PharmaCore: The Automatic Generation of 3D Structure-Based Pharmacophore Models from Protein/Ligand Complexes.

In this work, we present PharmaCore: a new, completely automatic workflow aimed at generating three-dimensional (3D) structure-based pharmacophore models toward any target of interest. The proposed approach relies on using cocrystallized ligands to create the input files for generating the pharmacophore hypotheses, integrating not only the three-dimensional structural information on the ligand but also data concerning the binding mode of these molecules put in the protein cavity. We developed a Python library that, starting from the specific UniProt ID of the protein under investigation as the only element that requires user intervention, subsequently collects and aligns the corresponding structures bearing a known ligand in a fully automated fashion, bringing them all into the same coordinate system. The protocol includes a final phase in which the aligned small molecules are used to produce the pharmacophore hypotheses directly onto the protein structure using a specific software, e.g., Phase (Schrödinger LLC). To validate the entire procedure and highlight the possible applications in the field of drug discovery and repositioning, we first generated pharmacophores for soluble epoxide hydrolase (sEH) and compared with already-published ones. Then, we reproduced the binding profile of a reported selective binder of ATAD2 bromodomain (AM879), testing it against a panel of 1741 pharmacophores related to 16 epigenetic proteins and automatically generated with PharmaCore, finally disclosing putative unprecedented off-targets. The computational predictions were successfully validated with AlphaScreen assays, highlighting the applicability of the proposed workflow in drug discovery and repositioning. Finally, the process was also validated on tankyrase 2 and SARS-CoV-2 MPro, confirming the robustness of PharmaCore.

Repositioning of Small Molecules through the Inverse Virtual Screening in silico Tool: Case of Benzothiazole-Based Inhibitors of Soluble Epoxide Hydrolase (sEH).

Computational techniques accelerate drug discovery by identifying bioactive compounds for specific targets, optimizing molecules with moderate activity, or facilitating the repositioning of inactive items onto new targets. Among them, the Inverse Virtual Screening (IVS) approach is aimed at the evaluation of one or a small set of molecules against a panel of targets for addressing target identification. In this work, a focused library of benzothiazole-based compounds was re-investigated by IVS. Four items, originally synthesized and tested on bromodomain-containing protein 9 (BRD9) but yielding poor results, were critically re-analyzed, disclosing only a partial fit with 3D structure-based pharmacophore models, which, in the meanwhile, were developed for this target. Afterwards, these compounds were re-evaluated through IVS on a panel of proteins involved in inflammation and cancer, identifying soluble epoxide hydrolase (sEH) as a putative interacting target. Three items were subsequently confirmed as able to inhibit sEH activity, spanning from 70% up to 30% when tested at 10 µM. Finally, one benzothiazole-based compound emerged as the most promising inhibitor featuring an IC50 in the low micromolar range (IC50 = 6.62 ± 0.13 µM). Our data confirm IVS as a predictive tool for accelerating the target identification and repositioning processes.

Prodrug Approach to Exploit (S) Alanine as Arginine Mimic Moiety in the Development of Protein Arginine Methyltransferase 4 Inhibitors.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as an emerging target class in oncology and other diseases. A successful strategy to identify PRMT substrate-competitive inhibitors has been to exploit chemical scaffolds able to mimic the arginine substrate. (S)-Alanine amide moiety is a valuable arginine mimic for the development of potent and selective PRMT4 inhibitors; however, its high hydrophilicity led to derivatives with poor cellular outcomes. Here, we describe the development of PRMT4 inhibitors featuring a central pyrrole core and an alanine amide moiety. Rounds of optimization, aimed to increase lipophilicity and simultaneously preserve the inhibitory activity, produced derivatives that, despite good potency and physicochemical properties, did not achieve on-target effects in cells. On the other hand, masking the amino group with a NAD(P)H:quinone oxidoreductase 1 (NQO1)-responsive trigger group, led to prodrugs able to reduce arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155). These results indicate that prodrug strategies can be successfully applied to alanine-amide containing PRMT4 inhibitors and provide an option to enable such compounds to achieve sufficiently high exposures in vivo.

