A novel bioresponsive self-immolative spacer based on aza-quinone methide reactivity for the controlled release of thiols, phenols, amines, sulfonamides or amides.

A stimuli-sensitive linker is one of the indispensable components of prodrugs for cancer therapy as it covalently binds the drug and releases it upon external stimulation at the tumour site. Quinone methide elimination has been widely used as the key transformation to release drugs based on their nucleofugacity. The usual approach is to bind the drug to the linker as a carbamate and release it as a free amine after a self-immolative 1,6-elimination. Although this approach is very efficient, it is limited to amines (as carbamates), alcohols or phenols (as carbonates) or other acidic functional groups. We report here a self-immolative spacer capable of directly linking and releasing amines, phenols, thiols, sulfonamides and carboxyamides after a reductive stimulus. The spacer is based on the structure of (5-nitro-2-pyrrolyl)methanol (NPYM-OH), which was used for the direct alkylation of the functional groups mentioned above. The spacer is metabolically stable and has three indispensable sites for bioconjugation: the bioresponsive trigger, the conjugated 1,6 self-immolative system and a third arm suitable for conjugation with a carrier or other modifiers. Release was achieved by selective reduction of the nitro group over Fe/Pd nanoparticles (NPs) in a micellar aqueous environment (H2O/TPGS-750-M), or by NADH mediated nitroreductase activation. A DFT study demonstrates that, during the 1,6 elimination, the transition state formed from 5-aminopyrrole has a lower activation energy compared to other 5-membered heterocycles or p-aminobenzyl derivatives. The NPYM scaffold was validated by late-stage functionalisation of approved drugs such as celecoxib, colchicine, vorinostat or ciprofloxacin. A hypoxia-activated NPYM-based prodrug (HAP) derived from HDAC inhibitor ST7612AA1 was also produced, which was active in cancer cells under hypoxic conditions.

A blood test to monitor bee health across a European network of agricultural sites of different land-use by MALDI BeeTyping mass spectrometry.

There are substantial concerns about impaired honey bee health and colony losses due to several poorly understood factors. We used MALDI profiling (MALDI BeeTyping®) analysis to investigate how some environmental and management factors under field conditions across Europe affected the honey bee haemolymph peptidome (all peptides in the circulatory fluid), as a profile of molecular markers representing the immune status of Apis mellifera. Honey bees were exposed to a range of environmental stressors in 128 agricultural sites across eight European countries in four biogeographic zones, with each country contributing eight sites each for two different cropping systems: oilseed rape (OSR) and apple (APP). The full haemolymph peptide profiles, including the presence and levels of three key immunity markers, namely the antimicrobial peptides (AMPs) Apidaecin, Abaecin and Defensin-1, allowed the honey bee responses to environmental variables to be discriminated by country, crop type and site. When considering just the AMPs, it was not possible to distinguish between countries by the prevalence of each AMP in the samples. However, it was possible to discriminate between countries on the amounts of the AMPs, with the Swedish samples in particular expressing high amounts of all AMPs. A machine learning model was developed to discriminate the haemolymphs of bees from APP and OSR sites. The model was 90.6 % accurate in identifying the crop type from the samples used to build the model. Overall, MALDI BeeTyping® of bee haemolymph represents a promising and cost-effective "blood test" for simultaneously monitoring dozens of peptide markers affected by environmental stressors at the landscape scale, thus providing policymakers with new diagnostic and regulatory tools for monitoring bee health.

Novel indices reveal that pollinator exposure to pesticides varies across biological compartments and crop surroundings.

Declines in insect pollinators have been linked to a range of causative factors such as disease, loss of habitats, the quality and availability of food, and exposure to pesticides. Here, we analysed an extensive dataset generated from pesticide screening of foraging insects, pollen-nectar stores/beebread, pollen and ingested nectar across three species of bees collected at 128 European sites set in two types of crop. In this paper, we aimed to (i) derive a new index to summarise key aspects of complex pesticide exposure data and (ii) understand the links between pesticide exposures depicted by the different matrices, bee species and apple orchards versus oilseed rape crops. We found that summary indices were highly correlated with the number of pesticides detected in the related matrix but not with which pesticides were present. Matrices collected from apple orchards generally contained a higher number of pesticides (7.6 pesticides per site) than matrices from sites collected from oilseed rape crops (3.5 pesticides), with fungicides being highly represented in apple crops. A greater number of pesticides were found in pollen-nectar stores/beebread and pollen matrices compared with nectar and bee body matrices. Our results show that for a complete assessment of pollinator pesticide exposure, it is necessary to consider several different exposure routes and multiple species of bees across different agricultural systems.

