TAp73 represses NF-κB-mediated recruitment of tumor-associated macrophages in breast cancer.

Infiltration of tumor-promoting immune cells is a strong driver of tumor progression. Especially the accumulation of macrophages in the tumor microenvironment is known to facilitate tumor growth and to correlate with poor prognosis in many tumor types. TAp73, a member of the p53/p63/p73 family, acts as a tumor suppressor and has been shown to suppress tumor angiogenesis. However, what role TAp73 has in regulating immune cell infiltration is unknown. Here, we report that low levels of TAp73 correlate with an increased NF-κB-regulated inflammatory signature in breast cancer. Furthermore, we show that loss of TAp73 results in NF-κB hyperactivation and secretion of Ccl2, a known NF-κB target and chemoattractant for monocytes and macrophages. Importantly, TAp73-deficient tumors display an increased accumulation of protumoral macrophages that express the mannose receptor (CD206) and scavenger receptor A (CD204) compared to controls. The relevance of TAp73 expression in human breast carcinoma was further accentuated by revealing that TAp73 expression correlates negatively with the accumulation of protumoral CD163+ macrophages in breast cancer patient samples. Taken together, our findings suggest that TAp73 regulates macrophage accumulation and phenotype in breast cancer through inhibition of the NF-κB pathway.

Visible Light and Triselenium Dicyanide (TSD): New Horizons in the Synthesis of Organic Selenocyanates.

Herein, we describe a new method for the synthesis of α-carbonyl selenocyanates by reacting triselenium dicyanide (TSD) and styrenes under blue light irradiation and O2 atmosphere. The reactions are triggered by the formation of Se-centered radical species, followed by the addition/oxidation of the styrene π-bond. α-Carbonyl selenocyanates and α-hydroxy selenocyanates were obtained in moderate to excellent yields from aryl- and alkyl-substituted alkenes, respectively. It was demonstrated that α-carbonyl selenocyanates could be used as a synthetic platform in a multicomponent reaction strategy to prepare 2-phenylimidazo[1,2-a]pyridine derivatives, which were evaluated for their photophysical properties. Overall, this new method provides a useful tool for synthesizing α-carbonyl selenocyanates, and demonstrates their potential for use in the synthesis of other compounds, thus giving new synthetic opportunities to construct organic selenocyanate compounds.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

Microstructured Polymer System Containing Proanthocyanidin-Enriched Extract from Limonium brasiliense as a Prophylaxis Strategy to Prevent Recurrence of Porphyromonas gingivalis.

Periodontal diseases are a global oral health problem affecting almost 10% of the global population. Porphyromonas gingivalis is one of the main bacteria involved in the initiation and progression of inflammatory processes as a result of the action of the cysteine proteases lysin- and arginine-gingipain. Surelease/polycarbophil microparticles containing a lyophilized proanthocyanidin-enriched fraction from the rhizomes of Limonium brasiliense, traditionally named "baicuru" (ethyl acetate fraction), were manufactured. The ethyl acetate fraction was characterized by UHPLC by the presence of samarangenins A and B (12.10 ± 0.07 and 21.05 ± 0.44%, respectively) and epigallocatechin-3-O-gallate (13.44 ± 0.27%). Physiochemical aspects of Surelease/polycarbophil microparticles were characterized concerning particle size, zeta potential, entrapment efficiency, ethyl acetate fraction release, and mucoadhesion. Additionally, the presence of the ethyl acetate fraction-loaded microparticles was performed concerning potential influence on viability of human buccal KB cells, P. gingivalis adhesion to KB cells, gingipain activity, and P. gingivalis biofilm formation. In general, all Surelease/polycarbophil microparticles tested showed strong adhesion to porcine cheek mucosa (93.1 ± 4.2% in a 30-min test), associated with a prolonged release of the ethyl acetate fraction (up to 16.5 ± 0.8% in 24 h). Preincubation of KB cells with Surelease/polycarbophil microparticles (25 µg/mL) resulted in an up to 93 ± 2% reduced infection rate by P. gingivalis. Decreased activity of the P. gingivalis-specific virulence factors lysin- and arginine-gingipain proteases by Surelease/polycarbophil microparticles was confirmed. Surelease/polycarbophil microparticles decreased biofilm formation of P. gingivalis (97 ± 2% at 60 µg/mL). Results from this study prove the promising activity of Surelease/polycarbophil microparticles containing ethyl acetate fraction microparticles as a prophylaxis strategy to prevent the recurrence of P. gingivalis.

