, zebrafish embryos treated with 10 μM (S)-blebbistatin ((S)-1) all died after 36 h (entry 17).6 In sum, sensitivity to the cytotoxic effects caused by (S)blebbistatin ((S)-1) strongly depends on the species under investigation and the incubation time. The potential cytotoxicity of (S)-blebbistatin ((S)-1) complicates its use as a myosin II research tool. The phenotype caused by cytotoxicity may be falsely attributed to the inhibition of myosin II function. Two analogues with reduced cytotoxicity are available: (S)-4′-nitroblebbistatin ((S)-54) and (S)-4′-aminoblebbistatin ((S)-55).6,8 In HeLa cells, (S)-4′nitroblebbistatin ((S)-54) does not cause damage or cell death, which strongly contrasts the behavior of (S)-blebbistatin ((S)1, Table 6, entry 11). In zebrafish embryos, treatmentassociated mortality upon (S)-blebbistatin ((S)-1) administration (10 μM) was 100% after 36−72 h, whereas that of (S)4′-nitroblebbistatin-treated animals (10 μM) was at 25%, and that of (S)-4′-aminoblebbistatin-treated animals (20 μM) was at the level of the nontreated controls (0%). Importantly, both analogues mediate the same nonmuscle myosin II specific effects as (S)-blebbistatin: the compounds induce multi9423



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nuclearity of Dictyostelium discoideum (Dd) cells and engender a significant decrease in the blebbing indices of human melanoma (M2) cells at 5 μM within an hour. The cytotoxicity of (S)-blebbistatin thus seems to be caused by off-target phenomena. Further testing of this hypothesis is highly desirable. Still, in experiments where the cytotoxicity of (S)blebbistatin ((S)-1) obscures the data, (S)-4′-nitroblebbistatin ((S)-54) and (S)-4′-aminoblebbistatin ((S)-55) serve as validated alternatives. Light Sensitivity and Phototoxicity. Studies involving fluorescence have identified a further limitation of (S)blebbistatin ((S)-1): exposure to light with wavelengths below 490 nm (Table 7) causes rapid compound degradation and loss of myosin II inhibition. No significant inactivation occurs at 543 nm excitation (entry 11).16,17,19 Several groups have investigated these phenomena in more detail, mostly relying on qualitative changes in the absorption and emission spectra as a degradation indicator.4,6,18,20 Mikulich et al. reported that alteration of the absorption spectrum of (S)-blebbistatin coincides with oxidation of dihydrorhodamine (a probe for peroxynitrite anions, nitroso radicals, nitro radicals, hydroxyl radicals, and superoxide anions) but not of singlet-oxygen sensor green (a probe for singlet oxygen).15 Verhasselt et al. have observed that water is moreover required to initiate degradation.80,81 This has led to the hypothesis that in aqueous media and under blue-light irradiation, the photosensitizing properties of (S)-blebbistatin may be sufficient to generate hydroxyl or peroxy radicals, which in turn induce oxidative degradation of (S)-blebbistatin. The use of the term photostability can thus be questioned, as (S)-blebbistatin might rather be prone to oxidative degradation. The observation that analogues bearing electron-withdrawing groups do not decompose under identical conditions (see below) corroborates this hypothesis, but further testing is highly desirable. The destruction of (S)-blebbistatin ((S)-1) by blue light entails toxic effects in cells (demonstrated in HeLa, FEMX-1, LNCaP, Du145, U87, F11-hTERT, bovine aortic endothelial cells, human blood monocytes, and rat cardiac myocytes), which are distinct from the cytotoxic effects mentioned above.6,8,15,18−20,97 Cell death after illumination in the presence of (S)-blebbistatin ((S)-1) cannot be avoided by refreshing the cell-culture medium. The final degradation products are, however, nontoxic.18 The cells are thus irreversibly damaged by a short-lived product formed during the photochemical reaction via a yet unknown mechanism. Much effort has been put into the development of stable and nonphototoxic analogues, which were mostly evaluated by qualitative spectroscopic analysis. Lucas-Lopez et al. found that (S)-7-nitro-6 norblebbistatin ((S)-47) was stable upon exposure to 436 and 510 nm filtered light.4,77 Unfortunately, no phototoxicity data were reported. Képiró, Várkuti, et al.6−8 prepared (S)-4′-chloroblebbistatin ((S)-26), (S)-4′-nitroblebbistatin ((S)-54), and (S)-4′-aminoblebbistatin ((S)-55) as stable analogues under 480 nm irradiation. The phototoxicity of these compounds was evaluated against HeLa cell morphology. Remarkably, (S)-4′-nitroblebbistatin ((S)-54) and (S)-4′-aminoblebbistatin ((S)-55) proved well-tolerated, whereas treatment with (S)-4′-chloroblebbistatin ((S)-26) caused extensive cell death. The tempting conclusion that a reduction in photosensitivity is not necessarily linked with a reduction in phototoxicity should be regarded with caution,