Distribution of infectious and parasitic agents among three sentinel bee species across European agricultural landscapes.

Infectious and parasitic agents (IPAs) and their associated diseases are major environmental stressors that jeopardize bee health, both alone and in interaction with other stressors. Their impact on pollinator communities can be assessed by studying multiple sentinel bee species. Here, we analysed the field exposure of three sentinel managed bee species (Apis mellifera, Bombus terrestris and Osmia bicornis) to 11 IPAs (six RNA viruses, two bacteria, three microsporidia). The sentinel bees were deployed at 128 sites in eight European countries adjacent to either oilseed rape fields or apple orchards during crop bloom. Adult bees of each species were sampled before their placement and after crop bloom. The IPAs were detected and quantified using a harmonised, high-throughput and semi-automatized qPCR workflow. We describe differences among bee species in IPA profiles (richness, diversity, detection frequencies, loads and their change upon field exposure, and exposure risk), with no clear patterns related to the country or focal crop. Our results suggest that the most frequent IPAs in adult bees are more appropriate for assessing the bees' IPA exposure risk. We also report positive correlations of IPA loads supporting the potential IPA transmission among sentinels, suggesting careful consideration should be taken when introducing managed pollinators in ecologically sensitive environments.

Multipurpose Iron-Chelating Ligands Inspired by Bioavailable Molecules.

Because of their capacity to bind metals, metal chelators are primarily employed for therapeutic purposes, but they can also find applications as colorimetric reagents and cleaning solutions as well as in soil remediation, electroplating, waste treatment, and so on. For instance, iron-chelation therapy, which is used to treat iron-overload disorders, involves removing excess iron from the blood through the use of particular molecules, like deferoxamine, that have the ability to chelate the metal. The creation of bioinspired and biodegradable chelating agents is a crucial objective that draws inspiration from natural products. In this context, starting from bioavailable molecules such as maltol and pyrogallol, new molecules have been synthetized and characterized by potentiometry, infrared spectroscopy and cyclic voltammetry. Finally, the ability of these to bind iron has been investigated, and the stability constants of ferric complexes are measured using spectrophotometry. These compounds offer intriguing scaffolds for an innovative class of versatile, multipurpose chelating agents.

Pesticide use negatively affects bumble bees across European landscapes.

Sustainable agriculture requires balancing crop yields with the effects of pesticides on non-target organisms, such as bees and other crop pollinators. Field studies demonstrated that agricultural use of neonicotinoid insecticides can negatively affect wild bee species1,2, leading to restrictions on these compounds3. However, besides neonicotinoids, field-based evidence of the effects of landscape pesticide exposure on wild bees is lacking. Bees encounter many pesticides in agricultural landscapes4-9 and the effects of this landscape exposure on colony growth and development of any bee species remains unknown. Here we show that the many pesticides found in bumble bee-collected pollen are associated with reduced colony performance during crop bloom, especially in simplified landscapes with intensive agricultural practices. Our results from 316 Bombus terrestris colonies at 106 agricultural sites across eight European countries confirm that the regulatory system fails to sufficiently prevent pesticide-related impacts on non-target organisms, even for a eusocial pollinator species in which colony size may buffer against such impacts10,11. These findings support the need for postapproval monitoring of both pesticide exposure and effects to confirm that the regulatory process is sufficiently protective in limiting the collateral environmental damage of agricultural pesticide use.

Neither sulfoxaflor, Crithidia bombi, nor their combination impact bumble bee colony development or field bean pollination.