Candida auris in Greek healthcare facilities: Active surveillance results on first cases and outbreaks from eleven hospitals within Attica region.

BACKGROUND: Candida auris was sporadically detected in Greece until 2019. Thereupon, there has been an increase in isolations among inpatients of healthcare facilities. AIM: We aim to report active surveillance data on MALDI-TOF confirmed Candida auris cases and outbreaks, from November 2019 to September 2021. METHODS: A retrospective study on hospital-based Candida auris data, over a 23-month period was conducted, involving 11 hospitals within Attica region. Antifungal susceptibility testing and genotyping were conducted. Case mortality and fatality rates were calculated and p-values less than 0.05 were considered statistically significant. Infection control measures were enforced and enhanced. RESULTS: Twenty cases with invasive infection and 25 colonized were identified (median age: 72 years), all admitted to hospitals for reasons other than fungal infections. Median hospitalisation time until diagnosis was 26 days. Common risk factors among cases were the presence of indwelling devices (91.1 %), concurrent bacterial infections during hospitalisation (60.0 %), multiple antimicrobial drug treatment courses prior to hospitalisation (57.8 %), and admission in the ICU (44.4 %). Overall mortality rate was 53 %, after a median of 41.5 hospitalisation days. Resistance to fluconazole and amphotericin B was identified in 100 % and 3 % of tested clinical isolates, respectively. All isolates belonged to South Asian clade I. Outbreaks were identified in six hospitals, while remaining hospitals detected sporadic C. auris cases. CONCLUSION: Candida auris has proven its ability to rapidly spread and persist among inpatients and environment of healthcare facilities. Surveillance focused on the presence of risk factors and local epidemiology, and implementation of strict infection control measures remain the most useful interventions.

Transitions of blood immune endotypes and improved outcome by anakinra in COVID-19 pneumonia: an analysis of the SAVE-MORE randomized controlled trial.

BACKGROUND: Endotype classification may guide immunomodulatory management of patients with bacterial and viral sepsis. We aimed to identify immune endotypes and transitions associated with response to anakinra (human interleukin 1 receptor antagonist) in participants in the SAVE-MORE trial. METHODS: Adult patients hospitalized with radiological findings of PCR-confirmed severe pneumonia caused by SARS-CoV-2 and plasma-soluble urokinase plasminogen activator receptor levels of ≥ 6 ng/ml in the SAVE-MORE trial (NCT04680949) were characterized at baseline and days 4 and 7 of treatment using a previously defined 33-messenger RNA classifier to assign an immunological endotype in blood. Endpoints were changes in endotypes and progression to severe respiratory failure (SRF) associated with anakinra treatment. RESULTS: At baseline, 23.2% of 393 patients were designated as inflammopathic, 41.1% as adaptive, and 35.7% as coagulopathic. Only 23.9% were designated as the same endotype at days 4 and 7 compared to baseline, while all other patients transitioned between endotypes. Anakinra-treated patients were more likely to remain in the adaptive endotype during 7-day treatment (24.4% vs. 9.9%; p < 0.001). Anakinra also protected patients with coagulopathic endotype at day 7 against SRF compared to placebo (27.8% vs. 55.9%; p = 0.013). CONCLUSION: We identify an association between endotypes defined using blood transcriptome and anakinra therapy for COVID-19 pneumonia, with anakinra-treated patients shifting toward endotypes associated with a better outcome, mainly the adaptive endotype. Trial registration ClinicalTrials.gov, NCT04680949, December 23, 2020.

Identification and Development of BRD9 Chemical Probes.

