Acrylonitrile derivatives: In vitro activity and mechanism of cell death induction against Trypanosoma cruzi and Leishmania amazonensis.

Leishmaniasis and Chagas disease are parasitic infections that affect millions of people worldwide, producing thousands of deaths per year. The current treatments against these pathologies are not totally effective and produce some side effects in the patients. Acrylonitrile derivatives are a group of compounds that have shown activity against these two diseases. In this work, four novels synthetic acrylonitriles were evaluated against the intracellular form and extracellular forms of L. amazonensis and T. cruzi. The compounds 2 and 3 demonstrate to have good selectivity indexes against both parasites, specifically the compound 3 against the amastigote form (SI = 6 against L. amazonensis and SI = 7.4 against T. cruzi). In addition, the parasites treated with these two compounds demonstrate to produce a programmed cell death, since they were positive for the events studied related to this type of death, including chromatin condensation, accumulation of reactive oxygen species and alteration of the mitochondrial membrane potential. In conclusion, this work confirms that acrylonitriles is a source of possible new compounds against kinetoplastids, however, more studies are needed to corroborate this activity.

Epidemiology of Melanoma.

Melanoma accounts for 1.7% of global cancer diagnoses and is the fifth most common cancer in the US. Melanoma incidence is rising in developed, predominantly fair-skinned countries, growing over 320% in the US since 1975. However, US mortality has fallen almost 30% over the past decade with the approval of 10 new targeted or immunotherapy agents since 2011. Mutations in the signaling-protein BRAF, present in half of cases, are targeted with oral BRAF/MEK inhibitor combinations, while checkpoint inhibitors are used to restore immunosurveillance likely inactivated by UV radiation. Although the overall 5-year survival has risen to 93.3% in the US, survival for stage IV disease remains only 29.8%. Melanoma is most common in white, older men, with an average age of diagnosis of 65. Outdoor UV exposure without protection is the main risk factor, although indoor tanning beds, immunosuppression, family history and rare congenital diseases, moles, and obesity contribute to the disease. Primary prevention initiatives in Australia implemented since 1988, such as education on sun-protection, have increased sun-screen usage and curbed melanoma incidence, which peaked in Australia in 2005. In the US, melanoma incidence is not projected to peak until 2022-2026. Fewer than 40% of Americans report practicing adequate protection (sun avoidance from 10 a.m.-4 p.m. and regular application of broad-spectrum sunscreen with an SPF > 30). A 2-4-fold return on investment is predicted for a US sun-protection education initiative. Lesion-directed skin screening programs, especially for those at risk, have also cost-efficiently reduced melanoma mortality.

Role of intracellular signaling pathways and their inhibitors in the treatment of inflammation.

Inflammation is not only a defense mechanism of the innate immune system against invaders, but it is also involved in the pathogenesis of many diseases such as atherosclerosis, thrombosis, diabetes, epilepsy, and many neurodegenerative disorders. The World Health Organization (WHO) reports worldwide estimates of people (9.6% in males and 18.0% in females) aged over 60 years, suffering from symptomatic osteoarthritis, and around 339 million suffering from asthma. Other chronic inflammatory diseases, such as ulcerative colitis and Crohn's disease are also highly prevalent. The existing anti-inflammatory agents, both non-steroidal and steroidal, are highly effective; however, their prolonged use is marred by the severity of associated side effects. A holistic approach to ensure patient compliance requires understanding the pathophysiology of inflammation and exploring new targets for drug development. In this regard, various intracellular cell signaling pathways and their signaling molecules have been identified to be associated with inflammation. Therefore, chemical inhibitors of these pathways may be potential candidates for novel anti-inflammatory drug approaches. This review focuses on the anti-inflammatory effect of these inhibitors (for JAK/STAT, MAPK, and mTOR pathways) describing their mechanism of action through literature search, current patents, and molecules under clinical trials.

A novel C-terminal Hsp90 inhibitor KU758 synergizes efficacy in combination with BRAF or MEK inhibitors and targets drug-resistant pathways in BRAF-mutant melanomas.