MNK2 governs the macrophage antiinflammatory phenotype.

Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.

A hybrid of 1-deoxynojirimycin and benzotriazole induces preferential inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE).

The synthesis of four heterodimers in which the copper(I)-catalysed azide-alkyne cycloaddition was employed to connect a 1-deoxynojirimycin moiety with a benzotriazole scaffold is reported. The heterodimers were investigated as inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The heterodimers displayed preferential inhibition (> 9) of BuChE over AChE in the micromolar concentration range (IC50 = 7-50 µM). For the most potent inhibitor of BuChE, Cornish-Bowden plots were used, which demonstrated that it behaves as a mixed inhibitor. Modelling studies of the same inhibitor demonstrated that the benzotriazole and 1-deoxynojirimycin moiety is accommodated in the peripheral anionic site and catalytic anionic site, respectively, of AChE. The binding mode to BuChE was different as the benzotriazole moiety is accommodated in the catalytic anionic site.

Thermoresponsive Hydrogel-Loading Aluminum Chloride Phthalocyanine as a Drug Release Platform for Topical Administration in Photodynamic Therapy.

Phthalocyanine aluminum chloride (Pc) is a clinically viable photosensitizer (PS) to treat skin lesions worsened by microbial infections. However, this molecule presents a high self-aggregation tendency in the biological fluid, which is an in vivo direct administration obstacle. This study proposed the use of bioadhesive and thermoresponsive hydrogels comprising triblock-type Pluronic F127 and Carbopol 934P (FCarb) as drug delivery platforms of Pc (FCarbPc)-targeting topical administration. Carbopol 934P was used to increase the F127 hydrogel adhesion on the skin. Rheological analyses showed that the Pc presented a low effect on the hydrogel matrix, changing the gelation temperature from 27.2 ± 0.1 to 28.5 ± 0.9 °C once the Pc concentration increases from zero to 1 mmol L-1. The dermatological platform showed matrix erosion effects with the release of loaded Pc micelles. The permeation studies showed the excellent potential of the FCarb platform, which allowed the partition of the PS into deeper layers of the skin. The applicability of this dermatological platform in photodynamic therapy was evaluated by the generation of reactive species which was demonstrated by chemical photodynamic efficiency assays. The low effect on cell viability and proliferation in the dark was demonstrated by in vitro assays using L929 fibroblasts. The FCarbPc fostered the inhibition of Staphylococcus aureus strain, therefore demonstrating the platform's potential in the treatment of dermatological infections of microbial nature.

Tacrine-sugar mimetic conjugates as enhanced cholinesterase inhibitors.

We have used the Cu(i)-catalyzed azide-alkyne Huisgen cycloaddition reaction to obtain two families of bivalent heterodimers where tacrine is connected to an azasugar or iminosugar, respectively, via linkers of variable length. The heterodimers were investigated as cholinesterase inhibitors and it was found that their activity increased with the length of the linker. Two of the heterodimers were significantly stronger acetylcholinesterase inhibitors than the monomeric tacrine. Molecular modelling indicated that the longer heterodimers fitted better into the active gorge of acetylcholinesterase than the shorter counterparts and the former provided more efficient simultaneous interaction with the tryptophan residues in the catalytic anionic binding site (CAS) and the peripheral anionic binding site (PAS).

