Antibacterial Prodrugs to Overcome Bacterial Antimicrobial Resistance.

Antimicrobial resistance (AMR) is an increasingly concerning phenomenon that requires urgent attention because it poses a threat to human and animal health. Bacteria undergo continuous evolution, acquiring novel resistance mechanisms in addition to their intrinsic ones. Multidrug-resistant and extensively drug-resistant bacterial strains are rapidly emerging, and it is expected that bacterial AMR will claim the lives of 10 million people annually by 2050. Consequently, the urgent need for the development of new therapeutic agents with new modes of action is evident. The antibacterial prodrug approach, a strategy that includes drug repurposing and derivatization, integration of nanotechnology, and exploration of natural products, is highlighted in this review. Thus, this publication aims at compiling the most pertinent research in the field, spanning from 2021 to 2023, offering the reader a comprehensive insight into the AMR phenomenon and new strategies to overcome it.

Alkyl deoxyglycoside-polymyxin combinations against critical priority carbapenem-resistant gram-negative bacteria.

The escalating antimicrobial resistance crisis urges the development of new antibacterial treatments with innovative mechanisms of action, particularly against the critical priority carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA) and Enterobacteriaceae (CRE). Membrane-disrupting dodecyl deoxyglycosides have been reported for their interesting phosphatidylethanolamine-associated bactericidal activity against Gram-positive strains; however, their inability to penetrate the Gram-negative outer membrane (OM) renders them useless against the most challenging pathogens. Aiming to repurpose alkyl deoxyglycosides against Gram-negative bacteria, this study investigates the antimicrobial effects of five reference compounds with different deoxygenation patterns or anomeric configurations in combination with polymyxins as adjuvants for enhanced OM permeability. The generation of the lead 4,6-dideoxy scaffold was optimized through a simultaneous dideoxygenation step and applied to the synthesis of a novel alkyl 4,6-dideoxy C-glycoside 5, herein reported for the first time. When combined with subtherapeutic colistin concentrations, most glycosides demonstrated potent antimicrobial activity against several multidrug-resistant clinical isolates of CRAB, CRE and CRPA exhibiting distinct carbapenem resistance mechanisms, together with acceptable cytotoxicity against human HEK-293T and Caco-2 cells. The novel 4,6-dideoxy C-glycoside 5 emerged as the most promising prototype structure for further development (MIC 3.1 µg/mL when combined with colistin 0.5 µg/mL against CRPA or 0.25 µg/mL against several CRE and CRAB strains), highlighting the potential of C-glycosylation for an improved bioactive profile. This study is the first to show the potential of IM-targeting carbohydrate-based compounds for the treatment of infections caused by MDR Gram-negative pathogens of clinical importance.

Recent antibacterial carbohydrate-based prodrugs, drugs and delivery systems to overcome antimicrobial resistance.

Antimicrobial resistance is an increasing phenomenon that is threatening global health. Tuberculosis causative bacteria and several resistant and multidrug-resistant bacteria are widely spread and listed by the World Health Organization as global priorities for research and development. Hence, new antibacterial agents with new modes of action are urgently required. In this context, carbohydrate-based drugs have been extensively studied and used, presenting several benefits for therapeutical purposes. In this review, the latest efforts done in the carbohydrate-based antibacterial agents research field, reported from 2021 to 2023, are summarized. Carbohydrate-based prodrugs, drugs, and delivery systems are covered, highlighting derivatization of existing antibiotics, use of nanotechnology, and repurposing of available therapeutical agents as the most popular strategies used in antibacterial agents' development.

YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

Recent developments in CFTR modulator drugs have had a significant transformational effect on the treatment of individuals with Cystic Fibrosis (CF) who carry the most frequent F508del-CFTR mutation in at least one allele. However, the clinical effects of these revolutionary drugs remain limited by their inability to fully restore the plasma membrane (PM) stability of the rescued mutant channels. Here, we shed new light on the molecular mechanisms behind the reduced half-life of rescued F508del-CFTR at the PM of airway cells. We describe that YES1 protein kinase is enriched in F508del-CFTR protein PM complexes, and that its interaction with rescued channels is mediated and dependent on the adaptor protein YAP1. Moreover, we show that interference with this complex, either by depletion of one of these components or inhibiting YES1 activity, is sufficient to significantly improve the abundance and stability of modulator-rescued F508del-CFTR at the surface of airway cells. In addition, we found that this effect was mediated by a decreased phosphorylation of the scaffold protein SHC1, a key regulator of MAPK pathway activity. In fact, we showed that depletion of SHC1 or inhibition of MAPK pathway signaling was sufficient to improve rescued F508del-CFTR surface levels, whereas an ectopic increase in pathway activation downstream of SHC1, through the use of a constitutively active H-RAS protein, abrogated the stabilizing effect of YES1 inhibition on rescued F508del-CFTR. Taken together, our findings not only provide new mechanistic insights into the regulation of modulator-rescued F508del-CFTR membrane stability, but also open exciting new avenues to be further explored in CF research and treatment.