Many pollinators, including bumble bees, are in decline. Such declines are known to be driven by a number of interacting factors. Decreases in bee populations may also negatively impact the key ecosystem service, pollination, that they provide. Pesticides and parasites are often cited as two of the drivers of bee declines, particularly as they have previously been found to interact with one another to the detriment of bee health. Here we test the effects of an insecticide, sulfoxaflor, and a highly prevalent bumble bee parasite, Crithidia bombi, on the bumble bee Bombus terrestris. After exposing colonies to realistic doses of either sulfoxaflor and/or Crithidia bombi in a fully crossed experiment, colonies were allowed to forage on field beans in outdoor exclusion cages. Foraging performance was monitored, and the impacts on fruit set were recorded. We found no effect of either stressor, or their interaction, on the pollination services they provide to field beans, either at an individual level or a whole colony level. Further, there was no impact of any treatment, in any metric, on colony development. Our results contrast with prior findings that similar insecticides (neonicotinoids) impact pollination services, and that sulfoxaflor impacts colony development, potentially suggesting that sulfoxaflor is a less harmful compound to bee health than neonicotinoids insecticides.

Structural Characterization of Human Heat Shock Protein 90 N-Terminal Domain and Its Variants K112R and K112A in Complex with a Potent 1,2,3-Triazole-Based Inhibitor.

Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.

Quinolines and Oxazino-quinoline Derivatives as Small Molecule GLI1 Inhibitors Identified by Virtual Screening.

A virtual screening approach based on a five-feature pharmacophoric model for negative modulators of GLI1 was applied to databases of commercially available compounds. The resulting quinoline derivatives showed significant ability to reduce the GLI1 protein level and were characterized by submicromolar antiproliferative activity toward human melanoma A375 and medulloblastoma DAOY cell lines. Decoration of the quinoline ring and chemical rigidification to an oxazino-quinoline scaffold allowed us to deduce SAR considerations for future ligand optimization.

A pH-responsive crosslinker platform for antibody-drug conjugate (ADC) targeting delivery.

We report a new 1-6 self-immolative, traceless crosslinker derived from the natural product gallic acid. The linker acts through a pH-dependent mechanism for drug release. This 5-(hydroxymethyl)pyrogallol orthoester derivative (HMPO) was stable for 24 hours at pH values of 7.4 and 6.6 and in plasma, releasing molecules bound to the hydroxymethyl moiety under acid-dependent stimuli at pH 5.5. The linker was non-toxic and was used for the conjugation of Doxorubicin (Doxo) or Combretastatin A4 with Cetuximab. The ADCs formed showed their pH responsivity reducing cell viability of A431 and A549 cancer cells better than Cetuximab alone.

Pulmonary Function and Psychological Burden Three Months after COVID-19: Proposal of a Comprehensive Multidimensional Assessment Protocol.

Persisting limitations in respiratory function and gas exchange, cognitive impairment, and mental health deterioration have been observed weeks and months after acute SARS-CoV-2 (COVID-19). The present study aims at assessing the impairment at three-months in patients who successfully recovered from acute COVID-19. We collected data from May to July 2020. Patients underwent a multidimensional extensive assessment including pulmonary function test, psychological tests, thoracic echo scan, and functional exercise capacity. A total of 21 patients (M:13; Age 57.05 ± 11.02) completed the global assessment. A considerable proportion of patients showed symptoms of post-traumatic stress disorder (28.6%), moderate depressive symptoms (9.5%), and clinical insomnia (9.5%); 14.3% of patients exhibited moderate anxiety. A total of eleven patients (52.4%) showed impaired respiratory gas exchange capacity (P-DLCO, DLCO ≤ 79% pred). Compared to patients with normal gas exchange, the P-DLCO subgroup perceived a significant worsening in quality of life (QoL) after COVID-19 (p = 0.024), higher fatigue (p = 0.005), and higher impact of lung disease (p = 0.013). In P-DLCO subgroup, higher echo score was positively associated with hospitalization length of stay (p = 0.047), depressive symptoms (p = 0.042), fatigue (p = 0.035), impairment in mental health (p = 0.035), and impact of lung disease in health status (p = 0.020). Pulmonary function and echo scan lung changes were associated to worsened QoL, fatigue, and psychological distress symptoms.

Synthesis of 2-Substitued Indoles via Pd-Catalysed Cyclization in an Aqueous Micellar Medium.

