Correction: Reyes Romero et al. Computer- and NMR-Aided Design of Small-Molecule Inhibitors of the Hub1 Protein. Molecules 2022, 27, 8282.

In the published publication [...].

Structural and biological characterization of pAC65, a macrocyclic peptide that blocks PD-L1 with equivalent potency to the FDA-approved antibodies.

Recent advances in immuno-oncology have opened up new and impressive treatment options for cancer. Notwithstanding, overcoming the limitations of the current FDA-approved therapies with monoclonal antibodies (mAbs) that block the PD-1/PD-L1 pathway continues to lead to the testing of multiple approaches and optimizations. Recently, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD-L1 ranging from sub-micromolar to micromolar. In this study, we present the most potent non-antibody-based PD-1/PD-L1 interaction inhibitor reported to date. The structural and biological characterization of this macrocyclic PD-L1 targeting peptide provides the rationale for inhibition of both PD-1/PD-L1 and CD80/PD-L1 complexes. The IC50 and EC50 values obtained in PD-L1 binding assays indicate that the pAC65 peptide has potency equivalent to the current FDA-approved mAbs and may have similar activity to the BMS986189 peptide, which entered the clinical trial and has favorable safety and pharmacokinetic data. The data presented here delineate the generation of similar peptides with improved biological activities and applications not only in the field of cancer immunotherapy but also in other disorders related to the immune system.

Modeling Adsorption of CO2 in Rutile Metallic Oxide Surfaces: Implications in CO2 Catalysis.

CO2 is the most abundant greenhouse gas, and for this reason, it is the main target for finding solutions to climatic change. A strategy of environmental remediation is the transformation of CO2 to an aggregated value product to generate a carbon-neutral cycle. CO2 reduction is a great challenge because of the large C=O dissociation energy, ~179 kcal/mol. Heterogeneous photocatalysis is a strategy to address this issue, where the adsorption process is the fundamental step. The focus of this work is the role of adsorption in CO2 reduction by means of modeling the CO2 adsorption in rutile metallic oxides (TiO2, GeO2, SnO2, IrO2 and PbO2) using Density Functional Theory (DFT) and periodic DFT methods. The comparison of adsorption on different metal oxides forming the same type of crystal structure allowed us to observe the influence of the metal in the adsorption process. In the same way, we performed a comparison of the adsorption capability between two different surface planes, (001) and (110). Two CO2 configurations were observed, linear and folded: the folded conformations were observed in TiO2, GeO2 and SnO2, while the linear conformations were present in IrO2 and PbO2. The largest adsorption efficiency was displayed by the (001) surface planes. The CO2 linear and folded configurations were related to the interaction of the oxygen on the metallic surface with the adsorbate carbon, and the linear conformations were associated with the physisorption and folded configurations with chemisorption. TiO2 was the material with the best performance for CO2 interactions during the adsorption.

Grammatical evolution-based design of SARS-CoV-2 main protease inhibitors.

A series of SARS-CoV-2 main protease (SARS-CoV-2-Mpro) inhibitors were modeled using evolutive grammar algorithms. We have generated an automated program that finds the best candidate to inhibit the main protease, Mpro, of SARS-CoV-2. The candidates were constructed based on a pharmacophore model of the above-mentioned target; relevant moieties of such molecules were modified using data-basis sets with similar chemical behavior to the reference moieties. Additionally, we used the SMILES language to translate 3D chemical structures to 1D words; then, an evolutive grammar algorithm was used to explore the chemical space and obtain new candidates, which were evaluated via the binding energy of molecular coupling assays as an evaluation function. Finally, sixteen molecules were obtained in 3 runs of our program, three of which show promising binding properties as SARS-CoV-2-Mpro inhibitors. One of them, TTO, maintained its relevant binding properties during 100 ns molecular dynamics experiments. For this reason, TTO is the best candidate to inhibit SARS-CoV-2-Mpro. The software we developed for this contribution is available at the following URL: https://github.com/masotelof/GEMolecularDesign.

Computer- and NMR-Aided Design of Small-Molecule Inhibitors of the Hub1 Protein.

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein.

Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction.

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC50 value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.

Physicochemical and Microbiological Stability of Two Oral Solutions of Methadone Hydrochloride 10 mg/mL.

In this article, we studied physicochemical and microbiological stability and determined the beyond-use date of two oral solutions of methadone in three storage conditions. For this, two oral solutions of methadone (10 mg/mL) were prepared, with and without parabens, as preservatives. They were packed in amber glass vials kept unopened until the day of the test, and in a multi-dose umber glass bottle opened daily. They were stored at 5 ± 3 °C, 25 ± 2 °C and 40 ± 2 °C. pH, clarity, and organoleptic characteristics were obtained. A stability-indicating high-performance liquid chromatography method was used to determine methadone. Microbiological quality was studied and antimicrobial effectiveness testing was also determined following European Pharmacopoeia guidelines. Samples were analyzed at days 0, 7, 14, 21, 28, 42, 56, 70, and 91 in triplicate. After 91 days of storage, pH remained stable at about 6.5-7 in the two solutions, ensuring no risk of methadone precipitation. The organoleptic characteristics remained stable (colorless, odorless, and bitter taste). The absence of particles was confirmed. No differences were found with the use of preservatives. Methadone concentration remained within 95-105% in all samples. No microbial growth was observed. Hence, the two oral methadone solutions were physically and microbiologically stable at 5 ± 3 °C, 25 ± 2 °C, and 40 ± 2 °C for 91 days in closed and opened amber glass bottles.

Competition NMR for Detection of Hit/Lead Inhibitors of Protein-Protein Interactions.

Screening for small-molecule fragments that can lead to potent inhibitors of protein-protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low µM-mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak µM-mM ligand-protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex's protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

Autologous bone marrow-derived cells for venous leg ulcers treatment: a pilot study.

BACKGROUND: Chronic venous leg ulcers (VLUs) are a common problem in clinical practice and available treatments are not satisfactory. The use of adjuvant therapies in combination with lower limb compression may lead to improved healing rates. Chronic wounds are candidates for new strategies in the emergent field of regenerative medicine. Bone marrow-derived cells (BMDCs) contain cells and secrete cytokines known to participate in wound healing. Thus, BMDC therapy seems a logical strategy for the treatment of chronic wounds. Our objective was to evaluate feasibility, safety and initial clinical outcome of autologous BMDC therapy associated with standard treatment in patients with VLUs. METHODS: We conducted an open-label, single-arm, prospective pilot clinical trial in four patients with six chronic VLUs. The study protocol was approved by the institutional and national review boards and ethics committees. Bone marrow was harvest, processed and then administered by multiple injections into the ulcers. All patients received standard treatment and non-healing characteristics of the VLUs were confirmed at study entry. RESULTS: Ulcer size and wound pain evaluated 12 months after BMDC treatment were significantly reduced (P < 0.05). BMDC treatment was safe and well tolerated in long-term follow-up. DISCUSSION: Despite the low number of patients studied, our results showed that autologous BMDC treatment could be a useful, feasible and safe procedure to enhance ulcer healing. However, randomized controlled trials with more patients are needed to address this question and translate this approach into clinical practice.

The correlation of TNF alpha levels with the lipid profile of dengue patients.

Immunological factors, such as cytokines, have been proposed as a cause of changes in the lipid profile of dengue patients. We studied whether serum lipid levels and serum TNF-α levels are associated in a group of dengue patients from an endemic region in the Northwest of Mexico. We found statistically important differences in the serum lipid profile and the TNF-α levels of dengue patients compared with the control group, were observed. However, TNF-α levels did not correlate with the lipid profile of dengue patients.

N-acetylcysteine improves the quality of red blood cells stored for transfusion.

Storage inflicts a series of changes on red blood cells (RBC) that compromise the cell survival and functionality; largely these alterations (storage lesions) are due to oxidative modifications. The possibility of improving the quality of packed RBC stored for transfusion including N-acetylcysteine (NAC) in the preservation solution was explored. Relatively high concentrations of NAC (20-25 mM) were necessary to prevent the progressive leakage of hemoglobin, while lower concentrations (≥2.5 mM) were enough to prevent the loss of reduced glutathione during the first 21 days of storage. Peroxiredoxin-2 was also affected during storage, with a progressive accumulation of disulfide-linked dimers and hetero-protein complexes in the cytosol and also in the membrane of stored RBC. Although the presence of NAC in the storage solution was unable to avoid the formation of thiol-mediated protein complexes, it partially restored the capacity of the cell to metabolize H2O2, indicating the potential use of NAC as an additive in the preservation solution to improve RBC performance after transfusion.

Cross-cultural Adaptation and Validation of the Medication Regimen Complexity Index Adapted to Spanish.

BACKGROUND: The most widely used validated instrument to assess the complexity of medication regimens is the Medication Regimen Complexity Index (MRCI). OBJECTIVE: This study aimed to translate, adapt, and validate a reliable version of the MRCI adapted to Spanish (MRCI-E). METHODS: The cross-cultural adaptation process consisted of an independent translation by 3 clinical pharmacists and a backtranslation by 2 native English speakers. A reliability analysis was conducted on 20 elderly randomly selected patients. Two clinical pharmacists calculated the MRCI-E from discharge treatments and 2 months later. For the validity analysis, the sample was augmented to 60 patients. Convergent validity was assessed by analyzing the correlation between the number of medications; discriminant validity was stratified by gender; and predictive validity was determined by analyzing the ability to predict readmission and mortality at 3 and 6 months. RESULTS: The MRCI-E retained the original structure of 3 sections. The reliability analysis demonstrated an excellent internal consistency (Cronbach's α=0.83), and the intraclass correlation coefficient exceeded 0.9 in all cases. The correlation coefficient with the number of medications was 0.883 ( P<0.001). No significant differences were found when stratified by gender (3.6; 95%CI=-2.9 to 10.2; P=0.27). Patients who were readmitted at 3 months had a higher MRCI-E score (10.7; 95%CI=4.4 to 17.2; P=0.001). The differences remained significant in patients readmitted at 6 months, but differences in mortality were not detected. CONCLUSIONS: The MRCI-E retains the reliability and validity of the original index and provides a suitable tool to assess the complexity of medication regimens in Spanish.

Solubilizer Tag Effect on PD-L1/Inhibitor Binding Properties for m-Terphenyl Derivatives.

Although heavily studied, the subject of anti-PD-L1 small-molecule inhibitors is still elusive. Here we present a systematic overview of the principles behind successful anti-PD-L1 small-molecule inhibitor design on the example of the m-terphenyl scaffold, with a particular focus on the neglected influence of the solubilizer tag on the overall affinity toward PD-L1. The inhibitor developed according to the proposed guidelines was characterized through its potency in blocking PD-1/PD-L1 complex formation in homogeneous time-resolved fluorescence and cell-based assays. The affinity is also explained based on the crystal structure of the inhibitor itself and its costructure with PD-L1 as well as a molecular modeling study. Our results structuralize the knowledge related to the strong pharmacophore feature of the m-terphenyl scaffold preferential geometry and the more complex role of the solubilizer tag in PD-L1 homodimer stabilization.

Design of mobile and website health application devices for drug tolerability in hereditary fructose intolerance.

BACKGROUND: Hereditary fructose intolerance (HFI) is a rare metabolic disease caused by aldolase B deficiency. The aim of our study was to analyse excipient tolerability in patients with HFI and other related diseases and to design mobile and website health applications to facilitate the search for drugs according to their tolerance. RESULTS: A total of 555 excipients listed in the Spanish Medicines Agency database (July 2023) were classified as suitable for HFI patients, suitable with considerations ((glucose and glucose syrup, intravenous sucrose, oral mannitol, polydextrose, gums and carrageenans, ethanol, sulfite caramel and vanilla), not recommended (intravenous mannitol) and contraindicated (fructose, oral sucrose, invert sugar, sorbitol, maltitol, lactitol, isomaltitol, fruit syrups, honey, sucrose esters and sorbitol esters). Glucose and glucose syrup were classified as suitable with considerations due to its possible fructose content and their potential endogenous fructose production. For other related intolerances, wheat starch was contraindicated and oatmeal was not recommended in celiac disease; oral lactose and lactose-based coprocessed excipient (Cellactose®) were not recommended in lactose intolerance; and glucose, invert sugar and oral sucrose were not recommended in diabetes mellitus. The applications were named IntoMed®. Results are listed in order of tolerability (suitable drugs appear first and contraindicated drugs at the end), and they are accompanied by a note detailing their classified excipients. If a drug contains excipients within different categories, the overall classification will be the most restrictive. The apps are also able to classify substances with the same criteria if they act as active ingredients. The tools exhibited good usability (82.07 ± 13.46 points on the System Usability Scale [range: 0-100]) on a sample of HFI patients, their families and health care professionals. CONCLUSIONS: IntoMed® is a tool for finding information about the tolerability of drugs according to excipients for patients with HFI and other related intolerances, with good usability. It is a fast and reliable system that covers the current excipient legislation and expands on it with other specific information: HFI patients should be alert for excipients such as mannitol (especially in intravenous drugs), fruit syrups, honey, sulfite caramel or vanilla. Glucose might contain or produce fructose, and special precaution is needed because of potential errors in their composition.

Physicochemical stability of bortezomib solutions for subcutaneous administration.

For the majority of cytotoxic drug preparations, such as bortezomib, the unit dose information is not available. In addition, there is a lack of information on the physicochemical stability of the pharmaceutical preparation after opening; this information is crucial for its administration to patients in successive visits, and the per-patient cost can be affected. The purpose of our proposed physicochemical stability study is to determine the shelf life of the reconstituted liquid product under refrigeration and clinical practice conditions. This evaluation was extended to both vials and ready-to-use syringes prefilled with the contents of the open vial. The stability test design includes the specified storage conditions and the critical physicochemical parameters of reconstituted injectable bortezomib. Furthermore, this approach includes the determination of impurities, the monitoring of the purity of the mean peak using a photodiode array, the control of the mass balance, the monitoring of subvisible particles using a laser diffraction analyser, and the setting of stability specifications. For the chemical stability study, the amount of bortezomib and its degradation products were determined using a stability-indicating HPLC method. The physical inspection of the samples was performed throughout the stability study, and their pH values were also monitored. Bortezomib (2.5 mg/mL) in 0.9% sodium chloride remained stable for 7 days when stored in both polypropylene syringes and vials at 5 ± 3 °C (refrigeration) and shielded from light. Additionally, it exhibits stability for 24 h under storage conditions simulating clinical use (20-30 °C and protected from light). The proposed protocol provides the stability in the vials once reconstituted and in prefilled refrigerated syringes; this protocol can be used to reduce waste and increase cost savings.

Persistence in the Methadone Maintenance Program and Its Relationship with the Medication Regimen Complexity Index in Opioid-Dependent Patients.

It has been shown that the Medication Regimen Complexity Index (MRCI) is a useful and reliable tool for calculating the complexity of the pharmacotherapeutic regimen (CPR). Furthermore, a high MRCI is associated with lower adherence. However, the MRCI of opioid-dependent patients (ODP) has not been studied. The aim of this study is to calculate the Methadone Maintenance Program (MMP) persistence and the MRCI score in a ODP cohort. Second, to analyze its relationship and association with other variables. To accomplish this research, an observational study including adults with a confirmed diagnosis of opiate-dependency according to the DSM-5 in a MMP center was carried out. To define MMP-persistence, a group was created by the researchers who defined five weighted items according to their agreed importance. Our first contribution was to create a new definition of MMP-persistence. This study also identified age, comorbidities, and received methadone maintenance doses as successful predictors for MMP-persistence. We have also shown that the MRCI does not seem to be a useful tool to determine MMP-persistence, probably because there are multiple factors that influence it in addition to the CPR. It is necessary to continue searching for more precise selection and stratification tools for ODP to improve their persistence.

Chemical analysis and chemical imaging of fragrances and volatile compounds by low-temperature plasma ionization mass spectrometry.

RATIONALE: The rapid analysis of volatile compounds, such as fragrances, is important in many commercial industries. The various ambient ionization methods have until now been largely applied to non-volatile or low-volatile compounds with success, and this study develops a semi-quantitative method for volatile compounds in commercial cleaning products. METHODS: Low-temperature plasma (LTP) ionization was used to perform rapid analysis, determine limits of detection (LODs) and perform chemical imaging on eight fragrances. Several mass analyzers including an ion trap, a quadrupole and an orbitrap were used to rapidly screen volatile compounds from cloth, paper, and glass and determine compositions present in a commercial cleaning product. Peltier cooling was used in some cases to enhance the retention time of compounds on a surface. RESULTS: This LTP method allowed the detection of fragrances in low picogram absolute amounts from glass, paper and cloth. Quantitation was demonstrated for compounds in a commercial cleaning product 1 min after the product was applied to a vinyl tile surface. High-throughput analysis and simultaneous detection of multiple compounds in a mixture were demonstrated with analysis times of less than 1 min. Modest spatial resolution (better than 1 cm) was achieved with LTP ionization. CONCLUSIONS: A semi-quantitative method has been demonstrated for the routine analysis of volatile and semi-volatile compounds. This method would be useful in quality control and production environments to determine product persistence, location of analytes and to complement olfactory studies for determining concentrations in the ambient environment.

Cryopreservation of an artificial human oral mucosa stroma. A viability and rheological study.

The aim of this study was to evaluate the viability and biomechanical properties of artificial human oral mucosa stroma (HOMS) subjected to cryopreservation with different cryoprotectant solutions. Artificial HOMS based on a fibrin-agarose matrix with human gingival fibroblasts cultured 7 days in vitro were cryopreserved with three cryoprotectant solutions: (A) TC-199 Medium, DMSO 15%, albumin; (B) DMEM, FCS, DMSO 10%; (C) QC Medium, glycerol. As controls, artificial HOMS not subjected to cryopreservation (CF) and HOMS cryopreserved without cryoprotectant solution (CS) were used. Histological analysis by light microscopy showed that solutions A and B preserved a pattern of porosity similar to values in CF. Based on the number of intact cells in the fibrin-agarose matrix, substitutes preserved with solution B showed the best results. Cell proliferation detected with PCNA immunochemical methods showed that the cell proliferation index was highest in substitutes cryopreserved with solution B. The reculture method and cell viability analyses with Live & Dead(®) revealed increased number of viable in cells preserved with solution B. Artificial stroma substitutes in CS control samples showed the greatest alterations in microstructure and cell proliferation. Analysis of the biomechanical properties showed that substitutes cryopreserved with different solutions had adequate rheological parameters (yield stress, elastic modulus and viscous modulus) and were therefore suitable for use in regenerative medicine. These results establish effective methods of cryopreservation for all experimental situations and suggest that solution B (DMEM, FCS, DMSO 10%) was the best cryoprotectant for the cryopreservation of an artificial oral human mucosa substitute based on a fibrin-agarose matrix.

An in vitro biocompatibility study of conventional and resin-modified glass ionomer cements.

PURPOSE: To evaluate the biocompatibility of a glass-ionomer (GIC) and a resin-modified glass-ionomer cement (RM-GIC), cell viability was examined in a model of human gingival fibroblasts using morphological, biochemical, and ionic patterns by means of phase contrast microscopy, lactate dehydrogenase (LDH) release, and quantitative x-ray microanalysis (EPXMA). MATERIALS AND METHODS: The GIC Ketac-Molar Easymix (3M ESPE) and the RM-GIC Vitrebond (3M ESPE) were compared in human gingival fibroblasts exposed to the cements for 72 h. As controls, fibroblasts cultured with DMEM culture medium (negative control) and with 1% triton × (positive control) were used. RESULTS: Light microscopic findings showed greater morphological alterations in cells exposed to RM-GIC than to GIC. The relative percentage of LDH released from the cells to the supernatant was significantly higher in RMGIC cultures than in the control. Quantitative x-ray microanalysis showed that cultures exposed to RM-GIC were characterized by an increase in intracellular Na and a decrease in intracellular Cl and K. These changes in ion composition were significant compared to control and GIC cultures. CONCLUSION: The three indicators of cellular biocompatibility after 72 h of exposure showed that RM-GIC led to more marked alterations than GIC in human gingival fibroblasts.