Medicinal Chemistry and Use of Myosin II Inhibitor - ACS Publications

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The Medicinal Chemistry and Use of Myosin II Inhibitor (S)-Blebbistatin and Its Derivatives Bart I. Roman, Sigrid Verhasselt, and Christian Victor Stevens J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00503 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Journal of Medicinal Chemistry

The Medicinal Chemistry and Use of Myosin II Inhibitor (S)-Blebbistatin and Its Derivatives Bart I. Roman,a,b,*,# Sigrid Verhasselt,a,# Christian V. Stevensa,b,* a

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. b

Cancer Research Institute Ghent, De Pintelaan 185, 9000 Gent, Belgium. #

In equal contribution

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, stability issues. A large toolbox of analogs 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 analog for a given application. As the unmet need for high-potency analogs remains, we also propose starting points for medicinal chemists in search of nanomolar myosin II inhibitors.

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Introduction 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).

Scheme 1. Topics covered in this perspective.

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

The affinity of (S)-blebbistatin (S)-1 is not limited to particular myosin II isoforms: it covers cardiac-, skeletal- and smooth-muscle and non-muscle 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


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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 light sensitive and phototoxic.4,6,15-20 Due to 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 in cell biology studies or a starting point for the development of targeted therapeutic tools. 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, as well as its mechanism of action and 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 analogs. 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.

Myosins Myosins are cellular proteins that act as motors which consume 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 muscle-based movement is the most striking one.21-23 The myosin superfamily is divided into different classes, based on 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 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 non-covalently 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 regulatory light chains are encoded by three distinct genes.21,27-32

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 crosslinking of actin filaments.

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 that 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


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C-terminal non-helical tailpiece. The tail domain determines the cellular localization and is responsible for filament assembly (Figure 2B). The anti-parallel 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, loops) (Figure 3): an N-terminal subdomain, an upper 50-kDa 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 ATP binding site is mediated via the 50-kDa cleft through switch II. A seven-stranded β-sheet core and associated loops form a transducer region.21,23,27,33,35,36,38-44

Figure 3. Simplified schematic diagram of the myosin II head domain, which ends in the lever arm (ATP and actin binding site marked with *). Adapted from Sweeney et al.23

The 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 starts with myosin tightly bound to actin (A·M). Next, a sequence of conformational changes is triggered in the myosin head domain by contact of the γ-phosphate group of an ATP molecule with 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 (M·ATP, step a).23,27,33,38,43-47

Scheme 2. Schematic representation of the ATPase cycle. Adapted from Winkelmann et al.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 through 60° and priming of the lever arm (recovery stroke, orange arrows, step b). ATP is then hydrolyzed into adenosine diphosphate (ADP) and Pi (step c). At this stage, the hydrolysis products remain bound to the myosin head, forming a myosin·ADP·Pi complex that has still a 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 (formation of A·M·ADP, step d). 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 (green upward arrow, step e). At the end of the power stroke ADP is released (step f), which allows the start of a new cycle.27,33,38,41,45,46,50 Myosin II isoforms and physiological function


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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 smooth-muscle myosins and nonmuscle myosins, (iii) the myosins 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 non-muscle 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 Smoothmuscle 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 paralogs: 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 diseases72 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.

(S)-Blebbistatin: chemical and biological characterization Discovery Blebbistatin was discovered by Cheung et al. during a high-throughput screening (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 dissolved dimethyl sulfoxide (DMSO) sample of compound 2, however, showed no influence on NM IIA ATPase activity. Curiously, over the next two days, the latter DMSO solution of 2 had converted from colorless to bright yellow on standing in air, with a concomitant gain in NM IIA ATPase inhibition. This suggested degradation of molecule 2 in the original library as well, due to repeated freeze-thaw cycles. Further investigation led to the discovery of (±)-blebbistatin (±)-1, 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

Scheme 3. Discovery of (±)-blebbistatin (±)-1 as an oxidation product of compound 2.74,75

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 and CD (see Scheme 1 for numbering) of the scaffold were connected via condensation of amine 5 with amide 4, upon amide activation with POCl3 (step i).


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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, PCl5) did not give superior outcomes. Intramolecular cyclization of amidine 6 using excess LiHMDS resulted in quinolinone 8 (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 and 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 (oxaziridine 9, step iii),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%.

Scheme 4. Lucas-Lopez synthesis.4,77 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 °C 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. a

Determination of enantiomeric excess via chiral-HPLC analysis. b Upon recrystallization from CH3CN, with

6 [α]26 = -464°. D

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 preparation in 78% overall yield (Scheme 5) presently is the most efficient synthesis of (S)-blebbistatin (S)-1.

Scheme 5. Verhasselt synthesis.79-81 Reagents and conditions: i. 1) 2 equiv POCl3, dry CH2Cl2, rt, 24 h; 2) 1.05 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. a Determination of enantiomeric excess via chiral-HPLC analysis. b Upon recrystallization from CH3CN.

A disadvantage of the syntheses of Lucas-Lopez and Verhasselt is that scaffold construction starts from a D-ring-containing building block. This approach does not allow for a divergent synthesis of analogs 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 (analogs) 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 two-step hydroxylation sequence (which contrasts the direct introduction in the previous routes). CuI-catalyzed N-arylation of (±)-17 with iodobenzene using diamine 18 as a ligand and direct desilylation


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with tetra-n-butylammonium fluoride (TBAF) yielded (±)-blebbistatin derivatives (±)-16,25–30 in low to moderately high yields (Table 1).

Lawson route

O NH2 O

N

MeO

O 5 i

N

N MeO

OMe 10 (88%)

O OMe

11 (91%)

N

N H

ii

Na N

O iii Cl

O

N

O

OTIPS N H

vi

(±)-17 (50%) (overall yield 13%, 6 steps)

O

OTIPS

N

v

N

(±)-16 (80%)

O

OH

N

iv

N

(±)-15 (52%) OMe

OMe

12 (89%)

N

O

N

Cl O 13

Cl

N

N

(±)-14 (91%) OMe

OMe

Scheme 6. Synthesis of racemic precursor (±)-17.82 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. c. 2.5 equiv LiHMDS, dry THF, −78 °C to 0 °C, 3 h. d. 0.5 equiv sodium dichloroisocyanurate (13), THF/H2O (1:1), rt, 4 h. e. 1.85 equiv NaOH, H2O/THF (9:4), rt, 18 h. f. 4 equiv DIPEA, 3 equiv TIPSOTf, dry CH2Cl2, reflux, 6 h. g. 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 deprotection.82,a,b



a

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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.

b

Combined yield of steps i and ii;

intermediates were not purified.