Treatment of Polarized Cystic Fibrosis Airway Cells With HGF Prevents VX-661-Rescued F508del-CFTR Destabilization Caused by Prolonged Co-exposure to VX-770.

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in CFTR, the most frequent of which is F508del. F508del causes ER retention and degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life at the plasma membrane. Despite the recent successes with small-molecule CFTR modulator drugs, the folding-corrector/gating-potentiator drug combinations approved for CF individuals carrying F508del-CFTR have sometimes produced severe side effects. Previously, we showed that a prolonged, 15-days treatment of polarized bronchial epithelial monolayers with the VX-809+VX-770 combination resulted in epithelial dedifferentiation effects that we found were caused specifically by VX-809. Moreover, prolonged VX-770 exposure also led to the destabilization of VX-809-rescued F508del-CFTR. Notably, co-treatment with the physiological factor HGF prevented VX-809-mediated epithelial differentiation and reverted the destabilizing effect of VX-770 on VX-809-rescued CFTR. Here, we show that prolonged treatment with VX-661, a second-generation corrector developed based on VX-809 structure, does not perturb epithelial integrity of polarized bronchial epithelial monolayers. Yet, its efficacy is still affected by co-exposure to VX-770, the potentiator present in all VX-661-containing combination therapies approved in the United States and Europe for treatment of F508del-CFTR carriers. Importantly, we found that co-treatment with HGF still ameliorated the impact of VX-770 in F508del-CFTR functional rescue by VX-661, without increasing cell proliferation (Ki-67) or altering the overall expression of epithelial markers (ZO-1, E-cadherin, CK8, CK18). Our findings highlight the importance of evaluating the cellular effects of prolonged exposure to CFTR modulators and suggest that the benefits of adding HGF to current combination therapies should be further investigated.

Glucosylpolyphenols as Inhibitors of Aß-Induced Fyn Kinase Activation and Tau Phosphorylation: Synthesis, Membrane Permeability, and Exploratory Target Assessment within the Scope of Type 2 Diabetes and Alzheimer's Disease.

Despite the rapidly increasing number of patients suffering from type 2 diabetes, Alzheimer's disease, and diabetes-induced dementia, there are no disease-modifying therapies that are able to prevent or block disease progress. In this work, we investigate the potential of nature-inspired glucosylpolyphenols against relevant targets, including islet amyloid polypeptide, glucosidases, and cholinesterases. Moreover, with the premise of Fyn kinase as a paradigm-shifting target in Alzheimer's drug discovery, we explore glucosylpolyphenols as blockers of Aß-induced Fyn kinase activation while looking into downstream effects leading to Tau hyperphosphorylation. Several compounds inhibit Aß-induced Fyn kinase activation and decrease pTau levels at 10 µM concentration, particularly the per-O-methylated glucosylacetophloroglucinol and the 4-glucosylcatechol dibenzoate, the latter inhibiting also butyrylcholinesterase and ß-glucosidase. Both compounds are nontoxic with ideal pharmacokinetic properties for further development. This work ultimately highlights the multitarget nature, fine structural tuning capacity, and valuable therapeutic significance of glucosylpolyphenols in the context of these metabolic and neurodegenerative disorders.

Remote triggering of TGF-ß/Smad2/3 signaling in human adipose stem cells laden on magnetic scaffolds synergistically promotes tenogenic commitment.