Melanoma remains the most aggressive and fatal form of skin cancer, despite several FDA-approved targeted chemotherapies and immunotherapies for use in advanced disease. Of the 100 350 new patients diagnosed with melanoma in 2020 in the US, more than half will develop metastatic disease leading to a 5-year survival rate <30%, with a majority of these developing drug-resistance within the first year of treatment. These statistics underscore the critical need in the field to develop more durable therapeutics as well as those that can overcome chemotherapy-induced drug resistance from currently approved agents. Fortunately, several of the drug-resistance pathways in melanoma, including the proteins in those pathways, rely in part on Hsp90 chaperone function. This presents a unique and novel opportunity to simultaneously target multiple proteins and drug-resistant pathways in this disease via molecular chaperone inhibition. Taken together, we hypothesize that our novel C-terminal Hsp90 inhibitor, KU758, in combination with the current standard of care targeted therapies (e.g. vemurafenib and cobimetinib) can both synergize melanoma treatment efficacy in BRAF-mutant tumors, as well as target and overcome several major resistance pathways in this disease. Using in vitro proliferation and protein-based Western Blot analyses, our novel inhibitor, KU758, potently inhibited melanoma cell proliferation (without induction of the heat shock response) in vitro and synergized with both BRAF and MEK inhibitors in inhibition of cell migration and protein expression from resistance pathways. Overall, our work provides early support for further translation of C-terminal Hsp90 inhibitor and mitogen-activated protein kinase pathway inhibitor combinations as a novel therapeutic strategy for BRAF-mutant melanomas.

Activation of CD8+ T Cells Contributes to Antitumor Effects of CDK4/6 Inhibitors plus MEK Inhibitors.

Concurrent MEK and CDK4/6 inhibition shows promise in clinical trials for patients with advanced-stage mutant BRAF/NRAS solid tumors. The effects of CDK4/6 inhibitor (CDK4/6i) in combination with BRAF/MEK-targeting agents on the tumor immune microenvironment are unclear, especially in melanoma, for which immune checkpoint inhibitors are effective in approximately 50% of patients. Here, we show that patients progressing on CDK4/6i/MEK pathway inhibitor combinations exhibit T-cell exclusion. We found that MEK and CDK4/6 targeting was more effective at delaying regrowth of mutant BRAF melanoma in immunocompetent versus immune-deficient mice. Although MEK inhibitor (MEKi) treatment increased tumor immunogenicity and intratumoral recruitment of CD8+ T cells, the main effect of CDK4/6i alone and in combination with MEKi was increased expression of CD137L, a T-cell costimulatory molecule on immune cells. Depletion of CD8+ T cells or blockade of the CD137 ligand-receptor interaction reduced time to regrowth of melanomas in the context of treatment with CDK4/6i plus MEKi treatment in vivo Together, our data outline an antitumor immune-based mechanism and show the efficacy of targeting both the MEK pathway and CDK4/6.

Cardiovascular Adverse Events Associated With BRAF and MEK Inhibitors: A Systematic Review and Meta-analysis.