Due to 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 (analogs).

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 heavy-atom (bromine)-containing analogs in order to obtain better-quality crystals (Scheme 7).4 A 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 (Strategy A). The same problem was encountered on bromination of the intermediates en route to (−)-blebbistatin (−)-1. The synthesis of (−)-6-bromo-blebbistatin (−)-35 from brominated aniline 32 was also unsuccessful, due to the


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low

yield

of

amidine

33

(Strategy

B).

The

authors

successfully

prepared

(−)-O-(3-

bromobenzoyl)blebbistatin (−)-38 and (−)-O-(4-bromobenzoyl)blebbistatin (−)-39 through acylation of the chiral alcohol in (−)-blebbistatin (−)-1 with bromobenzoyl chlorides 36 and 37, respectively (Strategy C). Unfortunately, recrystallization did not yield sufficiently high-quality crystals for X-ray analysis.

Scheme 7. Failed attempts to determine the absolute stereochemical configuration of (S)-1.4 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. a Time and yield were not reported.

b

The enantiomeric excess was not reported, but [α]26 D was −568 and −607 after

recrystallization of (−)-38 and (−)-39, respectively, from EtOAc/hexane.

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 (step e, 99%, ee >99%). Lastly, chiral-HPLC, 1H NMR and



MS analysis proved that this sample of (S)-blebbistatin (S)-1 was identical to (−)-blebbistatin (−)-1, thereby finally confirming its absolute stereochemistry.

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 analysis.4 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 °C 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. a Determination of enantiomeric excess via chiral-HPLC analysis. b

c 83 Upon recrystallization from CH3CN, [α]26 D = −526. X-ray structure image was rendered in Mercury 3.9

from Cambridge Structural Database entry CCDC-238392,84 originally uploaded by Lucas-Lopez et al.4

Mechanism and structural basis of (non-muscle) 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 (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, Figure 4). This leads to a stabilized, long-lived (S)-blebbistatin·myosin·ADP·Pi complex, in


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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, step e) from occurring.16,85,86

Scheme 9. (Non-muscle) myosin II ATPase inhibition mechanism of (S)-blebbistatin (S)-1. 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 a weak actin affinity. This prevents Pi release and force generation (i.e. the power stroke) from occurring.

Figure 4. Co-crystal 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.



The co-crystal 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: 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 AB 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

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

Molecular targets of (S)-blebbistatin Multiple groups have examined the ATPase inhibitory potency of blebbistatin 1 (in racemic form or as (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


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micromolar range. An exception is Drosophila melanogaster NM II, which is uniquely insensitive 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 co-crystal 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 by residues bearing large, aromatic side chains (i.e. Tyr or Phe) that prevent (S)-blebbistatin (S)-1 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 phosphorylationdependent 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 Table 2. Half-maximum inhibitory concentration (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

Myosin type

IC50 b (µM)

Selected (S)-blebbistatin contact residuesa

Reference(s)

D. discoideum

II

4.9–13b

Gly240

Leu262

Ser456

Ile455

3,6,7,8,11,87

Acanthamoeba

II

83

Gly

Leu

Ser

Ile

3

H. sapiens

Non-muscle IIA

4–14b

Gly

Leu

Ala

Ile

2,3,4,89

H. sapiens

Non-muscle IIB

4.6b

Gly

Leu

Ala

Ile

89



Page 18 of 63

G. gallus

Non-muscle IIB

1.8

Gly

Leu

Ala

Ile

3

M. musculus

Non-muscle IIC

3.2b

Glu

Leu

Ala

Ile

89

G. gallus

Smooth-muscle IIc

5.5–23.5

Gly

Leu

Ala

Ile

10,89

A. irradians

Striated-muscle II

2.3

Gly

Leu

Ala

Ile

51

O. cuniculus

Skeletal-muscle II

0.22–4.32b

Gly

Leu

Ala

Ile

3,5,6,7,8,79-81

S. scrofa

β-Cardiac-muscle II

1.2

Gly

Leu

Ala

Ile

3

D. melanogaster

Non-muscle II

>200d

Gly

Leu

Ala

Met

88

R. norvegigus

Ib

>150d

Gly

Leu

Tyr

Ile

3

Acanthamoeba

Ic

>150d

Gly

Leu

Tyr

Ile

3

M. musculus

Va

>150d

Gly

Leu

Tyr

Ile

3,89

B. taurus

X

>150d

Gly

Leu

Phe

Ile

3

H. sapiens

XV

>100d

Gly

Leu

Tyr

Ile

3

a

Sequence alignments were performed on the UniProt website (accession numbers are P08799, P05659, P35579, P35580,

Q789A4, Q6URW6, P10587, P24733, Q28641, P79293, Q99323, Q05096, P10569, Q99104, P79114 and Q9UKN7, respectively).90 b 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. c The IC50 value for M. gallopavo3 and B. taurus9 smooth-muscle myosin II ATPase inhibition are 80 µM and 4.3–11 µM, respectively; these data are not included in the table, since no sequence information is available for these myosins.d No inhibition was observed at the highest concentration evaluated.

Strengths, deficiencies and improved analogs 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 cell membrane 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


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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 diseases72 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 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 analogs 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 which prevent its wide-spread use as a pharmacological tool in in vivo model systems. A considerable body of research has targeted the development of (S)-blebbistatin analogs 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 analogs The number of A-ring modified analogs thus far prepared is limited to nine compounds. A series of analogs with simple modifications was prepared by Lucas-Lopez et al. The position of the methyl position was varied in derivatives (S)-43–46,93 while the influence of a nitro group was evaluated in (S)-7-nitro6-norblebbistatin (S)-47 (Table 3).4,77 These molecules were prepared using the higher described LucasLopez 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



Page 20 of 63

enolate oxygen (7Li NMR observations). Also ring closure en route to (S)-45 proceeded sluggishly, due a steric clash between the methyl substituent and the nitrogen-coordinated lithium cation.

Figure 6. Co-crystal structure of (S)-blebbistatin (S)-1 bound to Dictyostelium discoideum myosin II (PDB: 1YV3)86 suggests additional π-π stacking interactions with Tyr261 through extension of the aromatic system at ring A. Legend: carbon, amino acids: grey; carbon, (S)-blebbistatin (S)-1: orange; oxygen: red; nitrogen: blue.