The development of BRD9 inhibitors involves the design and synthesis of molecules that can specifically bind the BRD9 protein, interfering with the function of the chromatin-remodeling complex ncBAF, with the main advantage of modulating gene expression and controlling cellular processes. Here, we summarize the work conducted over the past 10 years to find new BRD9 binders, with an emphasis on their structure-activity relationships, efficacies, and selectivities in preliminary studies. BRD9 is expressed in a variety of cancer forms, hence, its inhibition holds particular significance in cancer research. However, it is crucial to note that the expanding research in the field, particularly in the development of new degraders, may uncover new therapeutic potentials.

Chemoproteomics Reveals USP5 (Ubiquitin Carboxyl-Terminal Hydrolase 5) as Promising Target of the Marine Polyketide Gracilioether A.

Mass spectrometry-based chemical proteomic approaches using limited proteolysis have become a powerful tool for the identification and analysis of the interactions between a small molecule (SM) and its protein target(s). Gracilioether A (GeA) is a polyketide isolated from a marine sponge, for which we aimed to trace the interactome using this strategy. DARTS (Drug Affinity Responsive Target Stability) and t-LiP-MS (targeted-Limited Proteolysis-Mass Spectrometry) represented the main techniques used in this study. DARTS was applied on HeLa cell lysate for the identification of the GeA target proteins, and t-LiP-MS was employed to investigate the protein's regions involved in the binding with GeA. The results were complemented through the use of binding studies using Surface Plasmon Resonance (SPR) and in silico molecular docking experiments. Ubiquitin carboxyl-terminal hydrolase 5 (USP5) was identified as a promising target of GeA, and the interaction profile of the USP5-GeA complex was explained. USP5 is an enzyme involved in the pathway of protein metabolism through the disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. This activity is connected to different cellular functions concerning the maintenance of chromatin structure and receptors and the degradation of abnormal proteins and cancerogenic progression. On this basis, this structural information opens the way to following studies focused on the definition of the biological potential of Gracilioether A and the rational development of novel USP5 inhibitors based on a new structural skeleton.

NMR Metabolomics and Chemometrics of Commercial Varieties of Phaseolus vulgaris L. Seeds from Italy and In Vitro Antioxidant and Antifungal Activity.

The metabolite fingerprinting of four Italian commercial bean seed cultivars, i.e., Phaseolus Cannellino (PCANN), Controne (PCON), Vellutina (PVEL), and Occhio Nero (PON), were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate data analysis. The hydroalcoholic and organic extract analysis disclosed more than 32 metabolites from various classes, i.e., carbohydrates, amino acids, organic acids, nucleosides, alkaloids, and fatty acids. PVEL, PCON, and PCANN varieties displayed similar chemical profiles, albeit with somewhat different quantitative results. The PON metabolite composition was slightly different from the others; it lacked GABA and pipecolic acid, featured a higher percentage of malic acid than the other samples, and showed quantitative variations of several metabolites. The lipophilic extracts from all four cultivars demonstrated the presence of omega-3 and omega-6 unsaturated fatty acids. After the determination of the total phenolic, flavonoids, and condensed tannins content, in vitro antioxidant activity was then assessed using the DPPH scavenging activity, the ABTS scavenging assay, and ferric-reducing antioxidant power (FRAP). Compared to non-dark seeds (PCON, PCANN), brown seeds (PVEL, PON) featured a higher antioxidant capacity. Lastly, only PON extract showed in vitro antifungal activity against the sclerotia growth of S. rolfsii, by inhibiting halo growth by 75%.

The impact of frailty and illness perceptions on quality of life among people living with HIV in Greece: A network analysis.

OBJECTIVE: Despite the significant advances in healthcare, people living with HIV still face challenges that affect their quality of life (QoL), both in terms of their physical state as represented by frailty and of their illness perceptions (IP). The aim of this study was to unravel the associations between these constructs (QoL, frailty, IP). METHODS: This multicenter, cross-sectional study included 477 people living with HIV (93% male; median age = 43 years, IQR = 51.7) from six HIV clinics in Greece. Frailty phenotype, QoL and IP were assessed using Fried's criteria, EuroQoL (EQ-5D-5L) and Brief Illness Perception Questionnaire (BIPQ), respectively. Network analysis model was utilized. RESULTS: Among frailty criteria, exhaustion had the highest expected influence, while the strongest correlation concerns exhaustion and weak grip strength (pr = 0.14). Regarding the QoL items, usual activities displayed the highest expected influence. The correlations of pain/discomfort with mobility (pr = 0.31), and usual activities with self-care (pr = 0.34) were the strongest. For the BIPQ items, the strongest correlation was found between illness concern and emotional response (pr = 0.45), whereas the latter item was the one that displayed the highest expected influence. Three communities were formed: 1) personal control, treatment control and coherence, 2) the frailty items with mobility, self-care, usual activities, and pain/discomfort, and 3) the rest BIPQ items with anxiety/depression. Identity displayed the highest bridge strength, followed by pain/discomfort, usual activities and consequences. CONCLUSIONS: The interplay between QoL, frailty, and IP in people living with HIV requires clinical attention. Self-reported exhaustion, slow walking speed, and low physical activity affect the physical QoL dimensions, while anxiety/depression is strongly associated with illness-related concern and perceived emotional effects, leading to psychological distress. Symptom management can improve QoL, and information on the disease and treatment can enhance control over the disease. Developing interventions to address QoL, frailty, and IP is crucial.

2-Substituted 1,5-benzothiazepine-based HDAC inhibitors exert anticancer activities on human solid and acute myeloid leukemia cell lines.

Herein, we report the development of a new series of histone deacetylase inhibitors (HDACi) containing a 2-substituted 1,5-benzothiazepine scaffold. First, a virtual combinatorial library (∼1.6 × 103 items) was built according to a convenient synthetic route, and then it was submitted to molecular docking experiments on seven HDACs isoforms belonging to classes I and II. Integrated computational filters were used to select the most promising ones that were synthesized through an optimized approach, also amenable to generating both racemic and enantioenriched benzothiazepine-based derivatives. The obtained compounds showed potent HDAC inhibitory activity, especially those containing the sulphone moiety, endowed with IC50 in the nanomolar range. In addition, in vitro outcomes of our synthesized compounds demonstrated a cytotoxic effect on U937 and HCT116 cell lines and an arrest in the G2/M phase (13 ≤ IC50 ≤ 18 µM). Finally, Western blot analyses outlined the modulation of the histone acetyl markers such as H3K9/14, acetyl-tubulin, and the apoptotic indicator p21 in both cancer cell lines, disclosing a good HDAC inhibitor activity exerted by the designed items. Given the key role of HDACs in many cellular pathways, which makes these enzymes appealing and "hot" drug targets, our findings highlighted the importance of these 2-substituted 1,5-benzothiazepine-based compounds (both in the reduced and oxidized version) for the development of novel epidrugs.

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents.

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi-preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1-5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1-5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues.

Exploring the chemical space of functionalized [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the bromodomain of BRD9.

Here we report a detailed structure-activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1-4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported "pharm-druglike2" 3D structure-based pharmacophore model, introducing it as "pharm-druglike2.1". Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

Low pre-ART CD4 count is associated with increased risk of clinical progression or death even after reaching 500 CD4 cells/µL on ART.

INTRODUCTION: Clinical disadvantages of initiating ART at low CD4 counts have been clearly demonstrated but whether any excess risk remains even after reaching relatively high/safe CD4 levels remains unclear. We explore whether individuals starting ART with <500 CD4 cells/µL who increased their CD4 count above this level, have, from this point onwards, similar risk of clinical progression to serious AIDS/non-AIDS events or death with individuals starting ART with ≥500 CD4 cells/µL. METHODS: Data were derived from a multicenter cohort (AMACS). Adults, starting PI, NNRTI or INSTI based ART, in or after 2000 were eligible, provided they started ART with ≥500 ("High CD4") or started with CD4 <500 cells/µL but surpassed this threshold while on ART ("Low CD4"). Baseline was the date of ART initiation ("High CD4") or of first reaching 500 CD4 cells/µL ("Low CD4"). Survival analysis, allowing for competing risks, was used to explore the risk of progression to study's endpoints. RESULTS: The study included 694 persons in the "High CD4" and 3,306 in the "Low CD4" group. Median (IQR) follow-up was 66 (36, 106) months. In total, 257 events (40 AIDS related, 217 SNAEs) were observed. Rates of progression did not differ significantly between the two groups but the subgroup of those initiating ART with <200 CD4 cells/µL had significantly higher risk of progression after baseline, compared to those in the "High CD4" group. CONCLUSIONS: Individuals starting ART with <200 cells/µL remain on increased risk even after reaching 500 CD4 cells/µL. These patients should be closely followed.

Target identification by structure-based computational approaches: Recent advances and perspectives.

The use of computational techniques in the early stages of drug discovery has recently experienced a boost, especially in the target identification step. Finding the biological partner(s) for new or existing synthetic and/or natural compounds by "wet" approaches may be challenging; therefore, preliminary in silico screening is even more recommended. After a brief overview of some of the most known target identification techniques, recent advances in structure-based computational approaches for target identification are reported in this digest, focusing on Inverse Virtual Screening and its recent applications. Moreover, future perspectives concerning the use of such methodologies, coupled or not with other approaches, are analyzed.

Effectiveness of full (booster) COVID-19 vaccination against severe outcomes and work absenteeism in hospitalized patients with COVID-19 during the Delta and Omicron waves in Greece.

AIM: We estimated vaccine effectiveness (VE) of full (booster) vaccination against severe outcomes in hospitalized COVID-19 patients during the Delta and Omicron waves. METHODS: The study extended from November 15, 2021 to April 17, 2022. Full vaccination was defined as a primary vaccination plus a booster ≥ 6 months later. RESULTS: We studied 1138 patients (mean age: 66.6 years), of whom 826 (72.6 %) had ≥ 1 comorbidity. Of the 1138 patients, 75 (6.6 %) were admitted to intensive care unit (ICU), 64 (5.6 %) received mechanical ventilation, and 172 (15.1 %) died. There were 386 (33.9 %) fully vaccinated, 172 (15.1 %) partially vaccinated, and 580 (51 %) unvaccinated patients. Unvaccinated patients were absent from work for longer periods compared to partially or fully vaccinated patients (mean absence of 20.1 days versus 12.3 and 17.3 days, respectively; p-value = 0.03). Compared to unvaccinated patients, fully vaccinated patients were less likely to be admitted to ICU [adjusted relative risk (ARR: 0.49; 95 % CI: 0.29-0.84)], mechanically ventilated (ARR: 0.43; 95 % CI: 0.23-0.80), and die (ARR: 0.57; 95 % CI: 0.42-0.78), while they were hospitalized for significantly shorter periods (ARR: 0.79; 95 % CI: 0.70-0.89). The adjusted full VE was 48.8 % (95 % CI: 42.7 %-54.9 %) against ICU admission, 55.4 % (95 % CI: 52.0 %-56.2 %) against mechanical ventilation, and 22.6 % (95 % CI: 7.4 %-34.8 %) against death. For patients with ≥ 3 comorbidities, VE was 56.2 % (95 % CI: 43.9 %-67.1 %) against ICU admission, 60.2 % (95 % CI: 53.7 %-65.4 %) against mechanical ventilation, and 43.9 % (95 % CI: 19.9 %-59.7 %) against death. CONCLUSIONS: Full (booster) COVID-19 vaccination conferred protection against severe outcomes, prolonged hospitalization, and prolonged work absenteeism.

Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors.

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

Efficacy and safety of early soluble urokinase plasminogen receptor plasma-guided anakinra treatment of COVID-19 pneumonia: A subgroup analysis of the SAVE-MORE randomised trial.

Background: The SAVE-MORE trial demonstrated that anakinra treatment in COVID-19 pneumonia with plasma soluble urokinase plasminogen activator (suPAR) levels of 6 ng/mL or more was associated with 0.36 odds for a worse outcome compared to placebo when expressed by the WHO-Clinical Progression Scale (CPS) at day 28. Herein, we report the results of subgroup analyses and long-term outcomes. Methods: This prospective, double-blind, randomised clinical trial, recruited patients with a confirmed SARS-CoV-2 infection, in need of hospitalisation, lower respiratory tract infection and plasma suPAR ≥6 ng/mL from 37 academic and community hospitals in Greece and Italy. Patients were 1:2 randomised to subcutaneous treatment with placebo or anakinra (100 mg) once daily for 10 days. Pre-defined subgroups of Charlson's comorbidity index (CCI), sex, age, level of suPAR, and time from symptom onset were analysed for the primary endpoint (overall comparison of distribution of frequencies of the scores from the WHO-CPS between treatments on day 28), by multivariable ordinal regression analysis in the intention to treat (ITT) population. This trial is registered with the EU Clinical Trials Register (2020-005828-11) and ClinicalTrials.gov (NCT04680949). Findings: Patients were enrolled between 23 December 2020 and 31 March 2021; 189 patients in the placebo arm and 405 patients in the anakinra arm were the ITT population. Multivariable analysis showed that anakinra treatment was accompanied by significantly lower odds for worse outcome compared to placebo at day 28 for all studied subgroups (CCI ≥ 2, OR: 0.34, 95% confidence intervals [CI] 0.22-0.50; CCI < 2, OR: 0.38, 95% CI 0.21-0.68; suPAR > 9 ng/mL, OR: 0.35, 95% CI 0.19-0.66; suPAR 6-9 ng/mL, OR: 0.35, 95% CI 0.24-0.52; patients ≥65 years, OR: 0.41, 95% CI 0.25-0.66; and patients <65 years, OR: 0.29, 95% CI 0.19-0.45). The benefit was uniform, irrespective of the time from start of symptoms until the start of the study drug. At days 60 and 90, anakinra treatment had odds of 0.40 (95% CI 0.28-0.57) and 0.46 (95% CI 0.32-0.67) respectively, for a worse outcome compared to placebo. The costs of general ward stay, ICU stay, and drugs were lower with anakinra treatment. Interpretation: Anakinra represents an important therapeutic tool in the management of COVID-19 that may be administered in all subgroups of patients; benefits are maintained until day 90. Funding: Hellenic Institute for the Study of Sepsis; Swedish Orphan Biovitrum AB.

Interferon gamma-induced protein 10 (IP-10) for the early prognosis of the risk for severe respiratory failure and death in COVID-19 pneumonia.

OBJECTIVES: Elevated concentrations of soluble urokinase plasminogen activator receptor (suPAR) predict progression to severe respiratory failure (SRF) or death among patients with COVID-19 pneumonia and guide early anakinra treatment. As suPAR testing may not be routinely available in every health-care setting, alternative biomarkers are needed. We investigated the performance of C-reactive protein (CRP), interferon gamma-induced protein-10 (IP-10) and TNF-related apoptosis-inducing ligand (TRAIL) for predicting SRF or death in COVID-19. METHODS: Two cohorts were studied; one discovery cohort with 534 patients from the SAVE-MORE clinical trial; and one validation cohort with 364 patients from the SAVE trial including also 145 comparators. CRP, IP-10 and TRAIL were measured by the MeMed Key® platform in order to select the biomarker with the best prognostic performance for the early prediction of progression into SRF or death. RESULTS: IP-10 had the best prognostic performance: baseline concentrations 2000 pg/ml or higher predicted equally well to suPAR (sensitivity 85.0 %; negative predictive value 96.6 %). Odds ratio for poor outcome among anakinra-treated participants of the SAVE-MORE trial was 0.35 compared to placebo when IP-10 was 2,000 pg/ml or more. IP-10 could divide different strata of severity for SRF/death by day 14 in the validation cohort. Anakinra treatment decreased this risk irrespective the IP-10 concentrations. CONCLUSIONS: IP-10 concentrations of 2,000 pg/ml or higher are a valid alternative to suPAR for the early prediction of progression into SRF or death the first 14 days from hospital admission for COVID-19 and they may guide anakinra treatment. CLINICALTRIALS: gov, NCT04680949 and NCT04357366.

Identification of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based small molecules as selective BRD9 binders.

Targeting bromodomain-containing protein 9 (BRD9) represents a promising strategy for the development of new agents endowed with anticancer properties. With this aim, a set of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based compounds was investigated following a combined approach that relied on in silico studies, chemical synthesis, biophysical and biological evaluation of the most promising items. The protocol was initially based on molecular docking experiments, accounting a library of 1896 potentially synthesizable items tested in silico against the bromodomain of BRD9. A first set of 21 compounds (1-21) was selected and the binding on BDR9 was assessed through AlphaScreen assays. The obtained results disclosed compounds 17 and 20 able to bind BRD9 in the submicromolar range (IC50 = 0.35 ± 0.18 µM and IC50 = 0.14 ± 0.03 µM, respectively) showing a promising selectivity profile when tested against further nine bromodomains. Taking advantage of 3D structure-based pharmacophore models, additional 10 derivatives were selected in silico for the synthetic step and binding assessment, highlighting seven compounds (22, 23, 25, 26, 28, 29, 31) able to selectively bind BRD9 among different bromodomains. The ability of the identified BRD9 binders to cross artificial membranes in vitro was also assessed, revealing a very good passive permeability profile. Preliminary studies were carried out on a panel of healthy and cancer human cell lines to explore the biological behavior of the selected compounds, disclosing a moderate activity and significant selectivity profile towards leukaemia cells. These results highlighted the applicability of the reported multidisciplinary approach for accelerating the selection of promising items and for driving the chemical synthesis of novel selective BRD9 binders. Moreover, the low molecular weight of the reported 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based BRD9 binders suggests the possibility for further exploring the chemical space in order to obtain new analogues with improved potency.

Mapping frailty in people living with HIV: A nationwide study in Greece.

OBJECTIVES: Frailty is known to affect people living with HIV prematurely, compared to the ageing seronegative population. In this cross-sectional study, we aimed to assess frailty prevalence in people living with HIV in Greece and find associations of frailty criteria with clinical data. METHODS: Demographic and clinical data were collected from 477 participants in six HIV clinics. Fried's frailty phenotype was used to assess frailty prevalence, and participants were classified as frail, pre-frail or robust. Associations of several factors with overall frailty phenotype, as well as with frailty criteria, were explored. RESULTS: The median age was 43 years old (IQR = 51.5) and 444/477 (93%) were men. Most of the participants (429/477, 93.5%) had an undetectable HIV viral load, and a CD4 cell count over 500 cells/µl (366/477, 76.7%). Frailty assessment classified 285/477 (62.1%) as robust, 155/477 (33.8%) as pre-frail and 19/477 (4.1%) as frail. Weakness in grip strength was the most prevalent criterion (128/477, 26.8%), followed by exhaustion (46/477, 9.6%). Lower CD4 cell count, history of AIDS diagnosis, CNS disorders, psychiatric diagnoses, and polypharmacy were strongly associated with frailty. CONCLUSIONS: Although the prevalence of frailty in people living with HIV in Greece is uncommon, when combined with pre-frailty over a third of people are affected, which requires attention in clinical practice. The physical and psychological aspects of frailty highlight the need for a holistic approach to prevent or counteract it. The diverse associations of frailty criteria with HIV-related and non-HIV-related factors suggest a possible variation in people's different healthcare needs.