Development of tacrine clusters as positively cooperative systems for the inhibition of acetylcholinesterase.

The synthesis of four tetra-tacrine clusters where the tacrine binding units are attached to a central scaffold via linkers of variable lengths is described. The multivalent inhibition potencies for the tacrine clusters were investigated for the inhibition of acetylcholinesterase. Two of the tacrine clusters displayed a small but significant multivalent inhibition potency in which the binding affinity of each of the tacrine binding units increased up to 3.2 times when they are connected to the central scaffold.

Methylglyoxal disturbs DNA repair and glyoxalase I system in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a highly reactive aldehyde able to form covalent adducts with proteins and nucleic acids, disrupting cellular functions. In this study, we performed a screening of Saccharomyces cerevisiae (S. cerevisiae) strains to find out which genes of cells are responsive to MG, emphasizing genes against oxidative stress and DNA repair. Yeast strains were grown in the YPD-Galactose medium containing MG (0.5 to 12 mM). The tolerance to MG was evaluated by determining cellular growth and cell viability. The toxicity of MG was more pronounced in the strains with deletion in genes engaged with DNA repair checkpoint proteins, namely Rad23 and Rad50. MG also impaired the growth and viability of S. cerevisiae mutant strains Glo1 and Gsh1, both components of the glyoxalase I system. Differently, the strains with deletion in genes encoding for antioxidant enzymes were apparently resistant to MG. In summary, our data indicate that DNA repair and MG detoxification pathways are keys in the control of MG toxicity in S. cerevisiae.

Hetero-Diels-Alder Reactions of Quinone Methides: The Origin of the α-Regioselectivity of 3-Methylene-1,2,4-naphthotriones.

The regioselective formation of α- and ß-lapachone via hetero-Diels-Alder reactions was investigated by experimental and computational approaches. The experimentally observed α-selectivity was explored in detail, revealing that the lower energy barrier for the formation of α-lapachone is a result of lower Pauli repulsion throughout the reaction path, when compared to the ß-isomer. By comparing equivalent points on both α- and ß-lapachone potential energy surfaces (PES), according to the activation strain model (ASM) and energy decomposition analysis (EDA), we were able to demonstrate that the Pauli repulsion term increases more significantly when going from reactants to TSß than to TSα, resulting in lower interaction energy in the early stages of the reaction path and in a later transition state for ß-lapachone. Moreover, we confirmed that regio- and diastereoselectivity trends previously reported for other quinone methide intermediates are also observed for 3-methylene-1,2,4-naphthotriones, such as small endo/exo diastereoselectivity, as well as pronounced ortho/meta regioselectivity for reactions performed with dienophile containing electron-releasing groups. The results presented here provide a deeper understanding of the reactivity of quinone methide derivatives, aiding the future rational design of the reaction condition, structural modification of possible quinone methide intermediates, and the development of more selective synthetic routes for quinone derivatives.

COVID-19: molecular targets, drug repurposing and new avenues for drug discovery.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious infection that may break the healthcare system of several countries. Here, we aimed at presenting a critical view of ongoing drug repurposing efforts for COVID-19 as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. Finally, we also discuss patent protection issues, cost effectiveness and scalability of synthetic routes for some of the most studied repurposing candidates since these are key aspects to meet global demand for COVID-19 treatment.

Improvement of bone repair with l-PRF and bovine bone in calvaria of rats. histometric and immunohistochemical study.