Table 3. A-ring modification of (S)-blebbistatin (S)-1: synthesis and ATPase inhibitory activity.a

Product

R

and

Yieldb

Eec

ATPase inhibitory activity

(%)

(%)

D. discoideum

Human

Rabbit skeletal muscle

myosin II

NM IIA

myosin II

% inhibition

IC50

% inhibition at

IC50

at 50 µM

(µM)

50 µM

5 µM

(µM)

reference

(S)-1 4,77,81,94

6-Me

30-78

86/>99

92

7.1 ± 0.4

97 ± 2

93 ± 1

1.02 ± 0.05

(S)-43 93

5-Me

20

64/99

88

N.d.

95 ± 1

86 ± 1

N.d.

(S)-44 93

7-Me

19

90/>99

90

N.d.

94 ± 1

86 ± 1

N.d.

(S)-45 93

8-Me

31

86/>99

35

N.d.

72 ± 2

31 ± 9

N.d.

(S)-46 93

H

19

86/>99

88

N.d.

95 ± 1

84 ± 1

N.d.

(S)-47 4,77

7-NO2

3

76

N.d.

28 ± 3

N.d.

N.d.

N.d.

(S)-48 81,94

49

72/>99

N.d.

N.d.

N.d.

N.d.

7.97 ± 0.02

(S)-49 81,94

15

92/96

N.d.

N.d.

N.d.

N.d.

8.46 ± 1.22


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(S)-50 81,94

95

98

N.d.

N.d.

N.d.

N.d.

14.5 ± 2.2

(S)-51 81,94

96

98 d

N.d.

N.d.

N.d.

N.d.

>20 e

N.d.: not determined. 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. b For compounds (S)-1 and (S)-43-49: overall yield from relevant N-aryl pyrrolidinone and 2-aminobenzoate; for compounds (S)-50-51: yield of steps i and ii, respectively. c Determination of ee via chiral-HPLC analysis; higher values are upon recrystallization from CH3CN. d

Based on the result for (S)-50. e Concentrations exceeding 20 µM resulted in compound precipitation in the assay

buffer.

A series of analogs 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, Leu641 of the myosin II binding pocket (Figure 6), extension of the ring system at positions C6 and C7 was explored. (S)-Benzo[h]blebbistatin (S)-48 (Table 3) and (S)-(1H)pyrrolo[3,2-h]blebbistatin (S)-51, the corresponding indoline (S)-50 and 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 chiralHPLC analysis. Table 3 provides a comparison between the ATPase IC50 values of the A-ring-modified analogs 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 to steric hindrance between the 8-methyl substituent and residue Tyr634 in a co-crystallization 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 was moreover noted above 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 are synthetically best accessible on C6 and C7. No beneficial binding interactions were noted on linear



Page 22 of 63

extension of the tricyclic core scaffold. Overall, the works of Lucas-Lopez4,77 and Verhasselt781,94 indicate that there is little potential for ATPase potency enhancement via decoration of the A-ring of the (S)blebbistatin scaffold. D-ring-modified analogs Modification of the D-ring of (S)-blebbistatin (S)-1 (numbering see Scheme 1) has mainly focused on the 3’- and 4’-positions, as the co-crystal 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 analogs. 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, while larger amounts were prepared from 1-(4iodophenyl)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 analogs 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 higher (Scheme 8), Lucas-Lopez et al. synthesized enantiopure (S)-4’-bromoblebbistatin (S)-31.4


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Scheme 10. Preparation of 4’-substituted (S)-blebbistatin analogs. 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 a Yield was not reported. b Ee was not reported. c Reagent stoichiometry, yield and ee were not reported. d Overall yield from relevant N-aryl pyrrolidinone and 2-aminobenzoate via the Lucas-Lopez or Verhasselt sequence. e The reaction mixture initially consisted of 50 mol% of (S)-54 and 50 mol% of (S)-55. f Ee derived from ee of (S)-58. g The

reaction mixture initially consisted of 94 mol% of (S)-57 and 6 mol% of dibenzylated product.

Substitution at the 3’-position was explored by Verhasselt et al. in a series of analogs 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 LucasLopez 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 analogs (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.

Scheme 11. Synthesis of 3’-modified (S)-blebbistatin analogs.79-81 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. 2x4 equiv Et2NOH, dry CH2Cl2, reflux, 2x 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. a Overall yield from relevant N-aryl pyrrolidinone and 2-aminobenzoate via the Lucas-Lopez or Verhasselt sequence.

b

Determination of ee via chiral HPLC analysis of corresponding

methyl ester.

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 analog in (S)-2,3-dihydro-1H-pyrrolo[2,3-c’]blebbistatin


Page 24 of 63

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(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)-74,75 started from pyrrolidinone 71 and followed the same route. Amidine synthesis proved difficult due to 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 (step ii, 73%, ee >99%) and indole (S)-75 were subsequently obtained in high ee (step iii, 70%, ee >99%).

Scheme 12. Synthesis of (S)-2,3-dihydro-1H-pyrrolo[2,3-c’]blebbistatin (S)-74 and (S)-1H-pyrrolo[2,3c’]blebbistatin (S)-75.80,81 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. a Overall yield from 71. b After recrystallization from CH3CN.

As described earlier, Lawson et al. also synthesized a range of D-ring-modified blebbistatin analogs (±)15 and (±)-25–30 via their newly developed route (Scheme 6, Table 1, Table 4).82 An overview of all hitherto reported analogs is presented in Table 4.

Table 4. D-ring modification of (S)-blebbistatin (S)-1: known analogs and ATPase inhibitory activity.



Product

(S)-1

R

H

Eea

ATPase inhibitory activity: IC50 c

(%)

D. discoideum

Rabbit skeletal-muscle

myosin II (µM)

myosin II (µM)

2.5 – 6.5

0.11 – 2.16

>99

Page 26 of 63

Reference

3,5,6,7,8,11, 79-81,87

(±)-25

4’-Me

Rac.

N.d.

N.d.

82

(S)-26

4’-Cl

N.d.b

1.9 ± 0.1

N.d.

6,7

(S)-31

4’-Br

>99

N.d.

N.d.

4

(S)-52

4’-I

N.d.

N.d.

N.d.

7,11

(S)-53

4’-N3

N.d.

5.2 ± 0.3

N.d.

7,11

(S)-54

4’-NO2

>99

2.3 ± 0.1

0.40 – 0.40

6,7,80,81

(S)-57

4’-OH

>99

N.d.

5.47 ± 1.32

79-81

(S)-55

4’-NH2

N.d.

6.6 ± 2

1.0 – 5.4

8,80,81

(±)-15

4’-OMe

Rac.

N.d.

N.d.

82

(S)-56

4’-OAllyl

>99

N.d.

0.380 ± 0.003

80,81

(S)-58

4’-OBn

>99

N.d.

>40d

80,81

(±)-29

4’-Ph

Rac.

N.d.

N.d.

82

(±)-27

3’-CF3

Rac.

N.d.

N.d.

82

(S)-62

3’-OH

99

N.d.

19.3 ± 0.5

79-81

(S)-63

3’-NH2

>99

N.d.

14.1 ± 0.1

79-81

(S)-61

3’-CN

83

N.d.

48.5 ± 0.1

79-81

(S)-65

3’-COOH

96

N.d.

>100e

79-81


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(S)-64

3’-CONH2

98

N.d.

>100e

79-81

(S)-59

3’-OAllyl

>99

N.d.

9.41 ± 1.83

80,81

(S)-66

3’-OAcryloyl

>99

N.d.

57.6 ± 7.8

80,81

(S)-67

3’-OPropanoyl

99

N.d.

23.5 ± 2.5

80,81

(S)-60

3’-N(Allyl)2

>99

N.d.

>10.8f

80,81

(S)-68

3’-NHAcryloyl

99

N.d.

>100g

80,81

(S)-69

3’-NHPropanoyl

>99

N.d.

>100g

80,81

(S)-70

>99

N.d.

>40d

80,81

(S)-74

>99

N.d.

7.70 ± 0.19

80,81

(S)-75

>99

N.d.

7.20 ± 0.59

80,81

(±)-28

Rac.

N.d.

N.d.

82

(±)-30

Rac.

N.d.

N.d.

82

N.d.: Not determined. a Ee of the final product used for biological testing; Rac.: Racemic mixture. b Also prepared in racemic form by Lawson et al.82 c Average±SD for single literature source, range of averages for multiple sources. d

Highest compound concentration used was 40 µM. e Highest compound concentrations used was 100 µM. f Highest

compound concentration used was 10.8 µM, as concentrations higher than 10.8 µM resulted in compound precipitation in the assay buffer. g Highest compound concentration used was 100 µM.

A comprehensive overview of available myosin II ATPase inhibitory activity data of D-ring-modified blebbistatin analogs is presented in Table 4. Not all known analogs 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, while some alterations lead to a complete loss of activity. 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 towards a far larger influence of sterics than electronics.80 Small groups are equally well tolerated in the 4’-position as in the 3’-position, while 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. Other analogs (±)-6-Bromo-4’-ethoxy-blebbistatin (±)-76 was discovered by Cheung et al. in the same high-throughput screen as (±)-blebbistatin (±)-1 (Figure 7). It is also an oxidation product of a 4-aminoquinoline, i.e. molecule 77.1,2,74

Figure 7. 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 while (S)-


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blebbistatin (S)-1 concentrations can indeed initially reach 50 µM, the compound will thereupon precipitate towards equilibrium concentrations over a short time frame (±90 min).7,8 Besides causing fluorescent precipitates, the application of (S)-blebbistatin (S)-1 at concentrations exceeding its steady-state 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. Besides, blebbistatin precipitates redissolve only slowly and have the tendency to attach to plastic or glass surfaces. This affects wash-out in commonly used experimental set-ups 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 analogs with a higher aqueous solubility while retaining good cell membrane permeability. A first attempt was undertaken by LucasLopez et al. by introducing an electron-withdrawing nitro group on the scaffold in (S)-7-nitro6-norblebbistatin (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 to (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 to (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 analog 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 30fold higher water solubility than (S)-blebbistatin.79,81 The latter two molecules were further shown not to



Page 30 of 63

interfere in (fluorescence) readouts in cell-based systems. (S)-4’-Hydroxyblebbistatin (S)-57 was significantly less soluble than the aforementioned polar analogs.80,81 Verhasselt et al. performed a comprehensive follow-up study on the solubility and membrane permeability (Caco-2, A-B/B-A) of polar and apolar (S)-blebbistatin analogs (Table 5).80,81 Compounds (S)-55, (S)-62 and (S)-63 were shown to combine excellent aqueous solubility and a high cell membrane permeability. Remarkably, all evaluated (S)-blebbistatin analogs 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 read-outs 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 best accessible. Table 5. Potency and physicochemical properties of selected (S)-blebbistatin analogs.79-81

Compound

Relative

Solubilityb

Caco-2 permeability assayc

potency to

(µM)

Papp (10-6 cm s-1)

Recovery (%)

t1/2

A-B

B-A

A-B

B-A

(min)

60.5 ± 1.8

19.2 ± 0.3

66 ± 1

66 ± 4

14

(S)-1a (S)-1

1.0 ± 0.2

6.18 ± 0.08

Photostability

(S)-54

2.5 ± 0.2

31.7 ± 1.6d

32.5 ± 1.0

2.23 ± 0.02

34 ± 1

72 ± 1

>90e

(S)-55

0.19 ± 0.01

>200f

69.4 ± 3.0

31.1 ± 3.5

85 ± 1

82 ± 1

44

(S)-56

2.3 ± 0.3

2.75 ± 0.05

29.7 ± 0.6

7.58 ± 0.05

32 ± 1

65 ± 1

8.8

(S)-57

0.16 ± 0.04

82.4 ± 9.7

N.d.

N.d.

N.d.

N.d.

4.7

(S)-59

0.23 ± 0.05

2.83 ± 0.09

N.d.

N.d.

N.d.

N.d.

15

(S)-62

0.11 ± 0.01

193 ± 1

28.5 ± 0.3

17.2 ± 0.1

49 ± 1

70 ± 1

11

(S)-63

0.15 ± 0.01

186 ± 9

62.7 ± 3.1

26.2 ± 0.1

79 ± 1

74 ± 4

13


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N.d.: not determined. a Based on IC50 values of ATPase inhibition in rabbit skeletal muscle myosin II. b Steady-state solubility in PBS pH 7.4 buffer (2% (v/v) DMSO). c Caco-2 A-B (pH 7.4/7.4) and B-A (pH 7.4/7.4) permeability at 20 µM. d The saturation concentration for this compound was also determined by Várkuti et al., yet under different conditions: 3.3 ± 0.1 µM at 0.1 vol/vol% DMSO and 3.6 ± 0.2 µM at 1 vol/vol% DMSO.8 e Sample collected after 90 min indicated 33% degradation.

f

Highest 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 vol/vol% DMSO and 426 ± 1.7 µM at 1 vol/vol% DMSO.8

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 of (S)-blebbistatin (S)-1 induces 65–100% cell death in various human cell lines (entries 1,3,5,7,9) 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 of (S)-blebbistatin (S)-1 for 24 h was 20–45%, while no cytotoxic effects on PANC1 cells were observed at this concentration (entry 16).66 Finally, zebrafish embryos treated with 10 µM of (S)-blebbistatin (S)-1 all died after 36 h (entry 17).6 In sum, the sensitivity to cytotoxic effects caused by (S)-blebbistatin (S)-1 strongly depends on the species under investigation and the incubation time. Table 6. Cytotoxicity of (S)-blebbistatin (S)-1 depends on cell type, inhibitor concentration and incubation time.

