Computational Approaches Drive Developments in Immune-Oncology Therapies for PD-1/PD-L1 Immune Checkpoint Inhibitors

Funding Information: This research was funded by Fundação para a Ciência e Tecnologia (FCT) Portugal, grant number UIDB/50006/2020 (LAQV-REQUIMTE), UIDP/04378/2020 and UIDB/04378/2020 (UCIBIO) and LA/P/0140/2020 (i4HB), the European Commission GLYCOTwinning (GA 101079417), the EJPRD ProDGNE (EJPRD/0001/2020 EU 825575) and SI I&DT, DCMatters (AVISO Nº 17/SI/2019) REF 47212. F.P. gratefully acknowledges FCT for an Assistant Research Position (CEECIND/01649/2021). Publisher Copyright: © 2023 by the authors.Computational approaches in immune-oncology therapies focus on using data-driven methods to identify potential immune targets and develop novel drug candidates. In particular, the search for PD-1/PD-L1 immune checkpoint inhibitors (ICIs) has enlivened the field, leveraging the use of cheminformatics and bioinformatics tools to analyze large datasets of molecules, gene expression and protein–protein interactions. Up to now, there is still an unmet clinical need for improved ICIs and reliable predictive biomarkers. In this review, we highlight the computational methodologies applied to discovering and developing PD-1/PD-L1 ICIs for improved cancer immunotherapies with a greater focus in the last five years. The use of computer-aided drug design structure- and ligand-based virtual screening processes, molecular docking, homology modeling and molecular dynamics simulations methodologies essential for successful drug discovery campaigns focusing on antibodies, peptides or small-molecule ICIs are addressed. A list of recent databases and web tools used in the context of cancer and immunotherapy has been compilated and made available, namely regarding a general scope, cancer and immunology. In summary, computational approaches have become valuable tools for discovering and developing ICIs. Despite significant progress, there is still a need for improved ICIs and biomarkers, and recent databases and web tools have been compiled to aid in this pursuit.publishersversionpublishe

Short-Term Herbage Intake Rate in Temperate Pastures Grasses Grown in Pure or in Intercropping Stands

Oat and ryegrass pastures grown in intercropping systems are the most common forages used during the fall and winter in subtropical and in some temperate climate regions. Nevertheless, it must be taken into account that the way in which the different species of plants are presented to the animals may lead to consequences for the efficiency of the grazing process (Prache and Damasceno 2006). Moreover, in hetero-geneous environments, animals may reduce intake rate due to a partial preference for a specific species (Gonçalves et al. 2009). Regarding the pasture development stage, it is known that the decrease of the leaf/stem ratio causes the animal to reduce the instantaneous herbage intake rate due to the reduction of the bite depth because of the physical barrier imposed by the stem (Benvenutti et al. 2006; Drescher et al. 2006) and/or the search for a higher food quality, in this case, leaf lamina (Soder et al. 2009). However, the dynamics of changes in temperate sward structures grown in pure stands compared to inter-cropping systems and its consequence in heifers\u27 short-term herbage intake rate (STHIR) are scarce. The objective of this work was to assess the changes in the STHIR in pastures of oat, ryegrass and their intercrop during the growing season. The hypothesis tested was that cattle reduce the STHIR in intercropping pastures compared to pure stands, and as the grazing season progress

Tiller Size/Density Compensation in Temperate Climate Grasses Grown in Monoculture or Intercropping Systems under Rotational Grazing

From the standpoint of tiller population dynamics, it is well known that the size and numbers of tillers in forage grasses are inversely related, where a greater tiller population density (TPD) is associated with smaller tillers and vice versa (Sbrissia et al. 2003; Hernandez-Garay et al. 1999; Matthew et al. 1995). This relationship has traditionally been made with the self-thinning power law described by Yoda et al. (1963), which considers the leaf area index (LAI) of the pasture constant when the slope of the relationship between numbers and size of tillers, on a logarithmic scale, is approximately -3/2 (Matthew et al. 1995). Notably few studies have assessed this relationship in intercropping systems. Moreover, although studies that evaluated intercrops showed relationships that were nearly -3/2 for the individually analysed species (Yu et al. 2008; Nie et al. 1997;White and Harper 1970), Nie et al. (1997) suggested that all plants that occur in the grass field should be used to properly estimate self-thinning in mixed species pastures. Thus, the aim of the present study was to test the main hypothesis that the tiller size/density compensation mechanisms operate in the same way in mixed pastures of oat and Italian ryegrass under rotational grazing and that the plant communities adapt their population to maintain a relatively constant LAI

Reusability of L-asparaginase immobilized on silica-based supported ionic liquids