Injuries affecting load bearing tendon tissues are a significant clinical burden and efficient treatments are still unmet. Tackling tendon regeneration, tissue engineering strategies aim to develop functional substitutes that recreate native tendon milieu. Tendon mimetic scaffolds capable of remote magnetic responsiveness and functionalized magnetic nanoparticles (MNPs) targeting cellular mechanosensitive receptors are potential instructive tools to mediate mechanotransduction in guiding tenogenic responses. In this work, we combine magnetically responsive scaffolds and targeted Activin A type II receptor in human adipose stem cells (hASCs), under alternating magnetic field (AMF), to synergistically facilitate external control over signal transduction. The combination of remote triggering TGF-ß/Smad2/3 using MNPs tagged hASCs, through magnetically actuated scaffolds, stimulates overall expression of tendon related genes and the deposition of tendon related proteins, in comparison to non-stimulated conditions. Moreover, the phosphorylation of Smad2/3 proteins and their nuclear co-localization was also more evident. Overall, biophysical stimuli resulting from magnetic scaffolds and magnetically triggered cells under AMF stimulation modulate the mechanosensing response of hASCs towards tenogenesis, holding therapeutic promise. STATEMENT OF SIGNIFICANCE: The concept of magnetically-assisted tissue engineering may assist the development of innovative solutions to treat tendon disorders upon remote control of biological processes as cell migration or differentiation. Herein, we originally combine a fibrous aligned superparamagnetic scaffold, based on a biodegradable polymeric blend of starch and poly-ɛ-caprolactone incorporating magnetic nanoparticles (MNPs), and human adipose stem cells (hASCs) labelled with MNPs functionalized with anti-activin receptor type IIA (ActRIIA). Constructs were stimulated using alternating magnetic field (AMF), to activate the ActRIIA and subsequent induction of TGF-ß signaling, through Smad2/3 phosphorylation cascade, enhancing the expression of tendon-related markers. Altogether, these findings contribute with powerful bio-magnetic approaches to activate key tenogenic pathways, envisioning future translation of magnetic biomaterials into regenerative platforms for tendon repair.

Epoxylathyrane Derivatives as MDR-Selective Compounds for Disabling Multidrug Resistance in Cancer.

BACKGROUND: Multidrug resistance (MDR) has been regarded as one of the major hurdles for the successful outcome of cancer chemotherapy. The collateral sensitivity (CS) effect is one the most auspicious anti-MDR strategies. Epoxylathyrane derivatives 1-16 were obtained by derivatization of the macrocyclic diterpene epoxyboetirane A (17), a lathyrane-type macrocyclic diterpene isolated from Euphorbia boetica. Some of these compounds were found to strongly modulate P-glycoprotein (P-gp/ABCB1) efflux. PURPOSE: The main goal was to develop lathyrane-type macrocyclic diterpenes with improved MDR-modifying activity, by targeting more than one anti-MDR mechanism. STUDY DESIGN/METHODS: In this study, the potential CS effect of compounds 1-16 was evaluated against gastric (EPG85-257), pancreatic (EPP85-181), and colon (HT-29) human cancer cells and their drug-resistant counterparts, respectively selected against mitoxantrone (EPG85-257RNOV; EPP85-181RNOV; HT-RNOV) or daunorubicin (EPG85-257RDB; EPP85-181RDB; HT-RDB). The most promising compounds (8, 15, and 16) were investigated as apoptosis inducers, using the assays annexin V/PI and active caspase-3. RESULTS: The compounds were more effective against the resistant gastric cell lines, being the CS effect more significant in EPG85-257RDB cells. Taking together the IC50 values and the CS effect, compounds 8, 15, and 16 exhibited the best results. Epoxyboetirane P (8), with the strongest MDR-selective antiproliferative activity against gastric carcinoma EPG85-257RDB cells (IC50 of 0.72 µM), being 10-fold more active against this resistant subline than in sensitive gastric carcinoma cells. The CS effect elicited by compounds 15 and 16 appeared to be by inducing apoptosis via caspase-3 activation. Structure-activity relationships of the compounds were additionally obtained through regression models to clarify the structural determinants associated to the CS effect. CONCLUSIONS: This study reinforces the importance of lathyrane-type diterpenes as lead molecules for the research of MDR-modifying agents.

Magnetic biomaterials and nano-instructive tools as mediators of tendon mechanotransduction.

