Fosgonimeton attenuates amyloid-beta toxicity in preclinical models of Alzheimer's disease.

Positive modulation of hepatocyte growth factor (HGF) signaling may represent a promising therapeutic strategy for Alzheimer's disease (AD) based on its multimodal neurotrophic, neuroprotective, and anti-inflammatory effects addressing the complex pathophysiology of neurodegeneration. Fosgonimeton is a small-molecule positive modulator of the HGF system that has demonstrated neurotrophic and pro-cognitive effects in preclinical models of dementia. Herein, we evaluate the neuroprotective potential of fosgonimeton, or its active metabolite, fosgo-AM, in amyloid-beta (Aß)-driven preclinical models of AD, providing mechanistic insight into its mode of action. In primary rat cortical neurons challenged with Aß (Aß1-42), fosgo-AM treatment significantly improved neuronal survival, protected neurite networks, and reduced tau hyperphosphorylation. Interrogation of intracellular events indicated that cortical neurons treated with fosgo-AM exhibited a significant decrease in mitochondrial oxidative stress and cytochrome c release. Following Aß injury, fosgo-AM significantly enhanced activation of pro-survival effectors ERK and AKT, and reduced activity of GSK3ß, one of the main kinases involved in tau hyperphosphorylation. Fosgo-AM also mitigated Aß-induced deficits in Unc-like kinase 1 (ULK1) and Beclin-1, suggesting a potential effect on autophagy. Treatment with fosgo-AM protected cortical neurons from glutamate excitotoxicity, and such effects were abolished in the presence of an AKT or MEK/ERK inhibitor. In vivo, fosgonimeton administration led to functional improvement in an intracerebroventricular Aß25-35 rat model of AD, as it significantly rescued cognitive function in the passive avoidance test. Together, our data demonstrate the ability of fosgonimeton to counteract mechanisms of Aß-induced toxicity. Fosgonimeton is currently in clinical trials for mild-to-moderate AD (NCT04488419; NCT04886063).

ATH-1105, a small-molecule positive modulator of the neurotrophic HGF system, is neuroprotective, preserves neuromotor function, and extends survival in preclinical models of ALS.

Introduction: Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disorder, primarily affects the motor neurons of the brain and spinal cord. Like other neurodegenerative conditions, ongoing pathological processes such as increased inflammation, excitotoxicity, and protein accumulation contribute to neuronal death. Hepatocyte growth factor (HGF) signaling through the MET receptor promotes pro-survival, anti-apoptotic, and anti-inflammatory effects in multiple cell types, including the neurons and support cells of the nervous system. This pleiotropic system is therefore a potential therapeutic target for treatment of neurodegenerative disorders such as ALS. Here, we test the effects of ATH-1105, a small-molecule positive modulator of the HGF signaling system, in preclinical models of ALS. Methods: In vitro, the impact of ATH-1105 on HGF-mediated signaling was assessed via phosphorylation assays for MET, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Neuroprotective effects of ATH-1105 were evaluated in rat primary neuron models including spinal motor neurons, motor neuron-astrocyte cocultures, and motor neuron-human muscle cocultures. The anti-inflammatory effects of ATH-1105 were evaluated in microglia- and macrophage-like cell systems exposed to lipopolysaccharide (LPS). In vivo, the impact of daily oral treatment with ATH-1105 was evaluated in Prp-TDP43A315T hemizygous transgenic ALS mice. Results: In vitro, ATH-1105 augmented phosphorylation of MET, ERK, and AKT. ATH-1105 attenuated glutamate-mediated excitotoxicity in primary motor neurons and motor neuron- astrocyte cocultures, and had protective effects on motor neurons and neuromuscular junctions in motor neuron-muscle cocultures. ATH-1105 mitigated LPS-induced inflammation in microglia- and macrophage-like cell systems. In vivo, ATH-1105 treatment resulted in improved motor and nerve function, sciatic nerve axon and myelin integrity, and survival in ALS mice. Treatment with ATH-1105 also led to reductions in levels of plasma biomarkers of inflammation and neurodegeneration, along with decreased pathological protein accumulation (phospho-TDP-43) in the sciatic nerve. Additionally, both early intervention (treatment initiation at 1 month of age) and delayed intervention (treatment initiation at 2 months of age) with ATH-1105 produced benefits in this preclinical model of ALS. Discussion: The consistent neuroprotective and anti-inflammatory effects demonstrated by ATH-1105 preclinically provide a compelling rationale for therapeutic interventions that leverage the positive modulation of the HGF pathway as a treatment for ALS.

Fosgonimeton, a Novel Positive Modulator of the HGF/MET System, Promotes Neurotrophic and Procognitive Effects in Models of Dementia.