The synthesis of 2-substituted indoles starting from the corresponding unprotected 2-alkynylanilines was made possible in 3% TPGS-750-M water using Pd(OAc)2 alone as the catalyst. The reaction was sensitive to the heating mode respect to the nature of the starting material as, in many cases, convectional heating was better than microwave dielectric heating. The MW (microwave) delivery mode had also an influence in the formation of by-products and, consequently, product yields. A tandem Sonogashira-cyclisation reaction was also accomplished using Pd(OAc)2/Xphos in the nanomicellar water environment.

Antibody drug conjugates with hydroxamic acid cargos for histone deacetylase (HDAC) inhibition.

Antitumor hydroxamates SAHA and Dacinostat have been linked to cetuximab and trastuzumab through a non-cleavable linker based on the p-mercaptobenzyl alcohol structure. These antibody drug conjugates (ADCs) were able to inhibit HDAC in several tumour cell lines. The cetuximab based ADCs block human lung adenocarcinoma cell proliferation, demonstrating that bioconjugation with antibodies is a suitable approach for targeted therapy based on hydroxamic acid-containing drugs. This work also shows that ADC-based delivery might be used to overcome the classical pharmacokinetic problems of hydroxamic acids.

ErbB2 Targeted Epigenetic Modulation: Anti-tumor Efficacy of the ADC Trastuzumab-HDACi ST8176AA1.

Targeted therapy using monoclonal antibodies conjugated to toxins is gaining space in the treatment of cancer. Here, we report the anti-tumor effect of a new antibody drug conjugate (ADC) delivering a HDAC inhibitor to ErbB2+ solid tumors. Trastuzumab was partially reduced with tris [2-carboxyethyl] phosphine (TCEP) and conjugated to ST7464AA1, the active form of the prodrug HDAC inhibitor ST7612AA1, through a maleimide-thiol linker to obtain the Antibody Drug Conjugate (ADC) ST8176AA1. The average drug/antibody ratio (DAR) was 4.5 as measured by hydrophobic interaction chromatography (HIC). Binding of ST8176AA1 to ErbB2 receptor and internalization in tumor cells were investigated by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cytofluorimetry, and High Content Screening (HCS) Imaging. The biological activity of the ADC was evaluated in vitro and in vivo by measuring cell proliferation/cell cycle, apoptosis/DNA damage, tubulin, and histone acetylation and modulation of Epithelial/Mesenchymal Transition (EMT) markers. Receptor binding and internalization of ST8176AA1 were confirmed to be similar to trastuzumab. Higher anti-tumor activity of ST8176AA1 compared to trastuzumab was observed in vitro in tumor cell lines. Such higher activity correlated with increased acetylation of histones and alfa-tubulin as a consequence of HDAC inhibitor-mediated epigenetic modulation that also induced increased expression of ErbB2 and estrogen receptor in triple negative breast cancer cells. Consistently with in vitro data, ST8176AA1 exhibited higher tumor growth inhibition than trastuzumab in xenograft models of ovary and colon carcinoma and in two patient-derived xenograft (PDX) models of pancreatic carcinoma. Immunohistochemistry analysis of tumor masses showed lower expression of the proliferation marker Ki67 and higher expression of cleaved caspase-3 in mice treated with the ADC compared to those treated with trastuzumab and results correlated with increased acetylation of both histones and tubulin. Collectively, present data indicate that ADC ST8176AA1 can target epigenetic modulation to ErbB2+ tumors. Interestingly, the amount of HDACi estimated to be delivered at the ST8176AA1 effective dose would correspond to ~1/1,000 of ST7612AA1 effective dose. Therefore, ST8176AA1 is an attractive new therapeutic candidate because it exhibits increased anti-tumor potency compared to trastuzumab by exerting epigenetic modulation at a much safer dose compared to standard HDACi-based therapeutic protocols.

Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation.

We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC50 equal to 0.07 µM on NCI-H460 cells), a thiol-based molecule with a moderate toxicity in vivo. Two payloads were prepared using cleavable and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced sensible increment of histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible therapeutic applications beyond cancer.