Entry

Cell type

Concentration (µM)

Incubation time

Cytotoxicity (%)

Reference

1

U87

200

24 h

65

15



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2

U87

200

3h

40

15

3

Du145

200

24 h

90

15

4

Du145

200

3h

30

15

5

LNCaP

200

24 h

96

15

6

LNCaP

200

3h

10

15

7

F11-hTERT

200

24 h

85

15

8

F11-hTERT

200

3h

30

15

9

FEMX-1

200

24 h

100

15

10

FEMX-1

200

3h

30

15

11

HeLa

20

3 days

90

6

12

D. discoideum

20

3 days

0

6

13

MIAPaCa2

400

24 h

20

66

14

BxPC3

400

24 h

25

66

15

Capan2

400

24 h

45

66

16

PANC1

400

24 h

0

66

17

Zebrafish embryo

10

36 h

100

6

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 analogs

with

a

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, treatment-associated mortality on (S)-blebbistatin (S)-1 administration (10 µM) was 100 % after 36 h-72 h, while that of (S)-4’-nitroblebbistatin-treated animals (10 µM) was at 25% and that of (S)-4’-aminoblebbistatin (20 µM) was at the level of nontreated controls (0%). Importantly, both analogs mediate the same nonmuscle myosin II-specific effects as (S)-blebbistatin: the compounds induce multinuclearity 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)-


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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 Table 7. Light sensitivity of (S)-blebbistatin (S)-1 at various wavelengths.

Entry

Wavelength (nm)

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

Reference

1

295

Loss of inhibitory action on ATPase activity

16

2

351

Loss of inhibitory action on actin filament movement

19

3

365

Change in absorption spectrum

18

4

390–470

Change in absorption and emission spectrum and area% (HPLC)

15,80,81

5

425

Loss of inhibitory action on Pi release

16

6

436 + 510


4

7

450–490

Change in absorption and emission spectrum

18,20

8

458

Loss of inhibitory action on actin filament movement

19

9

480


6,8

10

488

Loss of inhibitory action on actin filament movement and

17,19

cytokinetic-ring contraction 11

543

None

19

Several groups have investigated these phenomena in more detail, mostly relying on qualitative changes in the absorption and emission spectrum 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 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 analogs bearing electron withdrawing groups do not decompose under identical conditions (see below) corroborate 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, 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 non-toxic.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 non-phototoxic analogs, 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-nm and 510-nm-filtered light.4,77 Unfortunately, no phototoxicity data were reported. Képiró and Várkuti et al. prepared (S)-4’-chloroblebbistatin (S)-26, (S)-4’-nitroblebbistatin (S)-54 and (S)-4’-aminoblebbistatin (S)-55 as stable analogs under 480-nm irradiation. The phototoxicity of these compounds was evaluated against HeLa cell morphology. Remarkably, (S)-4’-nitroblebbistatin (S)-54 or (S)-4’-aminoblebbistatin (S)-55 proved well-tolerated, while 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.


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Quantitative stability data under blue-light irradiation were generated by Verhasselt et al. for (S)blebbistatin and seven analogs (Table 5), under conditions relevant to common experimental set-ups (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 based on earlier reported qualitative data,8 was a factor two inferior to that of (S)-54. All other analogs performed similar or inferior (for (S)-4’-hydroxyblebbistatin (S)-57) to the parent compound (S)-blebbistatin (S)-1. Important other observations were a significant dependence of half-life on 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 analogs under blue light-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 the latter molecule in the absence of UV irradiation. On irradiation at 310 nm (a wavelength that is non-toxic to cells and tissues) it covalently crosslinks 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 value of more than 50 µM) targets of (S)-4’-azidoblebbistatin (S)-53 (and likely (S)-blebbistatin (S)-1).

Conclusion and perspectives for further research (S)-blebbistatin (S)-1 has been 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 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 analogs. Use of these novel derivatives has unfortunately been limited. As with all improved analogs of widely-used inhibitors, underlying reasons may be limited awareness by biologists and biochemists of the existence of these tools, neophobia or perceived unavailability. The confirmation of these analogs 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 8 presents an overview of the most performant (S)-blebbistatin derivatives for common applications.


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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 of the interaction of (S)-blebbistatin with various myosin motors during the entire ATPase cycle (Figure 8). From these insights, rational medicinal chemistry programs towards potent and selective analogs may be launched. Alternatively, high-throughput screens against different myosin isoforms can deliver hits (novel chemotypes) for chemical optimization efforts and, more importantly, novel drugable binding pockets.

Figure 8. (S)-Blebbistatin strengths and deficiencies, recommendations for the optimal use of the available improved analogs as tool compounds and suggestions for further research on myosin II inhibitors.



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 resume of the medicinal chemistry of (S)-blebbistatin, and that it will stimulate the search for yet superior and more selective inhibitors of myosin II isoforms.

Corresponding Author Information B.I.R.: [email protected], [email protected]; C.V.S.: [email protected]

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

Acknowledgements 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.

Abbreviations CCDC, Cambridge Crystallographic Data Centre; DMEM, Dulbecco’s Modified Eagle’s Medium; NM II , non-muscle myosin II; PMP, para-methoxyphenyl

Keywords


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(S)-Blebbistatin, myosin II inhibitor, synthesis, structure-activity relationship, structure-property relationship, physicochemical properties

Biographies Bart I. Roman (Kortrijk, 1984) is 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 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 the 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 post-doctoral 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 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, Fellow of the Royal Society of Chemistry and member of the American Chemical Society.

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Straight, A. F.; Cheung, A.; Limouze, J.; Chen, I.; Westwood, N. J.; Sellers, J. R.; Mitchison, T. J. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 2003, 299, 1743-1747.

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Limouze, J.; Straight, A. F.; Mitchison, T.; Sellers, J. R. Specificity of blebbistatin, an inhibitor of myosin II. J. Muscle Res. Cell Motil. 2004, 25, 337-341.

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Lucas-Lopez, C.; Patterson, S.; Blum, T.; Straight, A. F.; Toth, J.; Slawin, A. M. Z.; Mitchison, T. J.; Sellers, J. R.; Westwood, N. J. Absolute stereochemical assignment and fluorescence tuning of the small molecule tool, (–)-blebbistatin. Eur. J. Org. Chem. 2005, 1736-1740.

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Képiró. M. Azidation Technology: From Photoaffinity Labeling to Molecular Tattooing; Ph.D. Dissertation, Eötvös Loránd University, Budapest, Hungary, 2014.

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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.


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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. USA 2012, 109, 94029407. 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. Pflugers Arch. Eur. J. Physiol. 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, 455A455A. 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 2000, 1496, 3-22. 22. Mooseker, M. S.; Foth, B. J. The Structural and Functional Diversity of the Myosin Family of ActinBased Molecular Motors. In Myosins: a Superfamily of Molecular Motors, Coluccio, L. M., Ed.; Springer: 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. 24. Richards, T. A.; Cavalier-Smith, T. Myosin domain evolution and the primary divergence of eukaryotes. Nature 2005, 436, 1113-1118. 25. Foth, B. J.; Goedecke, M. C.; Soldati, T. New insights into myosin evolution and classification. Proc. Natl Acad. Sci. USA 2006, 103, 3681-3686. 26. Odronitz, F.; Kollmar, M. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species. Genome Biol. 2007, 8: R196. 27. Preller, M.; Manstein, D. J. Myosin structure, allostery, and mechano-chemistry. Structure 2013, 21, 1911-1922.


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28. Conti, M. A.; Kawamoto, S.; Adelstein, R. S. Non-Muscle Myosin II. In Myosins: a Superfamily of Molecular Motors, Coluccio, L. M., Ed.; Springer: The Netherlands, 2008; pp. 223-264. 29. Vicente-Manzanares, M.; Ma, X.; Adelstein, R. S.; Horwitz, A. R. Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat. Rev. Mol. Cell Biol. 2009, 10, 778-790. 30. Heissler, S. M.; Manstein, D. J. Nonmuscle myosin-2: mix and match. Cell. Mol. Life Sci. 2013, 70, 1-21. 31. Billington, N.; Wang, A.; Mao, J.; Adelstein, R. S.; Sellers. J. R. Characterization of three full-length human nonmuscle myosin II paralogs. J. Biol. Chem. 2013, 288, 33398-33410. 32. Betapudi. V. Life without double-headed non-muscle myosin II motor proteins. Frontiers in Chemistry 2014, 2: 45. 33. Holmes, K. C. Myosin Structure. In Myosins: a Superfamily of Molecular Motors, Coluccio, L. M., Ed.; Springer: The Netherlands, 2008; pp. 35-54. 34. Sandquist, J. C.; Means, A. R. The C-terminal tail region of nonmuscle myosin II directs isoformspecific distribution in migrating cells. Mol. Biol. Cell 2008, 19, 5156-5167. 35. Houdusse, A.; Kalabokis, V. N.; Himmel, D.; Szent-Gyorgyi, A. G.; Cohen, C. Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head. Cell 1999, 97, 459-470. 36. Reggiani, C.; Bottinelli, R. Myosin II: Sarcomeric Myosins, the Motors of Contraction in Cardiac and Skeletal Muscles. In Myosins: a Superfamily of Molecular Motors, Coluccio, L. M., Ed.; Springer: The Netherlands, 2008; pp. 125-169. 37. Aguilar-Cuenca, R.; Juanes-García, A.; Vicente-Manzanares, M. Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer. Cell. Mol. Life Sci. 2014, 71, 479-492. 38. Geeves, M. A.; Holmes, K. C. Structural mechanism of muscle contraction. Annu. Rev. Biochem. 1999, 68, 687-728.



39. Dominguez, R.; Freyzon, Y.; Trybus, K. M.; Cohen, C. Crystal Structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain. Cell 1998, 94, 559-571. 40. Burgess, S.; Walker, M.; Wang, F.; Sellers, J. R.; White, H. D.; Knight, P. J.; Trinick, J. The prepower stroke conformation of myosin V. J. Cell Biol. 2002, 159, 983-991. 41. Geeves, M. A. Review: The ATPase mechanism of myosin and actomyosin. Biopolymers 2016, 105, 483-491. 42. Rayment, I.; Rypniewski, W. R.; Schmidt-Base, K.; Smith, R.; Tomchick, D. R.; Benning, M. M.; Winkelmann, D. A.; Wesenberg, G.; Holden, H. M. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 1993, 261, 50-58. 43. von der Ecken, J.; Heissler, S. M.; Pathan-Chhatbar, S.; Manstein, D. J.; Raunser, S. Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution. Nature 2016, 534, 724-728. 44. Banerjee, C.; Hu, Z.; Huang, Z.; Warrington, J. A.; Taylor, D. W.; Trybus, K. M.; Lowey, S.; Taylor, K. A. The structure of the actin-smooth muscle myosin motor domain complex in the rigor state. J. Struct. Biol. 2017, 200, 325-333. 45. Takács, B.; Billington, N.; Gyimesi, M.; Kintses, B.; Málnási-Csizmadia, A.; Knight, P. J.; Kovács, M. Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke. Proc. Natl Acad. Sci. USA 2010, 107, 6799-6804. 46. Málnási-Csizmadia, A.; Pearson, D. S.; Kovács, M.; Woolley, R. J.; Geeves, M. A.; Bagshaw, C. R. Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue. Biochemistry 2001, 40, 12727-12737. 47. Winkelmann D. A.; Forgacs, E.; Miller, M. T.; Stock A. M. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity. Nat. Commun. 2015, 6, 7974.


