Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.

Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin-myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces binding of cMyBP-C to actin or myosin, is often decreased in heart failure (HF) patients, and is cardioprotective in model systems for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL), and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethyl rhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding and/or structural changes in the protein complex. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fskC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish feasibility for discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Behavioral variation in nesting phenology may offset sex-ratio bias in tuatara.

The nest environment for eggs of reptiles has lifelong implications for offspring performance and success, and, ultimately, for population viability and species distributions. However, understanding the various abiotic and biotic drivers of nesting is complex, particularly regarding variation in nesting behavior of females and consequences for sex ratios in species with temperature-dependent sex determination (TSD). We investigated how nest construction and nesting phenology affect the incubation environment of a reptile with TSD, the tuatara (Sphenodon punctatus), a species that is at risk from climate-mediated male bias in population sex ratios. Using longitudinal behavioral data, we addressed the following questions. (1) Does nesting behavior vary with seasonal or location cues? (2) Does variation in nesting phenology or nest construction affect the incubation environment? We aimed to investigate whether female tuatara could modify nesting behavior to respond to novel environments, including a warming climate, allowing for successful incubation and balanced population sex ratios, maintaining population viability throughout their historic range. We predicted that earlier nesting after warm winters increased the likelihood that females will be produced, despite the sex determining system where males are produced from warmer temperatures. Further research is needed to understand the extent to which nesting behavior varies by individual through time, and across the range of tuatara, and the importance of habitat variability in maintaining production of females under future climate warming.

Immunomodulatory effects of adipose tissue-derived stem cells on elastin scaffold remodeling in diabetes.

Diabetes is a major risk factor for the progression of vascular disease, contributing to elevated levels of glycoxidation, chronic inflammation and calcification. Tissue engineering emerges as a potential solution for the treatment of vascular diseases however there is a considerable gap in the understanding of how scaffolds and stem cells will perform in patients with diabetes. We hypothesized that adipose tissue-derived stem cells (ASCs) by virtue of their immunosuppressive potential would moderate the diabetes-intensified inflammatory reactions and induce positive construct remodeling. To test this hypothesis, we prepared arterial elastin scaffolds seeded with autologous ASCs and implanted them subdermally in diabetic rats and compared inflammatory markers, macrophage polarization, matrix remodeling, calcification and bone protein expression to control scaffolds implanted with and without cells in nondiabetic rats. ASC-seeded scaffolds exhibited lower levels of CD8+ T-cells and CD68+ pan-macrophages and higher numbers of M2 macrophages, smooth muscle cell-like and fibroblast-like cells. Calcification and osteogenic markers were reduced in ASCseeded scaffolds implanted in non-diabetic rats but remained unchanged in diabetes, unless the scaffolds were first pre-treated with penta-galloyl glucose (PGG), a known anti-oxidative elastin-binding polyphenol. In conclusion, autologous ASC seeding in elastin scaffolds is effective in combating diabetes-related complications. To prevent calcification, the oxidative milieu needs to be reduced by elastin-binding antioxidants such as PGG.