Importance: Cardiovascular adverse events (CVAEs) after treatment with BRAF and MEK inhibitors in patients with melanoma remain incompletely characterized. Objective: To determine the association of BRAF and MEK inhibitor treatment with CVAEs in patients with melanoma compared with BRAF inhibitor monotherapy. Data Sources: PubMed, Cochrane, and Web of Science were systematically searched for keywords vemurafenib, dabrafenib, encorafenib, trametinib, binimetinib, and cobinimetinib from database inception through November 30, 2018. Study Selection: Randomized clinical trials reporting on CVAEs in patients with melanoma being treated with BRAF and MEK inhibitors compared with patients with melanoma being treated with BRAF inhibitor monotherapy were selected. Data Extraction and Synthesis: Data assessment followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. Pooled relative risks (RRs) and 95% CIs were determined using random-effects and fixed-effects analyses. Subgroup analyses were conducted to assess study-level characteristics associated with CVAEs. Main Outcomes and Measures: The selected end points were pulmonary embolism, a decrease in left ventricular ejection fraction, arterial hypertension, myocardial infarction, atrial fibrillation, and QTc interval prolongation. All-grade and high-grade (≥3) CVAEs were recorded. Results: Overall, 5 randomized clinical trials including 2317 patients with melanoma were selected. Treatment with BRAF and MEK inhibitors was associated with an increased risk of pulmonary embolism (RR, 4.36; 95% CI, 1.23-15.44; P = .02), a decrease in left ventricular ejection fraction (RR, 3.72; 95% CI, 1.74-7.94; P < .001), and arterial hypertension (RR, 1.49; 95% CI, 1.12-1.97; P = .005) compared with BRAF inhibitor monotherapy. The RRs for myocardial infarction, atrial fibrillation, and QTc prolongation were similar between the groups. These results were consistent when assessing high-grade CVAEs (left ventricular ejection fraction: RR, 2.79; 95% CI, 1.36-5.73; P = .005; I2 = 29%; high-grade arterial hypertension: RR, 1.54; 95% CI, 1.14-2.08; P = .005; I2 = 0%), but RRs for high-grade pulmonary embolism were similar between groups. A higher risk of a decrease in left ventricular ejection fraction was associated with patients with a mean age younger than 55 years (RR, 26.50; 95% CI, 3.58-196.10; P = .001), and the associated risk of pulmonary embolism was higher for patients with a mean follow-up time longer than 15 months (RR, 7.70; 95% CI, 1.40-42.12; P = .02). Conclusions and Relevance: Therapy with BRAF and MEK inhibitors was associated with a higher risk of CVAEs compared with BRAF inhibitor monotherapy. The findings may help to balance between beneficial melanoma treatment and cardiovascular morbidity and mortality.

SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.

Neurofibromatosis type 1: State-of-the-art review with emphasis on pulmonary involvement.

Neurofibromatosis type 1 (NF-1), also known as von Recklinghausen's disease, is an autosomal dominant dysplasia of the ectoderm and mesoderm with a variable clinical expression, but near-complete penetrance before the age of 5 years. The estimated incidence is 1 in 3000 births. NF-1 is characterized by collections of neurofibromas, café-au-lait spots, axillary and inguinal freckling, and pigmented hamartomas in the iris (Lisch nodules). Pulmonary manifestations of NF-1, which usually include bilateral basal reticulations and apical bullae and cysts, are reported in 10-20% of adult patients. Clinically, neurofibromatosis-associated diffuse lung disease (NF-DLD) usually presents with nonspecific respiratory symptoms, including dyspnea on exertion, shortness of breath, and chronic cough or chest pain, at the time of diagnosis. Computed tomography (CT) is highly accurate for the identification and characterization of NF-DLD; it is the most reliable method for the diagnosis of this lung involvement. Various CT findings of NF-DLD, including cysts, bullae, ground-glass opacities, bibasilar reticular opacities, and emphysema, have been described in patients with NF-1. The typical CT pattern, however, is characterized by upper-lobe cystic and bullous disease, and basilar interstitial lung disease. Currently, the goal of NF-DLD treatment is the earliest possible diagnosis, focusing on symptom relief and interventions that positively alter the course of the disease, such as smoking cessation. The aim of this review is to describe the main clinical, pathological, and imaging aspects of NF-1, with a focus on pulmonary involvement.

Xanthogranulomatous reaction to trametinib for metastatic malignant melanoma.

Trametinib, a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, has demonstrated great promise in treating metastatic melanoma associated with BRAF V600E and V600K mutations; however, it also is highly associated with cutaneous adverse events (AEs). As both BRAF and MEK inhibitors become increasingly used to treat malignant melanoma, it is important to better characterize these AEs so that we can manage them. Herein, we present a case of a 66-year-old man who developed erythematous scaly papules on the face and bilateral upper extremities after beginning therapy with trametinib. The severity of the reaction worsened on trametinib monotherapy compared to combination therapy with a BRAF inhibitor. Biopsy revealed a xanthogranulomatous reaction.

Discovery of the cancer cell selective dual acting anti-cancer agent (Z)-2-(1H-indol-3-yl)-3-(isoquinolin-5-yl)acrylonitrile (A131).

Selective targeting of cancer cells over normal cells is a key objective of targeted therapy. However few approaches achieve true mechanistic selectivity resulting in debilitating side effects and dose limitation. In this work we describe the discovery of A131 (4a), a new agent with an unprecedented dual mechanism of action targeting both mitosis and autophagy. Compound 4a was first identified in a phenotypic screen in which HeLa cells treated with 4a manifested mitotic arrest along with formation of multiple vesicles. Further investigations showed that 4a causes an increase in mitotic marker pH3 and autophagy marker LC3. Importantly 4a induces cell death in cancer cells while sparing normal cells which regrow after 4a is removed. Dual activities against pH3 and LC3 markers are required for cancer cell selectivity. An extensive SAR investigation confirmed 4a as the optimal dual inhibitor with potency against a panel of 30 cancer cell lines (average antiproliferative GI50 1.5 µM). In a mouse model of paclitaxel-resistant colon cancer, 4a showed 74% tumor growth inhibition when administered at a dose of 20 mg/kg IP twice a day.

Efficacy of MEK inhibition in a K-Ras-driven cholangiocarcinoma preclinical model.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly malignancy with limited treatment options. Gain-of-function mutations in K-Ras is a very frequent alteration, occurring in ~15 to 25% of human iCCA patients. Here, we established a new iCCA model by expressing activated forms of Notch1 (NICD) and K-Ras (K-RasV12D) in the mouse liver (K-Ras/NICD mice). Furthermore, we investigated the therapeutic potential of MEK inhibitors in vitro and in vivo using human CCA cell lines and K-Ras/NICD mice, respectively. Treatment with U0126, PD901, and Selumetinib MEK inhibitors triggered growth restraint in all CCA cell lines tested, with the most pronounced growth suppressive effects being observed in K-Ras mutant cells. Growth inhibition was due to reduction in proliferation and massive apoptosis. Furthermore, treatment of K-Ras/NICD tumor-bearing mice with PD901 resulted in stable disease. At the molecular level, PD901 efficiently inhibited ERK activation in K-Ras/NICD tumor cells, mainly leading to increased apoptosis. Altogether, our study demonstrates that K-Ras/NICD mice represent a novel and useful preclinical model to study K-Ras-driven iCCA development and the effectiveness of MEK inhibitors in counteracting this process. Our data support the usefulness of MEK inhibitors for the treatment of human iCCA.

Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality.

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131's ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a Ras˧PIK3IP1˧PI3K signaling network.

Identification of (Z)-2,3-Diphenylacrylonitrileas Anti-Cancer Molecule in Persian Gulf Sea Cucumber Holothuria parva.

Hepatocellular carcinoma (HCC), also named cancerous hepatoma, is the most common type of malignant neoplasia of the liver. In this research, we screened the Persian Gulf sea cucumber Holothuria parva (H. parva) methanolic sub-fractions for the possible existence of selective toxicity on liver mitochondria isolated from an animal model of HCC. Next, we purified the most active fraction. Thus the structure of the active molecule was identified. HCC was induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) protocol. Rat liver mitochondria for evaluation of the selective cytotoxic effects of sub-fractions of H. parva were isolated and then mitochondrial parameters were determined. Our results showed that C1 sub-fraction of methanolic extract of H. parva considerably increased reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), swelling in mitochondria and cytochrome c release only on HCC liver mitochondria. Furthermore, the methanolic extract of H. parva was investigated furthermore and the active fraction was extracted. In this fraction, (Z)-2,3-diphenylacrylonitrile molecule, which is also known as α-cyanostilbene, was identified by mass analysis. This molecule increased ROS generation, collapse of MMP, swelling in mitochondria and finally cytochrome c release only on HCC liver mitochondria. The derivatives of (Z)-2,3-diphenylacrylonitrile in other natural products were also reported as an anti-cancer agent. These results suggest the eligibility of the (Z)-2,3-diphenylacrylonitrile as a complementary therapeutic agent for patients with HCC.

Trypanosoma cruzi cell death induced by the Morita-Baylis-Hillman adduct 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile).

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a serious health concern due to the lack of effective vaccines or satisfactory treatment. In the search for new compounds against this neglected disease, we have previously demonstrated that the compound 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile) (MBHA3), derived from the Morita-Baylis-Hillman reaction, effectively caused a loss of viability in both the epimastigote and trypomastigote forms. However, the mechanisms of parasite death elicited by MBHA3 remain unknown. The aim of this study was to better understand the morphophysiological changes and the mechanism of cell death induced by MBHA3 treatment on T. cruzi. To perform this analysis, we used confocal microscopy and flow cytometry to monitor the fluorescent probes such as annexin-V/propidium iodide (AV/PI), calcein-AM/ethidium homodimer (CA/EH), acridine orange (AO) and rhodamine 123 (Rho 123). Lower concentrations of MBHA3 led to alterations in the mitochondrial membrane potential and AO labeling, but did not decrease the viability of the epimastiogote forms, as determined by the CA/EH and AV/PI assays. Conversely, treatment with higher concentrations of MBHA3 led to extensive plasma membrane damage, loss of mitochondrion membrane potential, DNA fragmentation and acidification of the cytoplasm. Our findings suggest that at higher concentrations, MBHA3 induces T. cruzi epimastigote death by necrosis in a mitochondrion-dependent manner.

Synthesis and biological evaluation of sulfonyl acrylonitriles as novel inhibitors to peritoneal carcinomatosis.

The vast majority of cancer patients die from metastasis, the process by which cancer cells spread to secondary tissues through body fluids. Peritoneal carcinomatosis is a type of metastasis in which cancer cells gain access to the intra-abdominal cavity and then implant in the peritoneum, the thin tissue that lines the abdominal wall and internal organs. Unfortunately, peritoneal carcinomatosis can occur following surgical resection of intra-abdominal malignancies. We previously reported proapoptotic activity of (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile (BAY 11-7085, 1) on colon and pancreatic cancer cells during adhesion and demonstrated that this compound could significantly inhibit peritoneal carcinomatosis in mice.(1,2) In order to determine the chemical basis of the anti-metastatic properties of BAY 11-7085, a series of analogs were synthesized and evaluated for their ability to induce apoptosis in pancreatic and ovarian cancer cells during adhesion to mesothelial cells, which line the surface of the peritoneum. The co-culture assay results were validated using a murine peritoneal carcinomatosis model. These analogs may greatly benefit patients undergoing surgical resections of colorectal, pancreatic, and ovarian cancers depending on their tolerability.

Therapeutic effect of (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) against Staphylococcus aureus infection in a murine model.

Sortase enzymes belong to a family of transpeptidases found in Gram-positive bacteria. Sortase is responsible for the reaction that anchors surface protein virulence factors to the peptidoglycan cell wall of the bacteria. The compound (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) has previously been reported as a novel sortase inhibitor in vitro, but the in vivo effects of DMMA have not been studied. Here, we evaluated the in vivo effects of DMMA against infection by wild-type and sortase A- and/or sortase B-deficient Staphylococcus aureus in Balb/c mice. With DMMA treatment, survival rates increased and kidney and joint infection rates decreased (p<0.01) in a dose-dependent manner. The rate of kidney infection was significantly reduced in the mice treated with sortase A knock-out S. aureus (p<0.01). These results indicate that by acting as a potent inhibitor of sortase A and moderate inhibitor of sortase B, DMMA can decrease kidney and joint infection rates and reduce mortality in mice infected with S. aureus. These findings suggest that DMMA is a promising therapeutic compound against Gram-positive bacteria.