OBJECTIVES: The effect of leucocyte- and platelet-rich fibrin (L-PRF), associated with DBBM (deproteinized bovine bone mineral; Bio-Oss®) was investigated and compared with autogenous bone graft as a standard material for filling bone defects. MATERIAL AND METHODS: A defect of 5 mm in diameter was performed in 40 calvaria of rats. The animals were divided into 5 groups and received blood clot (CO), autogenous bone (AUT), DBBM (BIO), L-PRF, or DBBM associated with L-PRF (BIO-LPRF). After 4 and 8 weeks, bone regeneration was assessed by histometric and immunohistochemical analyses. RESULTS: The highest mean percentage of bone formation found at 4 and 8 weeks was observed for the BIO-L-PRF group (54.0% ± 2.8 and 63.6% ± 2.2). The lowest mean percentage at 4 and 8 weeks was observed for the CO group (16.7% ± 2.5 and 20.5% ± 1.0). There was statistical similarity among the AUT, BIO, and L-PRF groups. The expressions OC, RUNX 2, and VEGF showed a favorable aspect in the formation of new bone for BIO-L-PRF. VEGF was the marker with the highest expression because it was related to the initial healing process, promoting the migration and proliferation of endothelial cells in the region of the defect. Even after weeks, VEGF maintained a moderate expression. CONCLUSIONS: The association of L-PRF with DBBM improved bone repair when these biomaterials were inserted into the defects of the calvaria of rats. CLINICAL RELEVANCE: This reinforces the good performance of bovine bone and L-PRF as filler materials, especially when associated.

The diverse mechanisms and anticancer potential of naphthoquinones.

Cancer is one of the leading causes of death around the world and although the different clinical approaches have helped to increase survival rates, incidence is still high and so its mortality. Chemotherapy is the only approach which is systemic, reaching cancer cells in all body tissues and the search for new potent and selective drugs is still an attractive field within cancer research. Naphthoquinones, natural and synthetic, have garnered much attention in the scientific community due to their pharmacological properties, among them anticancer action, and potential therapeutic significance. Many mechanisms of action have been reported which also depend on structural differences among them. Here, we describe some of the most relevant mechanisms of action reported so far for naphthoquinones and highlight novel targets which are being described in the literature. Furthermore, we gather some of the most impressive efforts done by researchers to harness the anticancer properties of these compounds through specifically designed structural modifications.

Maxillary Alveolar Ridge Atrophy Reconstructed With Autogenous Bone Graft Harvested From the Proximal Ulna.

Atrophy of the alveolar ridge requires bone grafting at the implant site for rehabilitation of the masticatory function with dental implants. Despite the advances in the development of bone substitutes, autogenous bone graft remains the "criterion standard" because of its osteogenic, osteoinductive, osteoconductive potential, and non-immunogenic properties. However, harvesting of autogenous bone is not exempt from donor site morbidity. In this context, the use of autogenous bone derived from the proximal ulna might be a viable resource to obtain corticocancellous bone graft, as the harvesting from this donor site is associated with low morbidity. Thus, this article aimed to describe a case in which a maxillary sinus floor augmentation was performed by means of autogenous bone graft harvested from the proximal ulna, as the donor site. An appositional bone block graft harvested from the same region was used to augment the residual alveolar ridge. Clinically, healing proceeded uneventfully with no major complications. After 8 months, a proper amount of bone was found at the implant site, allowing a prosthetically driven implant insertion and subsequent implant-supported rehabilitation. Results were clinically and radiographically stable after a 3-year follow-up. The present case report suggested that proximal ulna as a donor site should be considered as a safe and reliable alternative for alveolar ridge augmentation. Besides the case presentation, a comprehensive review of the literature was also provided.

Doxycycline and Autogenous Bone in Repair of Critical-Size Defects.

PURPOSE: The association of doxycycline (DOX) and autogenous bone on repair of critical-size defects was evaluated. MATERIALS AND METHODS: Fifty albino rats were divided into 5 groups (n = 10). A 5-mm diameter defect was treated with: control (CO)-blood clot; DOX in Natrosol (NAT)-10% gel; NAT-gel; particulate autogenous bone (PAB); and PAB + DOX - PAB associated with 10% DOX gel. The animals were euthanized at 4 and 8 weeks postoperatively. Histomorphometric analysis was performed to assess the percentage of new bone in the defect area. Statistical analysis of the results was performed using analysis of variance and the Tukey test (P < 0.05). RESULTS: The results showed that new bone formation was limited to the margins of the defect. At 4 and 8 weeks, the group PAB + DOX showed higher bone formation (38.59% and 47.86%, respectively), with statistical difference in comparison with the CO (19.52%) at 4 weeks and CO (18.80%), DOX (22.05%), and NAT (15.89%) at 8 weeks (P < 0.05). CONCLUSIONS: The association of 10% DOX with autogenous bone significantly improved bone healing in critical-size defects.