Tendon tissues connect muscle to bone allowing the transmission of forces resulting in joint movement. Tendon injuries are prevalent in society and the impact on public health is of utmost concern. Thus, clinical options for tendon treatments are in demand, and tissue engineering aims to provide reliable and successful long-term regenerative solutions. Moreover, the possibility of regulating cell fate by triggering intracellular pathways is a current challenge in regenerative medicine. In the last decade, the use of magnetic nanoparticles as nano-instructive tools has led to great advances in diagnostics and therapeutics. Recent advances using magnetic nanomaterials for regenerative medicine applications include the incorporation of magnetic biomaterials within 3D scaffolds resulting in mechanoresponsive systems with unprecedented properties and the use of nanomagnetic actuators to control cell signaling. Mechano-responsive scaffolds and nanomagnetic systems can act as mechanostimulation platforms to apply forces directly to single cells and multicellular biological tissues. As transmitters of forces in a localized manner, the approaches enable the downstream activation of key tenogenic signaling pathways. In this minireview, we provide a brief outlook on the tenogenic signaling pathways which are most associated with the conversion of mechanical input into biochemical signals, the novel bio-magnetic approaches which can activate these pathways, and the efforts to translate magnetic biomaterials into regenerative platforms for tendon repair.

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.

Design and Synthesis of CNS-targeted Flavones and Analogues with Neuroprotective Potential Against H2O2- and Aß1-42-Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells.

With the lack of available drugs able to prevent the progression of Alzheimer's disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the widely reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and their C-glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical properties. To complement our analysis, logD7.4 was determined. From all screened compounds, the p-morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma cells against both H2O2- and Aß1-42-induced cell death. Cholinesterase inhibition was also evaluated, and modest inhibitory activities were found. This work highlights the potential of C-glucosylflavones as neuroprotective agents, and presents the p-morpholinyl C-glucosylflavone 37, which did not show any cytotoxicity towards HepG2 and Caco-2 cells at 100 µM, as a new lead structure for further development against AD.

Prolonged co-treatment with HGF sustains epithelial integrity and improves pharmacological rescue of Phe508del-CFTR.

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in the CFTR chloride channel, the most frequent of which is Phe508del. Phe508del causes not only intracellular retention and premature degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life when experimentally rescued to the plasma membrane (PM). Despite recent successes in the functional rescue of several CFTR mutations with small-molecule drugs, the folding-corrector/gating-potentiator drug combinations approved for Phe508del-CFTR homozygous patients have shown only modest benefit. Several factors have been shown to contribute to this outcome, including an unexpected intensification of corrector-rescued Phe508del-CFTR PM instability after persistent co-treatment with potentiator drugs. We have previously shown that acute co-treatment with hepatocyte growth factor (HGF) can significantly enhance the chemical correction of Phe508del-CFTR. HGF coaxes the anchoring of rescued channels to the actin cytoskeleton via induction of RAC1 GTPase signalling. Here, we demonstrate that a prolonged, 15-day HGF treatment also significantly improves the functional rescue of Phe508del-CFTR by the VX-809 corrector/VX-770 potentiator combination, in polarized bronchial epithelial monolayers. Importantly, we found that HGF treatment also prevented VX-770-mediated destabilization of rescued Phe508del-CFTR and enabled further potentiation of the rescued channels. Most strikingly, prolonged HGF treatment prevented previously unrecognized epithelial dedifferentiation effects of sustained exposure to VX-809. This was observed in epithelium-like monolayers from both lung and intestinal origin, representing the two systems most affected by adverse symptoms in patients treated with VX-809 or the VX-809/VX-770 combination. Taken together, our findings strongly suggest that co-administration of HGF with corrector/potentiator drugs could be beneficial for CF patients.

Exploring Anti-Prion Glyco-Based and Aromatic Scaffolds: A Chemical Strategy for the Quality of Life.

Prion diseases are fatal neurodegenerative disorders caused by protein misfolding and aggregation, affecting the brain progressively and consequently the quality of life. Alzheimer's is also a protein misfolding disease, causing dementia in over 40 million people worldwide. There are no therapeutics able to cure these diseases. Cellular prion protein is a high-affinity binding partner of amyloid ß (Aß) oligomers, the most toxic species in Alzheimer's pathology. These findings motivate the development of new chemicals for a better understanding of the events involved. Disease control is far from being reached by the presently known therapeutics. In this review we describe the synthesis and mode of action of molecular entities with intervention in prion diseases' biological processes and, if known, their role in Alzheimer's. A diversity of structures is covered, based on glycans, steroids and terpenes, heterocycles, polyphenols, most of them embodying aromatics and a structural complexity. These molecules may be regarded as chemical tools to foster the understanding of the complex mechanisms involved, and to encourage the scientific community towards further developments for the cure of these devastating diseases.