however, as no quantitative photostability data on (S)-4′chloroblebbistatin ((S)-26) are available. Quantitative stability data under blue-light irradiation were generated by Verhasselt et al. for (S)-blebbistatin and seven analogues (Table 5), under conditions relevant to common experimental setups (100 μM in a 1:1 mixture of DMSO and DMEM supplemented with 20%, v/v, fetal calf serum).80,81 (S)-4′-Nitroblebbistatin ((S)-54) was found to be highly stable (33% degradation after 90 min of irradiation). The stability of (S)-4′-aminoblebbistatin ((S)-55), contrary to what was expected on the basis of earlier-reported qualitative data,8 was inferior to that of (S)-54 by a factor of two. All other analogues performed similarly or were inferior (for (S)-4′hydroxyblebbistatin ((S)-57)) to the parent compound, (S)blebbistatin ((S)-1). Other important observations were the significant dependence of half-life on the concentration and matrix. Pilot experiments in simple aqueous solutions thus cannot be extrapolated to cell work in complex biological media. In conclusion, electron-withdrawing groups result in improved stability of (S)-blebbistatin analogues under bluelight irradiation and likely a reduction in phototoxicity, though the underlying mechanisms remain unclear. Research applications requiring exposure to light with wavelengths below 490 nm are best conducted with (S)-4′-nitroblebbistatin ((S)-54). Irreversible Myosin II Inhibitors. The low water solubility of (S)-blebbistatin ((S)-1) prevents inhibition at high concentrations in aqueous media. This limits the complete inhibition of myosin II driven cellular processes and complicates the identification of low-affinity cellular targets.3 A photoreactive covalent myosin II inhibitor, (S)-4′azidoblebbistatin ((S)-53), has been developed to overcome these limitations.11 This molecule has a comparable water solubility to (S)-blebbistatin ((S)-1), and its inhibitor profile is identical to that of the latter molecule in the absence of UV irradiation. Upon irradiation at 310 nm (a wavelength that is nontoxic to cells and tissues), it covalently cross-links bound protein, while the activity of unbound inhibitor is eliminated. Dictyostelium discoideum myosin II, for example, was completely and covalently bound by (S)-4′-azidoblebbistatin ((S)-53) using multiple addition−irradiation cycles at low concentrations (10 μM). The compound has also aided in the identification of previously unknown low-affinity (i.e., EC50 values of more than 50 μM) targets of (S)-4′-azidoblebbistatin ((S)-53) and likely of (S)-blebbistatin ((S)-1).

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CONCLUSION AND PERSPECTIVES FOR FURTHER RESEARCH (S)-blebbistatin ((S)-1) was discovered as a degradation product during a HTS for inhibitors of NM IIA ATPase activity. It is a micromolar, uncompetitive inhibitor that stabilizes the myosin·ADP·Pi complex of the ATPase cycle. The chiral hydroxyl group is crucial for the selective activity of the (S)-enantiomer. Its cell-membrane permeability, rapid inhibition, and reversible effects have established (S)blebbistatin ((S)-1) as an important research tool in the understanding of the function of myosin II in both normal and aberrant biological processes. Unfortunately, the affinity of (S)-blebbistatin ((S)-1) is not limited to particular myosin II isoforms. The molecule also possesses other deficiencies that encumber its application, which include low (micromolar) potency, fluorescence interference, poor water solubility, cytotoxicity, blue-light 9424



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Figure 7. (S)-Blebbistatin strengths and deficiencies, recommendations for the optimal use of the available improved analogues as tool compounds, and suggestions for further research on myosin II inhibitors.

insights, rational-medicinal-chemistry programs toward potent and selective analogues may be launched. Alternatively, highthroughput screens against different myosin isoforms can deliver hits (novel chemotypes) for chemical-optimization efforts and, more importantly, novel druggable binding pockets. The rapidity with which (S)-blebbistatin has been embraced by the research community reflects the need for sophisticated small-molecule chemical and pharmacological tools to dissect and manipulate myosin II dependent phenomena. We hope that this work will serve as an accessible résumé of the medicinal chemistry of (S)-blebbistatin, and that it will stimulate the search for superior and more selective inhibitors of myosin II isoforms.