All types of dementia, including Alzheimer's disease, are debilitating neurodegenerative conditions marked by compromised cognitive function for which there are few effective treatments. Positive modulation of hepatocyte growth factor (HGF)/MET, a critical neurotrophic signaling system, may promote neuronal health and function, thereby addressing neurodegeneration in dementia. Here, we evaluate a series of novel small molecules for their ability to (1) positively modulate HGF/MET activity, (2) induce neurotrophic changes and protect against neurotoxic insults in primary neuron culture, (3) promote anti-inflammatory effects in vitro and in vivo, and (4) reverse cognitive deficits in animal models of dementia. Through screening studies, the compound now known as fosgonimeton-active metabolite (fosgo-AM) was identified by use of immunocytochemistry to be the most potent positive modulator of HGF/MET and was selected for further testing. Primary hippocampal neurons treated with fosgo-AM showed enhanced synaptogenesis and neurite outgrowth, supporting the neurotrophic effects of positive modulators of HGF/MET. Additionally, fosgo-AM protected against neurotoxic insults in primary cortical neuron cultures. In vivo, treatment with fosgo-AM rescued cognitive deficits in the rat scopolamine amnesia model of dementia. Although fosgo-AM demonstrated several procognitive effects in vitro and in vivo, a prodrug strategy was used to enhance the pharmacological properties of fosgo-AM, resulting in the development of fosgonimeton (ATH-1017). The effect of fosgonimeton on cognition was confirmed in a lipopolysaccharide (LPS)-induced neuroinflammatory mouse model of dementia. Together, the results of these studies support the potential of positive modulators of HGF/MET to be used as novel therapeutics and suggest the drug candidate fosgonimeton might protect against neurodegeneration and be therapeutic in the management of Alzheimer's disease and other types of dementia.

Dilated cardiomyopathy mutation (R174W) in troponin T attenuates the length-mediated increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Alterations in length-dependent activation (LDA) may constitute a mechanism by which cardiomyopathy mutations lead to deleterious phenotypes and compromised heart function, because LDA underlies the molecular basis by which the heart tunes myocardial force production on a beat-to-beat basis (Frank-Starling mechanism). In this study, we investigated the effect of DCM-linked mutation (R173W) in human cardiac troponin T (TnT) on myofilament LDA. R173W mutation is associated with left ventricular dilatation and systolic dysfunction and is found in multiple families. R173W mutation is in the central region (residues 80-180) of TnT, which is known to be important for myofilament cooperativity and cross-bridge (XB) recruitment. Steady-state and dynamic contractile parameters were measured in detergent-skinned guinea pig left ventricular muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or mutant TnT (TnTR174W; guinea pig analog of human R173W mutation) at two different sarcomere lengths (SL): short (1.9 µm) and long (2.3 µm). TnTR174W decreased pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at long than at short SL; for example, pCa50 decreased by 0.12 pCa units at long SL and by 0.06 pCa units at short SL. Differential changes in pCa50 at short and long SL attenuated the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTR174W fibers; ΔpCa50 was 0.10 units in TnTWT fibers but only 0.04 units in TnTR174W fibers. Furthermore, TnTR174W blunted the SL-dependent increase in the magnitude of XB recruitment. Our observations suggest that the R173W mutation in human cardiac TnT may impair Frank-Starling mechanism.NEW & NOTEWORTHY This work characterizes the effect of dilated cardiomyopathy mutation in cardiac troponin T (TnTR174W) on myofilament length-dependent activation. TnTR174W attenuates the length-dependent increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Developmental increase in ß-MHC enhances sarcomere length-dependent activation in the myocardium.

Shifts in myosin heavy chain (MHC) isoforms in cardiac myocytes have been shown to alter cardiac muscle function not only in healthy developing hearts but also in diseased hearts. In guinea pig hearts, there is a large age-dependent shift in MHC isoforms from 80% α-MHC/20% ß-MHC at 3 wk to 14% α-MHC/86% ß-MHC at 11 wk. Because kinetic differences in α- and ß-MHC cross-bridges (XBs) are known to impart different cooperative effects on thin filaments, we hypothesize here that differences in α- and ß-MHC expression in guinea pig cardiac muscle impact sarcomere length (SL)-dependent contractile function. We therefore measure steady state and dynamic contractile parameters in detergent-skinned cardiac muscle preparations isolated from the left ventricles of young (3 wk old) or adult (11 wk old) guinea pigs at two different SLs: short (1.9 µm) and long (2.3 µm). Our data show that SL-dependent effects on contractile parameters are augmented in adult guinea pig cardiac muscle preparations. Notably, the SL-mediated increase in myofilament Ca2+ sensitivity (ΔpCa50) is twofold greater in adult guinea pig muscle preparations (ΔpCa50 being 0.11 units in adult preparations but only 0.05 units in young preparations). Furthermore, adult guinea pig cardiac muscle preparations display greater SL-dependent changes than young muscle preparations in (1) the magnitude of length-mediated increase in the recruitment of new force-bearing XBs, (2) XB detachment rate, (3) XB strain-mediated effects on other force-bearing XBs, and (4) the rate constant of force redevelopment. Our findings suggest that increased ß-MHC expression enhances length-dependent activation in the adult guinea pig cardiac myocardium.

Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity.

Recent clinical studies have revealed a new hypertrophic cardiomyopathy-associated mutation (F87L) in the central region of human cardiac troponin T (TnT). However, despite its implication in several incidences of sudden cardiac death in young and old adults, whether F87L is associated with cardiac contractile dysfunction is unknown. Because the central region of TnT is important for modulating the muscle length-mediated recruitment of new force-bearing cross-bridges (XBs), we hypothesize that the F87L mutation causes molecular changes that are linked to the length-dependent activation of cardiac myofilaments. Length-dependent activation is important because it contributes significantly to the Frank-Starling mechanism, which enables the heart to vary stroke volume as a function of changes in venous return. We measured steady-state and dynamic contractile parameters in detergent-skinned guinea pig cardiac muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or the guinea pig analogue (TnTF88L) of the human mutation at two different sarcomere lengths (SLs): short (1.9 µm) and long (2.3 µm). TnTF88L increases pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at short SL than at long SL; for example, pCa50 increases by 0.25 pCa units at short SL and 0.17 pCa units at long SL. The greater increase in pCa50 at short SL leads to the abolishment of the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTF88L fibers, ΔpCa50 being 0.10 units in TnTWT fibers but only 0.02 units in TnTF88L fibers. Furthermore, at short SL, TnTF88L attenuates the negative impact of strained XBs on force-bearing XBs and augments the magnitude of muscle length-mediated recruitment of new force-bearing XBs. Our findings suggest that the TnTF88L-mediated effects on cardiac thin filaments may lead to a negative impact on the Frank-Starling mechanism.

Omecamtiv Mecarbil Abolishes Length-Mediated Increase in Guinea Pig Cardiac Myofiber Ca2+ Sensitivity.

Omecamtiv mecarbil (OM) is a pharmacological agent that augments cardiac contractile function by enhancing myofilament Ca2+ sensitivity. Given that interventions that increase myofilament Ca2+ sensitivity have the potential to alter length-dependent activation (LDA) of cardiac myofilaments, we tested the influence of OM on this fundamental property of the heart. This is significant not only because LDA is prominent in cardiac muscle but also because it contributes to the Frank-Starling law, a mechanism by which the heart increases stroke volume in response to an increase in venous return. We measured steady-state and dynamic contractile indices in detergent-skinned guinea pig (Cavia porcellus) cardiac muscle fibers in the absence and presence of 0.3 and 3.0 µM OM at two different sarcomere lengths (SLs), short SL (1.9 µm) and long SL (2.3 µm). Myofilament Ca2+ sensitivity, as measured by pCa50 (-log of [Ca2+]free concentration required for half-maximal activation), increased significantly at both short and long SLs in OM-treated fibers when compared to untreated fibers; however, the magnitude of increase in pCa50 was twofold greater at short SL than at long SL. A consequence of this greater increase in pCa50 at short SL was that pCa50 did not increase any further at long SL, suggesting that OM abolished the SL dependency of pCa50. Furthermore, the SL dependency of rate constants of cross-bridge distortion dynamics (c) and force redevelopment (ktr) was abolished in 0.3-µM-OM-treated fibers. The negative impact of OM on the SL dependency of pCa50, c, and ktr was also observed in 3.0-µM-OM-treated fibers, indicating that cooperative mechanisms linked to LDA were altered by the OM-mediated effects on cardiac myofilaments.

L71F mutation in rat cardiac troponin T augments crossbridge recruitment and detachment dynamics against α-myosin heavy chain, but not against ß-myosin heavy chain.

The N-terminal extension of human cardiac troponin T (TnT), which modulates myofilament Ca2+ sensitivity, contains several hypertrophic cardiomyopathy (HCM)-causing mutations including S69F. However, the functional consequence of S69F mutation is unknown. The human analog of S69F in rat TnT is L71F (TnTL71F). Because the functional consequences due to structural changes in the N-terminal extension are influenced by the type of myosin heavy chain (MHC) isoform, we hypothesized that the TnTL71F-mediated effect would be differently modulated by α- and ß-MHC isoforms. TnTL71F and wild-type rat TnT were reconstituted into de-membranated muscle fibers from normal (α-MHC) and propylthiouracil-treated rat hearts (ß-MHC) to measure steady-state and dynamic contractile parameters. The magnitude of the TnTL71F-mediated attenuation of Ca2+-activated maximal tension was greater in α- than in ß-MHC fibers. For example, TnTL71F attenuated maximal tension by 31% in α-MHC fibers but only by 10% in ß-MHC fibers. Furthermore, TnTL71F reduced myofilament Ca2+ sensitivity by 0.11 pCa units in α-MHC fibers but only by 0.05 pCa units in ß-MHC fibers. TnTL71F augmented rate constants of crossbridge recruitment and crossbridge detachment dynamics in α-MHC fibers but not in ß-MHC fibers. Collectively, our data demonstrate that TnTL71F induces greater contractile deficits against α-MHC than against ß-MHC background.