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication.

Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response.

Epoxylathyrol Derivatives: Modulation of ABCB1-Mediated Multidrug Resistance in Human Colon Adenocarcinoma and Mouse T-Lymphoma Cells.

Epoxyboetirane A (1), a macrocyclic diterpene that was found to be inactive as an ABCB1 modulator, was submitted to several chemical transformations, aimed at generating a series of compounds with improved multidrug resistance (MDR)-modifying activity. Overall, 23 new derivatives were prepared, in addition to the already reported epoxylathyrol (2) and methoxyboetirol (3). Their anti-MDR potential was assessed through both functional and chemosensitivity assays on resistant human colon adenocarcinoma and human ABCB1-gene transfected L5178Y mouse lymphoma cells. Structure-activity relationship analysis showed that different substitution patterns led to distinct ABCB1 inhibitory activities, although intrinsic cellular characteristics seemed to influence the modulatory behavior. A considerable enhancement in MDR-modifying activity was observed for aromatic compounds in both cell lines, particularly in 3,17-disubstituted esters derived from 3, a Payne-rearranged Michael adduct of 2. All compounds tested were revealed to interact synergistically with doxorubicin, and ATPase inhibition by three representative MDR-modifying compounds was also investigated. On account of its outstanding ABCB1 inhibitory activity at 0.2 µM and overall remarkable bioactive profile, methoxyboetirane B (22) was found to be a new promising lead for MDR-reversing anticancer drug development.

Exploiting the therapeutic potential of 8-ß-d-glucopyranosylgenistein: synthesis, antidiabetic activity, and molecular interaction with islet amyloid polypeptide and amyloid ß-peptide (1-42).

8-ß-d-Glucopyranosylgenistein (1), the major component of Genista tenera, was synthesized and showed an extensive therapeutical impact in the treatment of STZ-induced diabetic rats, producing normalization of fasting hyperglycemia and amelioration of excessive postprandial glucose excursions and and increasing ß-cell sensitivity, insulin secretion, and circulating insulin within 7 days at a dose of 4 (mg/kg bw)/day. Suppression of islet amyloid polypeptide (IAPP) fibril formation by compound 1 was demonstrated by thioflavin T fluorescence and atomic force microscopy. Molecular recognition studies with IAPP and Aß1-42 employing saturation transfer difference (STD) confirmed the same binding mode for both amyloid peptides as suggested by their deduced epitope. Insights into the preferred conformation in the bound state and conformers' geometry resulting from interaction with Aß1-42 were also given by STD, trNOESY, and MM calculations. These studies strongly support 8-ß-d-glucopyranosylgenistein as a promising molecular entity for intervention in amyloid events of both diabetes and the frequently associated Alzheimer's disease.

The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq.

The transient existence of small RNAs free of binding to the RNA chaperone Hfq is part of the normal dynamic lifecycle of a sRNA. Small RNAs are extremely labile when not associated with Hfq, but the mechanism by which Hfq stabilizes sRNAs has been elusive. In this work we have found that polynucleotide phosphorylase (PNPase) is the major factor involved in the rapid degradation of small RNAs, especially those that are free of binding to Hfq. The levels of MicA, GlmY, RyhB, and SgrS RNAs are drastically increased upon PNPase inactivation in Hfq(-) cells. In the absence of Hfq, all sRNAs are slightly shorter than their full-length species as result of 3'-end trimming. We show that the turnover of Hfq-free small RNAs is growth-phase regulated, and that PNPase activity is particularly important in stationary phase. Indeed, PNPase makes a greater contribution than RNase E, which is commonly believed to be the main enzyme in the decay of small RNAs. Lack of poly(A) polymerase I (PAP I) is also found to affect the rapid degradation of Hfq-free small RNAs, although to a lesser extent. Our data also suggest that when the sRNA is not associated with Hfq, the degradation occurs mainly in a target-independent pathway in which RNase III has a reduced impact. This work demonstrated that small RNAs free of Hfq binding are preferably degraded by PNPase. Overall, our data highlight the impact of 3'-exonucleolytic RNA decay pathways and re-evaluates the degradation mechanisms of Hfq-free small RNAs.