Hedgehog pathway inhibitors of the acylthiourea and acylguanidine class show antitumor activity on colon cancer in vitro and in vivo.

Small series of acylguanidine and acylthiourea derivatives were synthesized in gram-scale and assayed for their ability to modulate the Hh signalling pathway. In vitro studies showed a low micromolar inhibitory activity toward tumor cell lines, while the oral administration revealed an excellent ADME profile in vivo. Compound 5 emerged as the most active and safe inhibitor of colon cancer cells both in vitro and in a xenograft mouse model. Based on these data, 5 could be prioritized to further development with the perspective of clinical studies.

Benzodiazepine Scaffold as Drug-like Molecular Simplification of FR235222: A Chemical Tool for Exploring HDAC Inhibition.

BACKGROUND: Synthesis, computational study and biological evaluation of peptidomimetic analogues of FR235222 (3), a natural immunosuppressant and HDAC inhibitor, have been reported. These new compounds, bearing α-hydroxyketone moiety, as more stable zinc binding group (ZBG), were evaluated in vitro as HDAC inhibitors against the human HDACs isoforms 1-9 and in cellular antiproliferative assays on U937 human leukemia cell line. The 1,4-benzodiazepin-2,5-dione (BDZ), capping group and the natural ZBG, (S,R)-2-amino-9-hydroxy-8-oxodecanoic acid (Ahoda), were evaluated in order to probe HDAC inhibition and/or paralogue selectivity. Some of the new derivatives showed an interesting activity against a number of HDAC isozymes. The observed activity profile was rationalized by a computational assisted SAR study, in order to understand how the BDZ classes interact with the enzyme into the catalytic pocket. Despite its poor solubility, compound 17b showed significant antiproliferative profile and HDAC inhibition activity. RESULT: In order to assess how the solubility issue could have affected the biological outcome, bioassay conditions were reproduced and quantification of precipitated particulate material was evaluated by turbidimetric and NMR studies together with physicochemical descriptors prediction. Thus, BDZ 17b has been chosen to be promising lead compounds for further optimization, in order to elucidate molecule- enzyme surface recognition.

Investigating the Neuroprotective Effects of Turmeric Extract: Structural Interactions of ß-Amyloid Peptide with Single Curcuminoids.

A broad biophysical analysis was performed to investigate the molecular basis of the neuroprotective action of Curcuma longa extracts in Alzheimer's disease. By combining circular dichroism and electron paramagnetic resonance experiments with molecular modeling calculations, the minor components of Curcuma longa extracts, such as demethoxycurcumin (2, DMC), bisdemethoxycurcumin (3, BDMC) and cyclocurcumin (4, CYC), were analyzed in a membrane environment mimicking the phospholipid bilayer. Our study provides the first evidence on the relative role of single curcuminoids interacting with Aß-peptide. When the CYC and curcumin metabolite tetrahydrocurcumin (5, THC) were inserted into an anionic lipid solution, a significant modification of the Aß CD curves was detected. These data were implemented by EPR experiments, demonstrating that CYC reaches the inner part of the bilayer, while the other curcuminoids are localized close to the membrane interface. Computational studies provided a model for the curcuminoid-Aß interaction, highlighting the importance of a constrained "semi-folded" conformation to interact with Aß analogously to the pattern observed in α-helical coiled-coil peptide structures. This combined approach led to a better understanding of the intriguing in vitro and in vivo activity of curcuminoids as anti-Alzheimer agents, paving a new path for the rational design of optimized druggable analogues.

Lactam based 7-amino suberoylamide hydroxamic acids as potent HDAC inhibitors.

A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, without substituent in position 7. In general, this modification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH group orientation did not strongly influence the inhibition, the contemporary modification of the suberoylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that showed IC50 values between 2 and 10nM against all Class I HDAC isoforms, demonstrating it to be a large spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC50=0.5µM against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far.

Domino reactions triggered by hydroformylation.

HF reaction represents a selective method for the synthesis of aldehydes starting from alkenes. Because of versatile aldehydes reactivity, it is possible to perform different domino protocols based on contemporary HF, including Michael's reaction, reductive amination, cyclopropanation, lactonization, and many others. This overview reports on the last 5 years' results obtained on this field.