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48. Kiani, F. A.; Fischer, S. Catalytic strategy used by the myosin motor to hydrolyze ATP. Proc. Natl Acad. Sci. USA 2014, 111, E2947-E2956. 49. Kiani, F. A.; Fischer, S. SPLINTS: small-molecule protein ligand interface stabilizers. Curr. Opin. Struct. Biol. 2015, 31, 115-122. 50. Málnási-Csizmadia, A.; Dickens, J. L.; Zeng, W.; Bagshaw, C. R. Switch movements and the myosin crossbridge stroke. J. Muscle Res. Cell. Motil. 2005, 26, 31-37. 51. Cremo, C. R.; Hartshorne, D. J. Smooth-Muscle Myosin II. In Myosins: a Superfamily of Molecular Motors, Coluccio, L. M., Ed.; Springer: The Netherlands, 2008; pp. 171-222. 52. Sjuve, R.; Haase, H.; Ekblad, E.; Malmqvist, U.; Morano, I.; Arner, A. Increased expression of nonmuscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder. Cell Tissue Res. 2001, 304, 271-278. 53. Yuen, S.L.; Ogut, O.; Brozovich, F.V. Nonmuscle myosin is regulated during smooth muscle contraction. Am. J. Physiol. Heart Circ. Physiol. 2009, 297, H191-H199. 54. Conti, M. A.; Adelstein, R. S. Nonmuscle myosin II moves in new directions. J. Cell Sci. 2008, 121, 11-18. 55. Ma, X.; Adelstein, R.S. The role of vertebrate nonmuscle Myosin II in development and human disease. Bioarchitecture 2014, 4, 88-102. 56. Arii, J.; Goto, H.; Suenaga, T.; Oyama, M.; Kozuka-Hata, H.; Imai, T.; Minowa, A.; Akashi, H.; Arase, H.; Kawaoka, Y.; Kawaguchi, Y. Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1. Nature 2010, 467, 859-862. 57. Antoine, T.; Shukla, D. Inhibition of myosin light chain kinase can be targeted for the development of new therapies against herpes simplex virus type-1 infection. Antivir. Ther. 2014, 19, 15-29.



58. Kadiu, I.; Gendelman, H.E. Human immunodeficiency virus type 1 endocytic trafficking through macrophage bridging conduits facilitates spread of infection. J. Neuroimmune Pharmacol. 2011, 6, 658-675. 59. Sun, Y.; Qi, Y.; Liu, C.; Gao, W.; Chen, P.; Fu, L.; Peng, B.; Wang, H.; Jing, Z.; Zhong, G.; Li, W. Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome Virus. J. Virol. 2014, 88, 237-248. 60. Kumakura, M.; Kawaguchi, A.; Nagata, K. Actin-myosin network is required for proper assembly of influenza virus particles. Virology 2015, 476, 141-150. 61. Liu, Z.; van Grunsven, L. A.; Van Rossen, E.; Schroyen, B.; Timmermans, J.-P.; Geerts, A.; Reynaert, H. Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells. Br. J. Pharmacol. 2010, 159, 304-315. 62. Atluri, K.; De Jesus, A. M.; Chinnathambi, S.; Brouillette, M. J.; Martin, J. A.; Salem, A. K.; Sander, E. A. Blebbistatin-loaded poly(D,L-lactide-co-glycolide) particles for treating arthrofibrosis. ACS Biomater. Sci. Eng. 2016, 2, 1097–1107. 63. Zhang, M.; Rao, P. V. Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes. Invest. Ophthalmol. Vis. Sci. 2005, 46, 4130-4138. 64. Young, E. J.; Blouin, A. M.; Briggs, S. B.; Sillivan, S. E.; Lin, L.; Cameron, M. D.; Rumbaugh, G.; Miller, C. A. Nonmuscle myosin IIB as a therapeutic target for the prevention of relapse to methamphetamine use. Mol. Psychiatry 2016, 21, 615-623. 65. Young, E. J.; Briggs, S. B.; Rumbaugh, G.; Miller, C. A. Nonmuscle myosin II inhibition disrupts methamphetamine-associated memory in females and adolescents. Neurobiol. Learn. Mem. 2017, 139, 109-116. 66. Duxbury, M. S.; Ashley, S. W.; Whang, E. E. Inhibition of pancreatic adenocarcinoma cellular invasiveness by blebbistatin: a novel myosin II inhibitor. Biochem. Biophys. Res. Commun. 2004, 313, 992-997.


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67. Derycke, L.; Stove, C.; Vercoutter-Edouart, A.-S.; De Wever, O.; Dollé, L.; Colpaert, N.; Depypere, H.; Michalski, J.-C.; Bracke, M. The role of non-muscle myosin IIA in aggregation and invasion of human MCF-7 breast cancer cells. Int. J. Dev. Biol. 2011, 55, 835-840. 68. Poincloux, R.; Collin, O.; Lizárraga, F.; Romao, M.; Debray, M.; Piel, M.; Chavrier, P. Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel. Proc. Natl Acad. Sci. USA 2011, 108, 1943-1948. 69. Beadle, C.; Assanah, M. C.; Monzo, P.; Vallee, R.; Rosenfeld, S. S.; Canoll, P. The role of myosin II in glioma invasion of the brain. Mol. Biol. Cell 2008, 19, 3357-3368. 70. Ivkovic, S.; Beadle, C.; Noticewala, S.; Massey, S. C.; Swanson, K. R.; Toro, L. N.; Bresnick, A. R.; Canoll, P.; Rosenfeld, S. S. Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple mitogens. Mol. Biol. Cell 2012, 23, 533-542. 71. Wigton, E. J.; Thompson, S. B.; Long, R. A.; Jacobelli, J. Myosin-IIA regulates leukemia engraftment and brain infiltration in a mouse model of acute lymphoblastic leukemia. J. Leukoc. Biol. 2016, 100, 143-153. 72. Brozovich, F. V.; Nicholson, C. J.; Degen, C. V.; Gao, Y. Z.; Aggarwal, M.; Morgan, K. G. Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders. Pharmacol. Rev. 2016, 68, 476-532. 73. Sirigu, S.; Hartman, J. J.; Planelles-Herrero, V. J.; Ropars, V.; Clancy, S.; Wang, X.; Chuang, G.; Qian, X.; Lu, P.-P.; Barrett, E.; Rudolph, K.; Royer, C.; Morgan, B. P.; Stura, E. A.; Malik, F. I.; Houdusse, A. M. Highly selective inhibition of myosin motors provides the basis of potential therapeutic application. Proc. Nat. Acad. Sci. U. S. A. 2016, 113, E7448-E7455. 74. Cheung, A.; Dantzig, J. A.; Hollingworth, S.; Baylor, S. M.; Goldman, Y. E.; Mitchison, T. J.; Straight, A. F. A small-molecule inhibitor of skeletal muscle myosin II. Nat. Cell Biol. 2002, 4, 8388. 75. Mitchison, T. J. Probing cell division with "chemical genetics". Harvey Lect. 2002, 98, 19-40.



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76. Westwood, N. J. Chemical genetics: how does it function? Philos. Trans. A Math. Phys. Eng. Sci. 2004, 362, 2761-2774. 77. Patterson, S.; Lucas-Lopez, C.; Westwood, N. J. Selective Chemical Intervention in Biological Systems: The Small Molecule Tool, (S)-(-)-Blebbistatin. In Proceedings of the International Beilstein workshop: The Chemical Theatre of Biological Systems; May 24-28, 2004, Bozen, Italy; pp. 147-166. 78. Davis, F. A.; Weismiller, M. C.; Murphy, C. K.; Reddy, R. T.; Chen, B.-C. Chemistry of oxaziridines.

18.

Synthesis

and

enantioselective

oxidations

of

the

[(8,8-

dihalocamphoryl)sulfonyl]oxaziridines. J. Org. Chem. 1992, 57, 7274-7285. 79. Verhasselt, S.; Roman, B. I.; De Wever, O.; Van Hecke, K.; Van Deun, R.; Bracke, M. E.; Stevens, C. V. Discovery of (S)-3’-hydroxyblebbistatin and (S)-3’-aminoblebbistatin: polar myosin II inhibitors with superior research tool properties. Org. Biomol. Chem. 2017, 15, 2104-2118. 80. Verhasselt, S.; Roman, B. I.; Bracke, M. E.; Stevens, C. V. Improved synthesis and comparative analysis of the tool properties of new and existing D-ring modified (S)-blebbistatin analogs. Eur. J. Med. Chem., 2017, 136, 85-103 81. Verhasselt, S. Development of Novel Blebbistatin Derivatives in the Quest for Improved Non-Muscle Myosin II Inhibitors; Ph.D. Dissertation, Ghent University, Ghent, Belgium, 2017. 82. Lawson, C. P. A. T.; Slawin, A. M. Z.; Westwood, N. J. Application of the copper catalysed Narylation of amidines in the synthesis of analogues of the chemical tool, blebbistatin. Chem. Commun. 2011, 47, 1057-1059. 83. Mercury, version 3.9 (Build RC1), Cambridge Crystallographic Data Centre: Cambridge, UK, 2016. 84. Lucas-Lopez, C.; Patterson, S.; Blum, T.; Straight, A. F.; Toth, J.; Slawin, A. M. Z.; Mitchison, T. J.; Sellers, J. R.; Westwood, N. J. CCDC 238392: Experimental Crystal Structure Determination, 2014, DOI: 10.5517/cc8022d (accessed Marc 17, 2017). 85. Kovács, M.; Tóth, J.; Hetényi, C.; Málnási-Csizmadia, A.; Sellers, J. R. Mechanism of blebbistatin inhibition of myosin II. J. Biol. Chem. 2004, 279, 35557-35563.


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Table of Contents Graphic


Page 52 of 63

850

120 myosin II

750

100

blebbistatin

80

650

60 550

40

450 350 1995

20

2000

2005 Year


2010

0 2015

Number of publications on blebbistatin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Number of publications on myosin II


Page 53 of 63

A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

head/motor domain

essential light chains regulatory light chains

Journal of Medicinal Chemistry neck domain tail domain or lever arm

heavy chains α-helical coiled-coil non-helical tail region tail region

B

actin filaments


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B

actin filaments


Page 54 of 63

upper 50-kDa

loop 1 Page 55 subdomain of 63 Journal of Medicinal Chemistry

β7

switch I P-loop

*ATP *binding site

β6 1 *actin switch II β5 2 binding site 50-kDa N-terminal subdomain β4 3 cleft β3 seven-stranded 4 β2 β-sheet 5 β1 lower 50-kDa 6 SH1-helix subdomain 7 ACS Paragon Plus Environment 8 lever arm relay helix 9 10 converter subdomain

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Page 56 of 63

head domain of Dictyostelium discoideum myosin II

ADP·vanadate

50-kDa cleft

(S)-blebbistatin ACS Paragon Plus Environment

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Gln637

Tyr261 Gly240

Leu262 Leu641

Ile455 Val630 Thr474

Tyr634

Glu467

Cys470

Ser456

Ile471 Phe466 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31


Page 58 of 63

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(S)-Blebbistatin

(S)-1

Strengths + myosin II selective + cell membrane permeable + rapid inhibition + reversible inhibitor

Deficiencies - low selectivity between myosin II isoforms - low potency - poorly water soluble - precipitates interfere in fluorescent readouts

Improved analogs: recommendations for use as tool compounds General application in (cell-based) assays Use (S)-3’-hydroxyblebbistatin (S)-62, (S)-3’-aminoblebbistatin (S)-63 or (S)-4’-aminoblebbistatin (S)-55 + highly superior water solubility + no interfere in fluorescent readouts + (S)-62 is synthetically best accessible

(S)-54 Képiró et al.6

(S)-55 Várkuti et al.8

(S)-62 Verhasselt et al.79,81 (S)-63 Verhasselt et al.79,81

Cytotoxicity issues Use (S)-4’-nitroblebbistatin (S)-54 or (S)-4’-aminoblebbistatin (S)-55 + non-toxic in HeLa cells (for (S)-54) + reduced (for (S)-54) or no (for (S)-55) toxicity in zebrafish embryos

Applications requiring exposure to light at wavelengths below 490 nm Use (S)-4’-nitroblebbistatin (S)-54 + non-photosensitive + non-phototoxic ACS Paragon Plus Environment

- cytotoxic in various (model) systems - blue-light sensitive - phototoxic

Unmet needs & starting points for further research towards pharmacological tools - low potency of (S)-blebbistatin - low selectivity between myosin II isoforms

Better understanding of structure-activity relationship high-resolution in vitro and in silico studies of the entire chemo-mechanical cycle High-throughput screening against multiple myosin II isoforms discovery of novel chemotypes & drugable binding pocket


LITERATURE OVERVIEW

BIOLOGY

Page 60 of 63

SYNTHESIS

Author’s perspective

DISCOVERY

CHARACTERIZATION (ACTIVITY, PROPERTIES) RATIONAL DESIGN Suggestions for further improvement of potency & properties

SUPERIOR ANALOGS


Recommendations for optimal use

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ATP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

A·M


actin dissociation a

ADP release f

Page 62 of 63

M·ATP b lever arm priming

ATPase A·M·ADP

cycle

M·ATP

power stroke e

c ATP hydrolysis Pi

A·M·ADP

actin binding Pi release d

B·M·ADP·Pi


myosin head domain and lever arm (M) actin (A) (S)-blebbistatin (B)

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LITERATURE OVERVIEW

BLEBBISTATIN SERIES

* Discovery

AUTHOR’S PERSPECTIVE * Recommendations for optimal

* Biology

use of existing analogs in

* Synthesis

biochemical work

* Characterization

* Suggestions for breakthroughs

* Rational design of superior

towards improved potency &

analogs

Myosin II inhibitor ACS Paragon Plus Environment

physicochemical properties

Medicinal Chemistry and Use of Myosin II Inhibitor - ACS Publications

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