Getting site-specific with actomyosin inhibitors

Abstract

Actin and myosin play important roles in many devastating diseases and thus are attractive targets for small-molecule therapy. In this issue of JBC, Guhathakurta et al. have developed a high-throughput screening assay to find small molecules that interfere with the actomyosin interaction. They utilized time-resolved FRET (TR-FRET) and a unique donor–acceptor pair (filamentous actin and a peptide that binds near the myosin-binding site on actin) to find novel molecules that interfere with the actomyosin ATPase and alter the structure of actin filaments. These findings demonstrate the power and potential of high-throughput TR-FRET in monitoring molecular interactions.

Introduction

The contractile proteins, actin and myosin, are responsible for producing the force and motion that drive many different biological functions including muscle contraction, cell movement, cell division, intracellular transport, and endocytosis/exocytosis (1). The ability to modulate actomyosin-based motility with therapeutic agents may be a method for treating devastating diseases such as heart failure, cancer, and diabetes. Indeed, recent findings have demonstrated promising results with small-molecule drugs targeted to cardiac myosin for treating heart failure (2, 3). However, the process of screening for new molecules can be extremely challenging and requires an assay that is highly specific to reduce false-positives and off-target effects. New data from Guhathakurta et al. (4) provide a compelling example in that regard, using a sophisticated fluorescence assay to monitor compound displacement of an actin-binding peptide to identify molecules that interfere with the actomyosin interaction (Fig. 1). This study provides a new platform for scientists in the actin field and beyond to interrogate protein–protein and other biomolecular interactions.

Figure 1.

Cartoon diagram of the FRET assay developed by Guhathakurta et al. (4). The actin filament is depicted with dark blue boxes, and the yellow star indicates the donor fluorescence label (fluorescein), while the myosin is shown in green with its two associated light chains (essential light chain, purple; regulatory light chain, blue). The N-terminal extension on the essential light chain is shown in red, and the acceptor fluorescence label (dabcyl) is depicted in light blue. When the acceptor-labeled N-terminal extension peptide (ANT) is bound to actin, they observed high FRET, while, upon displacement with a small molecule (orange), a reduction in FRET was observed. The method was used to screen hundreds of compounds for interactions with actin, and ten promising compounds were screened for their impact on actomyosin ATPase and actin structural dynamics.

Efforts to screen for compounds that inhibit the actomyosin interaction have mostly been limited to examining the actomyosin ATPase directly, which is quite time-consuming and can lead to compounds that bind actin or myosin. Therefore, a more specific approach is desirable. Time-resolved FRET (TR-FRET)² has many advantages as a method of screening for compounds that alter protein–protein interactions or important conformational changes in a target biomolecule (5). FRET monitors the distance between a donor and acceptor fluorescent probe and is typically quantitated by monitoring the steady-state fluorescence of the donor/acceptor, the fluorescence lifetime of the donor, or, in rare cases, the anisotropy of the donor. Monitoring FRET with steady-state fluorescence can create problems with signal-to-noise, precision, sample-to-sample variability, and interference from fluorescent compounds. However, fluorescence lifetime measurements, as detected in TR-FRET, are not dependent on the fluorophore concentration, and contaminating fluorescence can be easily separated out in the analysis. Historically, TR-FRET measurements required a relatively long acquisition time (∼10 s) limiting its utility in screening thousands of compounds. The approach of measuring fluorescence lifetimes with direct waveform recording (DWR) allows extremely rapid (∼0.1 ms) and precise measurements to be made with greatly improved signal-to-noise ratios (5). Therefore, DWR TR-FRET has outstanding potential to screen for novel compounds, provided that a FRET biosensor that accurately reports on the structure–function of the biomolecule of interest can be developed.

Guhathakurta et al. (4) have developed a unique assay using DWR TR-FRET to examine compounds that interfere with the actomyosin interaction. Myosins expressed in muscle are composed of a heavy chain and two associated light chains (essential and regulatory light chain), and each heavy chain dimerizes and assembles into thick filaments in muscle. In previous work, it was demonstrated that certain isoforms of skeletal and cardiac muscle myosin have an essential light chain that contains a long N-terminal extension (NTE) that modulates contraction by interacting with actin (6–8), and the first few residues of the NTE are critical for the interaction. Guhathakurta et al. (4) labeled actin with fluorescein (donor) and a 12-amino-acid peptide derived from the NTE with dabcyl (ANT), a nonfluorescent acceptor, and found a FRET efficiency similar to what they observed in previous work with an intact actomyosin complex (7). Importantly, in the presence of unlabeled myosin, the FRET efficiency was reduced significantly, suggesting that the ANT binds to the myosin-binding site on actin and can be displaced by myosin. Thus, the actin–ANT FRET pair could be used to find compounds that interfere with the myosin-binding site on actin. They screened over 727 compounds and found 10 that greatly altered the FRET efficiency. They then examined the remaining compounds for the ability to inhibit actin-activated myosin ATPase activity and found that most compounds met their criteria. They went on to demonstrate that the compounds of interest alter the structure of F-actin by performing phosphorescence anisotropy experiments. Overall, they concluded that their highly specific assay combined with TR-FRET was an extremely powerful method of finding novel compounds that alter actomyosin interactions and actin structural dynamics.

The ability to inhibit actomyosin interactions could prove to be extremely useful in treating various disease conditions. For example, inherited forms of heart failure are known to be caused by mutations in myosin and its binding partner myosin-binding protein C (9). The mutations that cause hypertrophic cardiomyopathy (HCM) are proposed to cause an increase in force generation by various proposed mechanisms. Therefore, interfering with the actomyosin interaction in these diseased muscles is proposed to reduce the impact of the “gain of function” mutations and prevent the development of hypertrophy. Indeed, one compound that interacts specifically with cardiac myosin and inhibits actin-activated myosin ATPase is entering Phase 3 clinical trials for the treatment of HCM (2). Other research groups are seeking to modulate actomyosin interactions in nonmuscle cells to treat cancer, neuronal disorders, and vascular disease (10).

The new screen described by Guhathakurta et al. (4) could be extended to search for small molecules that interfere with the actomyosin interaction in a more physiological environment. For example, a skinned muscle fiber or myofibril preparation could be utilized in which the actin is labeled at Cys-374, as in the current study, and the actin–ANT TR-FRET could be monitored in the presence of various small molecules. This would allow assessment of the feasibility of the small molecules in treating muscle diseases in the presence of the many regulatory proteins associated with actin and myosin in a muscle fiber. The authors point out that since actin is crucial for many cellular processes, it will be important for future studies to screen for small molecules that are specific for the actin isoform being targeted (e.g. muscle versus nonmuscle).

In summary, Guhathakurta et al. (4) have demonstrated an extremely powerful method of using TR-FRET to screen for compounds that interact with a target biomolecule. A secondary assay is required to validate the ability of the compounds to impact function (e.g. actomyosin ATPase), but that is feasible because the number of promising compounds can be dramatically narrowed by the TR-FRET screen. Future studies will be challenged to utilize similar technology to develop compounds that allosterically enhance actomyosin-based force generation for the treatment of diseases associated with depressed contractile function. It will also be exciting to see if this approach can be used to find novel compounds that specifically interfere with other actin-binding proteins to probe their biological function and role in disease conditions.