A next-generation BRAF inhibitor overcomes resistance to BRAF inhibition in patients with BRAF-mutant cancers using pharmacokinetics-informed dose escalation.

RAF inhibitors have transformed treatment for BRAF V600-mutant cancer patients, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAF V600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAF V600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in BRAF-mutant cancer patients refractory to approved RAF inhibitors.

Clinicopathological investigation of secretory carcinoma cases including a successful treatment outcome using entrectinib for high-grade transformation: a case report.

BACKGROUND: Secretory carcinoma (SC) of the salivary gland is a recently described malignant tumor harboring characteristic ETV6-NTRK3 gene fusion. SC generally has a favorable clinical course, and is currently regarded as a low-grade carcinoma. However, a small subset of SCs demonstrates aggressive clinical features with histologically high-grade transformed morphology, the molecular pathogenesis of which has not yet been elucidated. In this study, we performed a clinicopathological and molecular genetic study of patients with SC of the head and neck displaying various clinical characteristics to investigate the differences of pathological and molecular genetics between low-grade and high-grade components of SC. CASE PRESENTATION: Three cases with SC of the head and neck, including a conventional low-grade SC and two high-grade transformed SCs are described. High-grade transformed SCs with histological features such as nuclear polymorphism, distinctive nucleoli and increased mitotic activity developed locoregional recurrence and distant metastasis. Immunohistochemical analysis revealed that low- and high-grade components showed different expression patterns for S-100 protein and mammaglobin, whereas all examined components were positive for p-STAT5. p53-positive cell population was markedly higher in one case with high-grade transformed SC. The proliferative activity of high-grade components was markedly increased, with the Ki-67 labeling index ranging up to 30-32%. A fluorescence in situ hybridization study with an ETV6 (12p13) break apart probe revealed split signals in the nuclei in all 3 cases. A targeted next-generation sequencing-based fusion assay demonstrated that all 6 clinical samples from the 3 patients showed the presence of the ETV6-NTRK3 fusion transcripts. One patient with high-grade transformed SC showed a dramatic clinical response to the pan-TRK inhibitor, entrectinib, for the treatment of locoregional recurrence and pulmonary metastasis. CONCLUSIONS: High-grade transformed SC showed aggressive clinical and pathological features with increased Ki-67 labeling index. Molecular genetic study of gene rearrangement appears to be beneficial treatment as the presence of ETV6-NTRK3 translocation may represent a therapeutic target in SC, particularly the high-grade transformed type.

Calmidazolium Chloride and Its Complex with Serum Albumin Prevent Huntingtin Exon1 Aggregation.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing an expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules for HD, which is currently untreatable. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Because it is hydrophobic, CLC was adsorbed to the vial surface and could not sustain an inhibition effect for a longer duration. The use of bovine serum albumin (BSA) prevented CLC adsorption by forming a BSA-CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, the NT17 part of Httex1. Furthermore, biocompatible CLC-loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells.

Monomerization and aggregation of ß-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation.

ß-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of ß-lactoglobulin at pH7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of ß-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of ß-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~55Å, ~29Å and ~37Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57kcalmol-1 and 8.93kcalmol-1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of ß-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~77Å and ~117Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69kcalmol-1 and 9.09kcalmol-1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.

Single Molecular Level Probing of Structure and Dynamics of Papain Under Denaturation.

INTRODUCTION: Papain is a cysteine protease enzyme present in papaya and known to help in digesting peptide. Thus the structure and function of the active site of papain is of interest. OBJECTIVE: The objective of present study is to unveil the overall structural transformation and the local structural change around the active site of papain as a function of chemical denaturant. METHODS: Papain has been tagged at Cys-25 with a thiol specific fluorescence probe N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA). Guanidine hydrochloride (GnHCl) has been used as the chemical denaturant. Steady state, time-resolved, and single molecular level fluorescence techniques was applied to map the change in the local environment. RESULTS: It is found that papain undergoes a two-step denaturation in the presence of GnHCl. Fluorescence correlation spectroscopic (FCS) data indicate that the size (hydrodynamic diameter) of native papain is ~36.8 Å, which steadily increases to ~53 Å in the presence of 6M GnHCl. FCS study also reveals that the conformational fluctuation time of papain is 6.3 µs in its native state, which decreased to 2.7 µs in the presence of 0.75 M GnHCl. Upon further increase in GnHCl concentration the conformational fluctuation time increase monotonically till 6 M GnHCl, where the time constant is measured as 14 µs. On the other hand, the measurement of ellipticity, hence the helical structure, by circular dichroism spectroscopy is found to be incapable to capture such structural transformation. CONCLUSION: It is concluded that in the presence of small amount of GnHCl the active site of papain takes up a more compact structure (although the overall size increases) than in the native state, which has been designated as the intermediate state.

Water-Soluble Components of Sesame Oil Reduce Inflammation and Atherosclerosis.

Atherosclerosis, a major form of cardiovascular disease, is now recognized as a chronic inflammatory disease. Nonpharmacological means of treating chronic diseases have gained attention recently. We previously reported that sesame oil aqueous extract (SOAE) has anti-inflammatory properties, both in vitro and in vivo. In this study, we have investigated the antiatherosclerotic properties of SOAE, and the mechanisms, through genes and inflammatory markers, by which SOAE might modulate atherosclerosis. Low-density lipoprotein receptor (LDL-R) knockout female mice were fed with either a high-fat (HF) diet or an HF diet supplemented with SOAE. Plasma lipids and atherosclerotic lesions were quantified after 3 months of feeding. Plasma samples were used for global cytokine array. RNA was extracted from both liver tissue and the aorta, and used for gene analysis. The high-fat diet supplemented with SOAE significantly reduced atherosclerotic lesions, plasma cholesterol, and LDL cholesterol levels in LDL-R(-/-) mice. Plasma inflammatory cytokines were reduced in the SOAE diet-fed animals, but not significantly, demonstrating potential anti-inflammatory properties of SOAE. Gene analysis showed the HF diet supplemented with SOAE reduced gene expression involved in inflammation and induced genes involved in cholesterol metabolism and reverse cholesterol transport, an anti-inflammatory process. Our studies suggest that a SOAE-enriched diet could be an effective nonpharmacological treatment for atherosclerosis by controlling inflammation and regulating lipid metabolism.

Atherosclerosis--do we know enough already to prevent it?

In this review, we have briefly summarized the characteristics of lipids and lipoproteins and the atherosclerotic process. The development of atherosclerosis is a continuous process that involves numerous cellular and acellular processes that influence the behavior of each other. These include oxidative stress, lipoprotein modifications, macrophage polarization, macrophage lipid accumulation, generation of pro- and anti-inflammatory components, calcification, cellular growth and proliferation, and plaque rupture. The precise role(s) of many of these are unknown. Understanding the events at each particular stage might shed more light onto the process as a whole and could potentially reveal targets for intervention. Therapeutic modalities that work at one stage may have little to no influence on other stages of the disease.

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading.

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.