Healing at implants installed concurrently to maxillary sinus floor elevation with Bio-Oss® or autologous bone grafts. A histo-morphometric study in rabbits.

OBJECTIVE: To compare the healing at elevated sinus floors augmented either with deproteinized bovine bone mineral (DBBM) or autologous bone grafts and followed by immediate implant installation. MATERIAL AND METHODS: Twelve albino New Zealand rabbits were used. Incisions were performed along the midline of the nasal dorsum. The nasal bone was exposed. A circular bony widow with a diameter of 3 mm was prepared bilaterally, and the sinus mucosa was detached. Autologous bone (AB) grafts were collected from the tibia. Similar amounts of AB or DBBM granules were placed below the sinus mucosa. An implant with a moderately rough surface was installed into the elevated sinus bilaterally. The animals were sacrificed after 7 (n = 6) or 40 days (n = 6). RESULTS: The dimensions of the elevated sinus space at the DBBM sites were maintained, while at the AB sites, a loss of 2/3 was observed between 7 and 40 days of healing. The implants showed similar degrees of osseointegration after 7 (7.1 ± 1.7%; 9.9 ± 4.5%) and 40 days (37.8 ± 15%; 36.0 ± 11.4%) at the DBBM and AB sites, respectively. Similar amounts of newly formed mineralized bone were found in the elevated space after 7 days at the DBBM (7.8 ± 6.6%) and AB (7.2 ± 6.0%) sites while, after 40 days, a higher percentage of bone was found at AB (56.7 ± 8.8%) compared to DBBM (40.3 ± 7.5%) sites. CONCLUSIONS: Both Bio-Oss® granules and autologous bone grafts contributed to the healing at implants installed immediately in elevated sinus sites in rabbits. Bio-Oss® maintained the dimensions, while autologous bone sites lost 2/3 of the volume between the two periods of observation.

Sinus mucosa elevation using Bio-Oss® or Gingistat® collagen sponge: an experimental study in rabbits.

OBJECTIVE: To describe the sequential healing in augmented sinus cavities with deproteinized bovine bone mineral (DBBM) granules or collagen sponges. MATERIAL AND METHODS: Twenty albino New Zealand rabbits were included in the study. An incision was performed along the midline of the nasal dorsum, and the nasal bone was exposed. Circular bony windows with a diameter of 5 mm were prepared bilaterally. After elevation of the mucosa, the two sites were randomly filled with either DBBM or a collagen sponge. Five animals each were randomly sacrificed after 7, 14, 21 and 40 days, respectively. RESULTS: At both sides, new bone was found forming from the resident pristine bone. During the first stage of healing, DBBM granules were surrounded by a denser connective tissue that was attached to the biomaterial surface and that was progressively mineralized. At the collagen sponge side, the biomaterial was almost completely resorbed and the space was reduced by to two-thirds already after 21 days. At both sides, after 40 days, mineralized bone and marrow spaces were occupying large areas of the elevated space. CONCLUSIONS: New bone was found forming from the pristine bony walls of the sinus and extending toward the most peripheral regions in both sites. While DBBM particles yielded osteoconductivity and were able to preserve over time the space within the elevated mucosa, the collagen sponge failed the goal of maintaining the space.

Insights into cytochrome bc1 complex binding mode of antimalarial 2-hydroxy-1,4-naphthoquinones through molecular modelling.

BACKGROUND: Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES: The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS: We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS: Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS: Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.