sensitivity, and phototoxicity. Further elucidation of the precise chemical and biochemical nature of the cytotoxicity and phototoxicity of (S) blebbistatin ((S)-1) remains necessary to further validate the large body of research that has been conducted using this tool compound. As discussed above, the majority of the deficiencies of (S) blebbistatin ((S)-1) have been mended over recent years by the establishment of SPRs and the ensuing synthesis of superior analogues. Use of these novel derivatives has unfortunately been limited. As with all improved analogues of widely used inhibitors, underlying reasons may be limited awareness of the existence of these tools among biologists and biochemists, neophobia, or perceived unavailability. The confirmation of these analogues as bona fide tools for the study of myosin II in this perspective and the clear references to their synthetic protocols should remove the aforementioned barriers from the field of myosin research and beyond. To further increase awareness and as a guidance to researchers currently using (S)-blebbistatin as a tool, Figure 7 presents an overview of the most performant (S)-blebbistatin derivatives for common applications. The lack of selectivity among myosin II isoforms and low potency of (S)-blebbistatin remain unresolved. Despite significant exploration of the structure−activity landscape for rings A and D, the (S)-blebbistatin series thus remains unfit as a starting point for the development of targeted therapeutic tools against particular myosin II isoforms. As indicated above, the selectivity and affinity of the series cannot be rationalized from visual analysis of the residues lining the binding pocket. This has also been observed for other inhibitors of myosin motors.73 Underlying factors, such as the kinetics of the chemo-mechanical cycle and the residues influencing the associated geometries, are likely implicated in myosin ligand discrimination.79,81 We therefore call for high-resolution biochemical and computational (molecular-dynamics) studies on the interaction of (S)-blebbistatin with various myosin motors during the entire ATPase cycle (Figure 7). From these

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] and [email protected] (B.I.R.). *E-mail: [email protected] (C.V.S.). ORCID

Bart I. Roman: 0000-0002-9407-6721 Christian V. Stevens: 0000-0003-4393-5327 Author Contributions §

B.I.R. and S.V. contributed equally to this work.

Notes

The authors declare no competing financial interest. Biographies Bart I. Roman (Kortrijk, 1984) has a M.Sc. in Chemical Engineering (2006) and holds a Ph.D. in Applied Biological Sciences: Chemistry (2012, with Christian Stevens, Ghent University). In 2014, he conducted a postdoctoral stay at the lab of Phil Baran (the Scripps Research Institute). He is currently working as a senior postdoc in the SynBioC research group (Ghent University). Bart’s interests lie in the discovery of innovative (bio)chemical technology to interrogate, understand, and pharmacologically modulate cellular and molecular 9425



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(6) Képiró, M.; Várkuti, B. H.; Végner, L.; Vörös, G.; Hegyi, G.; Varga, M.; Málnási-Csizmadia, A. para-Nitroblebbistatin, the noncytotoxic and photostable myosin II inhibitor. Angew. Chem., Int. Ed. 2014, 53, 8211−8215. (7) Képiró, M. Azidation Technology: From Photoaffinity Labeling to Molecular Tattooing. Ph.D. Dissertation, Eötvös Loránd University, Budapest, Hungary, 2014. (8) Várkuti, B. H.; Képiró, M.; Horváth, I. Á .; Végner, L.; Ráti, S.; Zsigmond, Á .; Hegyi, G.; Lenkei, Z.; Varga, M.; Málnási-Csizmadia, A. A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative. Sci. Rep. 2016, 6, 26141. (9) Eddinger, T. J.; Meer, D. P.; Miner, A. S.; Meehl, J.; Rovner, A. S.; Ratz, P. H. Potent inhibition of arterial smooth muscle tonic contractions by the selective myosin II inhibitor, blebbistatin. J. Pharmacol. Exp. Ther. 2007, 320, 865−870. (10) Wang, H. H.; Tanaka, H.; Qin, X.; Zhao, T.; Ye, L.-H.; Okagaki, T.; Katayama, T.; Nakamura, A.; Ishikawa, R.; Thatcher, S. E.; Wright, G. L.; Kohama, K. Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction. Am. J. Physiol. Heart Circ. Physiol. 2008, 294, H2060− H2068. (11) Képiró, M.; Várkuti, B. H.; Bodor, A.; Hegyi, G.; Drahos, L.; Kovács, M.; Málnási-Csizmadia, A. Azidoblebbistatin, a photoreactive myosin inhibitor. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9402−9407. (12) Wong, K.; Van Keymeulen, A.; Bourne, H. R. PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells. J. Cell Biol. 2007, 179, 1141−1148. (13) Shiba, Y.; Fernandes, S.; Zhu, W.-Z.; Filice, D.; Muskheli, V.; Kim, J.; Palpant, N. J.; Gantz, J.; Moyes, K. W.; Reinecke, H.; Van Biber, B.; Dardas, T.; Mignone, J. L.; Izawa, A.; Hanna, R.; Viswanathan, M.; Gold, J. D.; Kotlikoff, M. I.; Sarvazyan, N.; Kay, M. W.; Murry, C. E.; Laflamme, M. A. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 2012, 489, 322−325. (14) Swift, L. M.; Asfour, H.; Posnack, N. G.; Arutunyan, A.; Kay, M. W.; Sarvazyan, N. Properties of blebbistatin for cardiac optical mapping and other imaging applications. Pfluegers Arch. 2012, 464, 503−512. (15) Mikulich, A.; Kavaliauskiene, S.; Juzenas, P. Blebbistatin, a myosin inhibitor, is phototoxic to human cancer cells under exposure to blue light. Biochim. Biophys. Acta, Gen. Subj. 2012, 1820, 870−877. (16) Ramamurthy, B.; Yengo, C. M.; Straight, A. F.; Mitchison, T. J.; Sweeney, H. L. Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIb. Biochemistry 2004, 43, 14832−14839. (17) Limouze, J.; Sakamoto, T.; Mitchison, T. J.; Straight, A. F.; Ostap, E. M.; Sellers, J. R. Blebbistatin, a myosin II inhibitor with interesting photochemical properties. Mol. Biol. Cell 2002, 13, 455A− 455A. (18) Kolega, J. Phototoxicity and photoinactivation of blebbistatin in UV and visible light. Biochem. Biophys. Res. Commun. 2004, 320, 1020−1025. (19) Sakamoto, T.; Limouze, J.; Combs, C. A.; Straight, A. F.; Sellers, J. R. Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light. Biochemistry 2005, 44, 584−588. (20) Bzymek, R.; Horsthemke, M.; Isfort, K.; Mohr, S.; Tjaden, K.; Müller-Tidow, C.; Thomann, M.; Schwerdtle, T.; Bähler, M.; Schwab, A.; Hanley, P. J. Real-time two- and three-dimensional imaging of monocyte motility and navigation on planar surfaces and in collagen matrices: roles of Rho. Sci. Rep. 2016, 6, 25016. (21) Sellers, J. R. Myosins: a diverse superfamily. Biochim. Biophys. Acta, Mol. Cell Res. 2000, 1496, 3−22. (22) Mooseker, M. S.; Foth, B. J. The Structural and Functional Diversity of the Myosin Family of Actin-Based Molecular Motors. In Myosins: a Superfamily of Molecular Motors; Coluccio, L. M., Ed.; Springer: Dordrecht, The Netherlands, 2008; pp 1−34. (23) Sweeney, H. L.; Houdusse, A. Structural and functional insights into the myosin motor mechanism. Annu. Rev. Biophys. 2010, 39, 539−557.

