Discovery of BLU-945, a Reversible, Potent, and Wild-Type-Sparing Next-Generation EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer.

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR mutant (L858R and ex19del)-driven non-small-cell lung cancer (NSCLC), most patients will eventually develop resistance to TKIs. In the case of first- and second-generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations.

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice.

Background: A hallmark of Alzheimer's disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aβ42 and Aβ40. Many drug discovery efforts have focused on decreasing the production of Aβ42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function.

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC.

Parvalbumin gene delivery improves diastolic function in the aged myocardium in vivo.

Abnormal relaxation of the heart, termed diastolic dysfunction, is a significant and growing problem that is a major cause of heart failure in the aged population. The potential of gene transfer of parvalbumin (Parv), a cytoplasmic calcium-binding protein, to improve diastolic function in the aged myocardium in vivo was evaluated. Despite evidence for an early developmental influence on the efficiency of Ad5 striated muscle transduction, results show that Ad5 gene transfer efficiency to adult cardiac myocytes in vitro is identical in young and old rats, suggesting that the basic processes of adenovirus binding and internalization are unaffected by aging.

Comparative analysis of parvalbumin and SERCA2a cardiac myocyte gene transfer in a large animal model of diastolic dysfunction.

Diastolic dysfunction results from impaired ventricular relaxation and is an important component of human heart failure. Genetic modification of intracellular calcium-handling proteins may hold promise to redress diastolic dysfunction; however, it is unclear whether other important aspects of myocyte function would be compromised by this approach. Accordingly, a large animal model of humanlike diastolic dysfunction was established through 1 yr of left ventricular (LV) pressure overload by descending thoracic aortic coarctation in canines.

Myofilament calcium sensitivity and cardiac disease: insights from troponin I isoforms and mutants.

The heightened Ca2+ sensitivity of force found with hypertrophic cardiomyopathy (HCM)-associated mutant cardiac troponin I (cTnIR145G; R146G in rodents) has been postulated to be an underlying cause of hypertrophic growth and premature sudden death in humans and in animal models of the disease. Expression of slow skeletal TnI (ssTnI), a TnI isoform naturally expressed in developing heart, also increases myofilament Ca2+ sensitivity, yet its expression in transgenic mouse hearts is not associated with overt cardiac disease. Gene transfer of TnI isoforms or mutants into adult cardiac myocytes is used here to ascertain if expression levels or functional differences between HCM TnI and ssTnI could help explain these divergent organ-level effects.

Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A.

Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI.

In vivo acceleration of heart relaxation performance by parvalbumin gene delivery.

Defective cardiac muscle relaxation plays a causal role in heart failure. Shown here is the new in vivo application of parvalbumin, a calcium-binding protein that facilitates ultrafast relaxation of specialized skeletal muscles. Parvalbumin is not naturally expressed in the heart.

Chimera analysis of troponin I domains that influence Ca(2+)-activated myofilament tension in adult cardiac myocytes.

The goal of this study was to investigate isoform-specific functional domains of the inhibitory troponin subunit, troponin I (TnI), as it functions within the intact myofilaments of adult cardiac myocytes. Adenovirus-mediated gene transfer was used to deliver and express a TnI chimera composed of the amino terminus of cardiac TnI (cTnI) and the carboxy terminus of slow skeletal TnI (ssTnI) in adult rat cardiac myocytes. The TnI chimera, designated N-card/slow-C TnI, was expressed and incorporated into myofilaments after gene transfer, without detectable changes in contractile protein stoichiometry or sarcomere architecture.

A nemaline myopathy mutation in alpha-tropomyosin causes defective regulation of striated muscle force production.

Nemaline myopathy (NM) is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent appearance of nemaline rods within the muscle fibers. Recently, a missense mutation inTPM3, which encodes the slow skeletal alpha-tropomyosin (alphaTm), was linked to NM in a large kindred with an autosomal-dominant, childhood-onset form of the disease. We used adenoviral gene transfer to fully differentiated rat adult myocytes in vitro to determine the effects of NM mutant human alphaTm expression on striated muscle sarcomeric structure and contractile function.

Direct, convergent hypersensitivity of calcium-activated force generation produced by hypertrophic cardiomyopathy mutant alpha-tropomyosins in adult cardiac myocytes.

Familial hypertrophic cardiomyopathy is a clinically and genetically diverse autosomal dominant disorder characterized by ventricular hypertrophy and myocyte disarray in the absence of known hypertrophic stimuli. It has been linked to many cardiac contractile proteins, including four point mutations in alpha-tropomyosin (Tm). Here we use adenoviral-mediated gene transfer into adult cardiac myocytes in vitro to show that all four hypertrophic cardiomyopathy alpha-Tm proteins can be expressed and incorporated into normal sarcomeric structures in cardiac myocytes at similar levels as normal alpha-Tm proteins after 5-6 days in culture.

Functional analysis of troponin I regulatory domains in the intact myofilament of adult single cardiac myocytes.

Troponin I is the putative molecular switch for Ca(2+)-activated contraction within the myofilament of striated muscles. To gain insight into functional troponin I domain(s) in the context of the intact myofilament, adenovirus-mediated gene transfer was used to replace endogenous cardiac troponin I within the myofilaments of adult cardiac myocytes with the slow skeletal isoform or a chimera of the slow skeletal and cardiac isoforms. Efficient expression and myofilament incorporation were observed in myocytes with each exogenous troponin I protein without detected changes in the stoichiometry of other contractile proteins and/or sarcomere architecture.

Thin filament protein dynamics in fully differentiated adult cardiac myocytes: toward a model of sarcomere maintenance.

Sarcomere maintenance, the continual process of replacement of contractile proteins of the myofilament lattice with newly synthesized proteins, in fully differentiated contractile cells is not well understood. Adenoviral-mediated gene transfer of epitope-tagged tropomyosin (Tm) and troponin I (TnI) into adult cardiac myocytes in vitro along with confocal microscopy was used to examine the incorporation of these newly synthesized proteins into myofilaments of a fully differentiated contractile cell. The expression of epitope-tagged TnI resulted in greater replacement of the endogenous TnI than the replacement of the endogenous Tm with the expressed epitope-tagged Tm suggesting that the rates of myofilament replacement are limited by the turnover of the myofilament bound protein.

Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes.

Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca2+ sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the alpha-MHC isoform to exclusively the beta-MHC isoform.

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins.

The direct effects of expressing hypertrophic cardiomyopathy-associated (HCM-associated) mutant troponin T (TnT) proteins on the force generation of single adult cardiac myocytes have not been established. Replication-defective recombinant adenovirus vectors were generated for gene transfer of HCM-associated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that the mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocyte.