Phenotyping of cancer-associated somatic mutations in the BCL2 transmembrane domain.

The BCL2 family of proteins controls cell death by modulating the permeabilization of the mitochondrial outer membrane through a fine-tuned equilibrium of interactions among anti- and pro-apoptotic members. The upregulation of anti-apoptotic BCL2 proteins represents an unfavorable prognostic factor in many tumor types due to their ability to shift the equilibrium toward cancer cell survival. Furthermore, cancer-associated somatic mutations in BCL2 genes interfere with the protein interaction network, thereby promoting cell survival. A range of studies have documented how these mutations affect the interactions between the cytosolic domains of BCL2 and evaluate the impact on cell death; however, as the BCL2 transmembrane interaction network remains poorly understood, somatic mutations affecting transmembrane regions have been classified as pathogenic-based solely on prediction algorithms. We comprehensively investigated cancer-associated somatic mutations affecting the transmembrane domain of BCL2 proteins and elucidated their effect on membrane insertion, hetero-interactions with the pro-apoptotic protein BAX, and modulation of cell death in cancer cells. Our findings reveal how specific mutations disrupt switchable interactions, alter the modulation of apoptosis, and contribute to cancer cell survival. These results provide experimental evidence to distinguish BCL2 transmembrane driver mutations from passenger mutations and provide new insight regarding selecting precision anti-tumor treatments.

Exploring the neuroprotective potential of antimicrobial peptides from Dinoponera quadriceps venom against pentylenetetrazole-induced seizures in vivo.

Epilepsy affects around 50 million people worldwide and 30% of patients have difficulty controlling the disease. The search for substances that can fill the existing gaps in the treatment of epilepsy is of great importance. Arthropod venoms are promising sources for this purpose due to the presence of small peptides that modulate the activity of ion channels and neuron receptors. The aim of this study was to investigate dinoponeratoxins from the Dinoponera quadriceps ant venom (M-PONTX-Dq3a, M-PONTX-Dq3b and M-PONTX-Dq3c) as potential anticonvulsants. We evaluated them in a seizure model induced by pentylenetetrazole (PTZ) in male swiss mice. Interestingly, intraperitoneal treatment with each peptide increased the time until the first seizure and the percentage of survival, with M-PONTX-Dq3b showing the best results. M-PONTX-Dq3a was discarded due to the appearance of some signs of toxicity with the increase in malondialdehyde (MDA) levels in the striatum. Both, M-PONTX-Dq3b and M-PONTX-Dq3c decreased iNOS and TNF-α in the hippocampus. Notably, M-PONTX-Dq3c treatment decreased the levels of MDA and nitrite in the cortex and hippocampus. Our results indicate that, M-PONTX-Dq3b and M-PONTX-Dq3c have anticonvulsant activity and exhibit anti-inflammatory effects in epilepsy, offering new perspectives for biopharmaceutical development.

Targeted Delivery of the Pan-Inflammasome Inhibitor MM01 as an Alternative Approach to Acute Lung Injury Therapy.

Acute lung injury (ALI) is a severe pulmonary disorder responsible for high percentage of mortality and morbidity in intensive care unit patients. Current treatments are ineffective, so the development of efficient and specific therapies is an unmet medical need. The activation of NLPR3 inflammasome during ALI produces the release of proinflammatory factors and pyroptosis, a proinflammatory form of cell death that contributes to lung damage spreading. Herein, it is demonstrated that modulating inflammasome activation through inhibition of ASC oligomerization by the recently described MM01 compound can be an alternative pharmacotherapy against ALI. Besides, the added efficacy of using a drug delivery nanosystem designed to target the inflamed lungs is determined. The MM01 drug is incorporated into mesoporous silica nanoparticles capped with a peptide (TNFR-MM01-MSNs) to target tumor necrosis factor receptor-1 (TNFR-1) to proinflammatory macrophages. The prepared nanoparticles can deliver the cargo in a controlled manner after the preferential uptake by proinflammatory macrophages and exhibit anti-inflammatory activity. Finally, the therapeutic effect of MM01 free or nanoparticulated to inhibit inflammatory response and lung injury is successfully demonstrated in lipopolysaccharide-mouse model of ALI. The results suggest the potential of pan-inflammasome inhibitors as candidates for ALI therapy and the use of nanoparticles for targeted lung delivery.

High-Capacity Mesoporous Silica Nanocarriers of siRNA for Applications in Retinal Delivery.

The main cause of subretinal neovascularisation in wet age-related macular degeneration (AMD) is an abnormal expression in the retinal pigment epithelium (RPE) of the vascular endothelial growth factor (VEGF). Current approaches for the treatment of AMD present considerable issues that could be overcome by encapsulating anti-VEGF drugs in suitable nanocarriers, thus providing better penetration, higher retention times, and sustained release. In this work, the ability of large pore mesoporous silica nanoparticles (LP-MSNs) to transport and protect nucleic acid molecules is exploited to develop an innovative LP-MSN-based nanosystem for the topical administration of anti-VEGF siRNA molecules to RPE cells. siRNA is loaded into LP-MSN mesopores, while the external surface of the nanodevices is functionalised with polyethylenimine (PEI) chains that allow the controlled release of siRNA and promote endosomal escape to facilitate cytosolic delivery of the cargo. The successful results obtained for VEGF silencing in ARPE-19 RPE cells demonstrate that the designed nanodevice is suitable as an siRNA transporter.

Combination of palbociclib with navitoclax based-therapies enhances in vivo antitumoral activity in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with a poor prognosis and limited effective therapeutic options. Induction of senescence, arrest of cell proliferation, has been explored as an effective method to limit tumor progression in metastatic breast cancer. However, relapses occur in some patients, possibly as a result of the accumulation of senescent tumor cells in the body after treatment, which promote metastasis. In this study, we explored the combination of senescence induction and the subsequent removal of senescent cells (senolysis) as an alternative approach to improve outcomes in TNBC patients. We demonstrate that a combination treatment, using the senescence-inducer palbociclib and the senolytic agent navitoclax, delays tumor growth and reduces metastases in a mouse xenograft model of aggressive human TNBC (hTNBC). Furthermore, considering the off-target effects and toxicity derived from the use of navitoclax, we propose a strategy aimed at minimizing the associated side effects. We use a galacto-conjugated navitoclax (nav-Gal) as a senolytic prodrug that can preferentially be activated by ß-galactosidase overexpressed in senescent cells. Concomitant treatment with palbociclib and nav-Gal in vivo results in the eradication of senescent hTNBC cells with consequent reduction of tumor growth, while reducing the cytotoxicity of navitoclax. Taken together, our results support the efficacy of combination therapy of senescence-induction with senolysis for hTNBC, as well as the development of a targeted approach as an effective and safer therapeutic opportunity.

Rational design of a trypanocidal peptide derived from Dinoponera quadriceps venom.

Chagas disease is caused by the parasite Trypanosoma cruzi and affects millions of people worldwide, having no effective cure. The main sanitary emergency is related to patients with chronic infection, which accumulate comorbidities causing patient death. However, actual chemotherapeutic treatments do not effectively address the chronic forms of the disease. Invertebrates are a relevant source of antimicrobial peptides (AMPs) as part of the innate immune system for their protection. The AMP M-PONTX-Dq3a, isolated from the Dinoponera quadriceps ant venom, has shown very effective antimicrobial and trypanocidal activities. Although M-PONTX-Dq3a has better activity that the current therapies, the peptide length has limited its possibilities to reach clinical application. In this investigation, we aimed to dissect the trypanocidal effect of M-PONTX-Dq3a fragments and to study the activity of substituted analogs, to improve not only peptide trypanocidal activity and bioavailability, but also production costs. Our studies have led to the identification of two smaller peptides, M-PONTX-Dq3a [1-15] and [Lys]3-M-PONTX-Dq3a [3-153-15 with similar trypanocidal activities that the parent peptide has against the three forms of T. cruzi benznidazole-resistant Y strain. Both peptides represent promising candidates to develop novel and effective trypanocidal bio-therapeutic agents, opening new avenues for the treatment of chronic patients.

Pharmacological senolysis reduces doxorubicin-induced cardiotoxicity and improves cardiac function in mice.

Many anticancer agents used in clinics induce premature senescence in healthy tissues generating accelerated aging processes and adverse side-effects in patients. Cardiotoxicity is a well-known limiting factor of anticancer treatment with doxorubicin (DOX), a very effective anthracycline widely used as antitumoral therapy in clinical practice, that leads to long-term morbidity and mortality. DOX exposure severely affects the population of cardiac cells in both mice and human hearts by inducing premature senescence, which may represent the molecular basis of DOX-induced cardiomyopathy. Here, we demonstrate that senescence induction in the heart contributes to impaired cardiac function in mice upon DOX treatment. Concomitant elimination of senescent cells with the senolytic Navitoclax in different formulations produces a significant decrease in senescence and cardiotoxicity markers together with the restoration of the cardiac function in mice followed by echocardiography. These results evidence the potential clinical use of senolytic therapies to alleviate cardiotoxicities induced in chemotherapy-treated patients.

Horseradish Peroxidase-Functionalized Gold Nanoconjugates for Breast Cancer Treatment Based on Enzyme Prodrug Therapy.

INTRODUCTION: Breast cancer has the highest mortality rate among cancers in women. Patients suffering from certain breast cancers, such as triple-negative breast cancer (TNBC), lack effective treatments. This represents a clinical concern due to the associated poor prognosis and high mortality. As an approach to succeed over conventional therapy limitations, we present herein the design and evaluation of a novel nanodevice based on enzyme-functionalized gold nanoparticles to efficiently perform enzyme prodrug therapy (EPT) in breast cancer cells. RESULTS: In particular, the enzyme horseradish peroxidase (HRP) - which oxidizes the prodrug indole-3-acetic acid (IAA) to release toxic oxidative species - is incorporated on gold nanoconjugates (HRP-AuNCs), obtaining an efficient nanoplatform for EPT. The nanodevice is biocompatible and effectively internalized by breast cancer cell lines. Remarkably, co-treatment with HRP-AuNCs and IAA (HRP-AuNCs/IAA) reduces the viability of breast cancer cells below 5%. Interestingly, 3D tumor models (multicellular tumor spheroid-like cultures) co-treated with HRP-AuNCs/IAA exhibit a 74% reduction of cell viability, whereas the free formulated components (HRP, IAA) have no effect. CONCLUSION: Altogether, our results demonstrate that the designed HRP-AuNCs nanoformulation shows a remarkable therapeutic performance. These findings might help to bypass the clinical limitations of current tumor enzyme therapies and advance towards the use of nanoformulations for EPT in breast cancer.

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity.

Inflammasomes are multiprotein complexes that represent critical elements of the inflammatory response. The dysregulation of the best-characterized complex, the NLRP3 inflammasome, has been linked to the pathogenesis of diseases such as multiple sclerosis, type 2 diabetes mellitus, Alzheimer's disease, and cancer. While there exist molecular inhibitors specific for the various components of inflammasome complexes, no currently reported inhibitors specifically target NLRP3PYD homo-oligomerization. In the present study, we describe the identification of QM380 and QM381 as NLRP3PYD homo-oligomerization inhibitors after screening small molecules from the MyriaScreen library using a split-luciferase complementation assay. Our results demonstrate that these NLRP3PYD inhibitors interfere with ASC speck formation, inhibit pro-inflammatory cytokine IL1-ß release, and decrease pyroptotic cell death. We employed spectroscopic techniques and computational docking analyses with QM380 and QM381 and the PYD domain to confirm the experimental results and predict possible mechanisms underlying the inhibition of NLRP3PYD homo-interactions.

Understanding MCL1: from cellular function and regulation to pharmacological inhibition.

Myeloid cell leukemia-1 (MCL1), an antiapoptotic member of the BCL2 family characterized by a short half-life, functions as a rapid sensor that regulates cell death and other relevant processes that include cell cycle progression and mitochondrial homeostasis. In cancer, MCL1 overexpression contributes to cell survival and resistance to diverse chemotherapeutic agents; for this reason, several MCL1 inhibitors are currently under preclinical and clinical development for cancer treatment. However, the nonapoptotic functions of MCL1 may influence their therapeutic potential. Overall, the complexity of MCL1 regulation and function represent challenges to the clinical application of MCL1 inhibitors. We now summarize the current knowledge regarding MCL1 structure, regulation, and function that could impact the clinical success of MCL1 inhibitors.

Gene-Directed Enzyme Prodrug Therapy by Dendrimer-Like Mesoporous Silica Nanoparticles against Tumor Cells.

We report herein a gene-directed enzyme prodrug therapy (GDEPT) system using gated mesoporous silica nanoparticles (MSNs) in an attempt to combine the reduction of side effects characteristic of GDEPT with improved pharmacokinetics promoted by gated MSNs. The system consists of the transfection of cancer cells with a plasmid controlled by the cytomegalovirus promoter, which promotes ß-galactosidase (ß-gal) expression from the bacterial gene lacZ (CMV-lacZ). Moreover, dendrimer-like mesoporous silica nanoparticles (DMSNs) are loaded with the prodrug doxorubicin modified with a galactose unit through a self-immolative group (DOXO-Gal) and modified with a disulfide-containing polyethyleneglycol gatekeeper. Once in tumor cells, the reducing environment induces disulfide bond rupture in the gatekeeper with the subsequent DOXO-Gal delivery, which is enzymatically converted by ß-gal into the cytotoxic doxorubicin drug, causing cell death. The combined treatment of the pair enzyme/DMSNs-prodrug are more effective in killing cells than the free prodrug DOXO-Gal alone in cells transfected with ß-gal.

The past, present, and future of breast cancer models for nanomedicine development.

Even given recent advances in nanomedicine development of breast cancer treatment in recent years and promising results in pre-clinical models, cancer nanomedicines often fail at the clinical trial stage. Limitations of conventional in vitro models include the lack of representation of the stromal population, the absence of a three-dimensional (3D) structure, and a poor representation of inter-tumor and intra-tumor heterogeneity. Herein, we review those cell culture strategies that aim to overcome these limitations, including cell co-cultures, advanced 3D cell cultures, patient-derived cells, bioprinting, and microfluidics systems. The in vivo evaluation of nanomedicines must consider critical parameters that include the enhanced permeability and retention effect, the host's immune status, and the site of tumor implantation. Here, we critically discuss the advantages and limitations of current in vivo models and report how the improved selection and application of breast cancer models can improve the clinical translation of nanomedicines.

BOK-MCL1 transmembrane interactions: a challenging target for cancer therapy.

Myeloid cell leukemia 1 (MCL1) gene amplification occurs in a wide range of human cancers and protein overexpression associates with malignant cell growth and evasion of apoptosis. We recently reported that disrupting the interaction between the transmembrane domains of MCL1 and BCL-2 related ovarian killer (BOK) induces cell death, thereby suggesting a new target site for anti-tumorigenic strategies.

Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis.

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

MUC1 Aptamer-Capped Mesoporous Silica Nanoparticles for Navitoclax Resistance Overcoming in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. In the last years, navitoclax has emerged as a possible treatment for TNBC. Nevertheless, rapid navitoclax resistance onset has been observed thorough Mcl-1 overexpression. As a strategy to overcome Mcl-1-mediated resistance, herein we present a controlled drug co-delivery system based on mesoporous silica nanoparticles (MSNs) targeted to TNBC cells. The nanocarrier is loaded with navitoclax and the Mcl-1 inhibitor S63845 and capped with a MUC1-targeting aptamer (apMUC1-MSNs(Nav/S63845)). The apMUC1-capped nanoparticles effectively target TNBC cell lines and successfully induce apoptosis, overcoming navitoclax resistance. Moreover, navitoclax encapsulation protects platelets against apoptosis. These results point apMUC1-gated MSNs as suitable BH3 mimetics nanocarriers in the targeted treatment of MUC1-expressing TNBC.

Preclinical antitumor efficacy of senescence-inducing chemotherapy combined with a nanoSenolytic.

The induction of senescence produces a stable cell cycle arrest in cancer cells, thereby inhibiting tumor growth; however, the incomplete immune cell-mediated clearance of senescent cells may favor tumor relapse, limiting the long-term anti-tumorigenic effect of such drugs. A combination of senescence induction and the elimination of senescent cells may, therefore, represent an efficient means to inhibit tumor relapse. In this study, we explored the antitumor efficacy of a combinatory senogenic and targeted senolytic therapy in an immunocompetent orthotopic mouse model of the aggressive triple negative breast cancer subtype. Following palbociclib-induced senogenesis and senolysis by treatment with nano-encapsulated senolytic agent navitoclax, we observed inhibited tumor growth, reduced metastases, and a reduction in the systemic toxicity of navitoclax. We believe that this combination treatment approach may have relevance to other senescence-inducing chemotherapeutic drugs and additional tumor types. SIGNIFICANCE: While the application of senescence inducers represents a successful treatment strategy in breast cancer patients, some patients still relapse, perhaps due to the subsequent accumulation of senescent cells in the body that can promote tumor recurrence. We now demonstrate that a combination treatment of a senescence inducer and a senolytic nanoparticle selectively eliminates senescent cells, delays tumor growth, and reduces metastases in a mouse model of aggressive breast cancer. Collectively, our results support targeted senolysis as a new therapeutic opportunity to improve outcomes in breast cancer patients.

Real-Time In†Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue.

In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR-FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide (S3). NB emission at 672 nm is highly quenched inside S3, yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of ß-galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK-Mel-103 and breast cancer 4T1 cells and in vivo in palbociclib-treated BALB/cByJ mice bearing breast cancer tumor.

A NIR light-triggered drug delivery system using core-shell gold nanostars-mesoporous silica nanoparticles based on multiphoton absorption photo-dissociation of 2-nitrobenzyl PEG.

Gold nanostars coated with a mesoporous silica shell and functionalized with poly(ethylene glycol) containing photolabile 2-nitrobenzyl moieties are able to release doxorubicin after NIR light irradiation at low power irradiance via a multiphoton absorption photo-dissociation process.

EU-OPENSCREEN: A Novel Collaborative Approach to Facilitate Chemical Biology.

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN's compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3 H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

Hybrid Mesoporous Nanocarriers Act by Processing Logic Tasks: Toward the Design of Nanobots Capable of Reading Information from the Environment.

Here, we present the design of smart nanodevices capable of reading molecular information from the environment and acting accordingly by processing Boolean logic tasks. As proof of concept, we prepared Au-mesoporous silica (MS) nanoparticles functionalized with the enzyme glucose dehydrogenase (GDH) on the Au surface and with supramolecular nanovalves as caps on the MS surface, which is loaded with a cargo (dye or drug). The nanodevice acts as an AND logic gate and reads information from the solution (presence of glucose and nicotinamide adenine dinucleotide (NAD+)), which results in cargo release. We show the possibility of coimmobilizing GDH and the enzyme urease on nanoparticles to mimic an INHIBIT logic gate, in which the AND gate is switched off by the presence of urea. We also show that such nanodevices can deliver cytotoxic drugs in cancer cells by recognizing intracellular NAD+ and the presence of glucose.