events driving malignant and neglected diseases. His work encompasses QSAR methodology, drug design, bio-organic synthesis, chemical biology, in vitro and in vivo biological evaluation, and natural products. Bart is a member of the Flemish Royal Society of Chemistry, the Société de Chimie Thérapeutique, and the American Chemical Society. Sigrid Verhasselt (1990) obtained a degree of Master of Science in Bioscience Engineering: Chemistry and Bioprocess Technology at Ghent University in 2013. She then started working at the Department of Green Chemistry and Technology (Ghent University) under the supervision of Prof. Dr. Ir. Christian Stevens, Dr. Ing. Bart Roman, and Prof. Dr. Marc Bracke. She performed research on the synthesis of blebbistatin derivatives, their evaluation as myosin II inhibitors, and their physicochemical characterization. She obtained a Ph.D. in Applied Biological Sciences: Chemistry from Ghent University in 2017. Sigrid’s research interests are situated in the fields of synthetic organic and medicinal chemistry. Prof. Dr. Ir. Christian V. Stevens (1965) is full professor at the Department of Green Chemistry and Technology of Ghent University, Belgium. He graduated in 1988 and obtained a Ph.D. in 1992 working with Prof. De Kimpe at Ghent University. He performed postdoctoral work at the University of Florida guided by Prof. Alan Katritzky (1992−1993). He became associate professor in 2000 and full professor in 2008. Chris has published over 230 international peer-reviewed papers and 14 patents and has received several prizes. His research interest focuses on heterocyclic chemistry, flow chemistry, and the use of renewable resources for industry. He is an active member of the Flemish Royal Society of Chemistry, a Fellow of the Royal Society of Chemistry, and a member of the American Chemical Society.

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ACKNOWLEDGMENTS B.I.R. and S.V. respectively thank the Fund for Scientific Research − Flanders (FWO-Vlaanderen) and the Special Research Fund of Ghent University (BOF-UGent) for financial support for this research. The authors thank Marc E. Bracke (Ghent University) for the helpful discussions and for proofreading this manuscript.

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ABBREVIATIONS CCDC, Cambridge Crystallographic Data Centre; DMEM, Dulbecco’s modified Eagle’s medium; NM II, nonmuscle myosin II; PMP, para-methoxyphenyl

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REFERENCES

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