L-asparaginase (ASNase) is an aminohydrolase enzyme used as an anticancer drug, e.g. in the treatment of acute lymphoblastic leukemia, in acrylamide reduction and in biosensing. Nevertheless, its low stability and thermolability, and susceptibility to proteases, hinder its application in the health and food industries. Hence, the improvement of its properties through efficient immobilization methods is in high demand. Thus, this work aims the development of silica-based supported ionic liquids (SILs) for the ASNase immobilization to improve its stability and enable its reusability. While activated silica with no ILs only kept total initial ASNase activity during the first cycle of reaction, SILs allowed 5 cycles of reaction, keeping 82% of initial ASNase activity, reinforcing their potential as alternative enzymatic supports.publishe

Sustainable lysis of Bacillus subtilis biomass to recover the biopharmaceutical L-asparaginase

The first-line biopharmaceutical used to treat Acute lymphoblastic leukemia (ALL), Oncaspar, is based on the enzyme L-asparaginase (ASNase), and has annual sales of ca. USD $100 million. In addition to other sources, genetically modified Bacillus subtilis is regarded as one of the most promising hosts for the ASNase production. The Aliivibrio fischeri ASNase type II, which has anti-tumour activity due its higher specific affinity for L-asparagine, expressed in B. subtillis is located in the periplasm. Therefore, cell lysis is required for the ASNase recovery. Nevertheless, typical cell lysis approaches, e.g. chemical methods with surfactants lead to some biocompatibility concerns and the need of extra purification steps. To overcome this drawback, in this work, ultrasound sonication (USS) conditions were studied to develop a greener and more biocompatible method for ASNase recovery from B. subtilis cell lysis. The USS cell lysis was optimized regarding the amplitude of USS pulse, number of lysis cycles and mass of cells/volume of solvent ratio. The identification and quantification of ASNase and major impurities present in the cell extract after lysis were investigated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high-performance liquid chromatography (SE-HPLC). ASNase activity was determined by monitoring the hydrolysis of the substrate, L-asparagine. The results obtained show that the ideal conditions for B. subtilis cell lysis are an amplitude of USS pulse of 60%, 40 cycles of lysis and 10 mL of phosphatebuffered saline (PBS) per 1 g of cells. Overall, an optimized sustainable B. subtilis cell lysis method was developed, avoiding the use of surfactants and with low energy consumption.publishe

Overview on protein extraction and purification using ionic-liquid-based processes

Proteins are one the most widely studied biomolecules with diverse functions and applications. Aiming at overcoming the current drawbacks of purification processes of proteins, the introduction of ionic liquids (ILs) has been a hot topic of research. ILs have been applied in the creation of aqueous biphasic systems (IL-based ABS), solid-phase extractions through poly(ionic liquid)s (PILs) and supported ionic-liquid phases (SILPs), and in the crystallization of proteins. In this sense, ILs have emerged as solvents, electrolytes or adjuvants, or as supported materials to tune the adsorption/affinity capacity aiming at developing an efficient, cost-effective, sustainable and green IL-based process for protein extraction. This review discusses different IL-based processes in the extraction and purification of proteins in the past years, namely IL-based aqueous biphasic systems (IL-based ABS), solid-phase extractions through PILs and SILPs, and protein crystallization. The type and structure of ILs applied and their influence in the different processes performance are also discussed.publishe

Enhanced enzyme reuse through the bioconjugation of L-asparaginase and silica-based supported ionic liquid-like phase materials

L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme’s single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase–SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10−3 mg of ASNase per mg of SSILLP material, and 60 min). The ASNase–SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.publishe

Carbon nanomaterials for the purification of antileukemic drugs

Getting older is the biggest risk factor for most fatal diseases, including cancer, heart disease and Alzheimer. To overcome such age-related society diseases, it is crucial to optimize the production and purification of biopharmaceuticals, such as nucleic acid-based products, antibodies and recombinant proteins and enzymes. Low cost production combined with high purity levels allow their routinely use by a widespread population. Continuous progresses have been made for the development of recombinant therapeutic enzymes. L-asparaginase (LA) is an antileukemic biopharmaceutical enzyme of current high-cost. LA is produced via fermentation and its purification usually comprises several steps that account up to 80% of its total production cost (1). This work aims to develop sustainable technologies to extract and purify LA. Reusable functionalized nanomaterials, namely carbon nanomaterials (CNTs), are used as cost-effective purification techniques for the target enzyme. Initially, the synthesis and modification of CNTs was performed. Different CNTs were obtained and used for the purification of LA. Commercial LA was used for the first purification tests, in order to understand the behaviour of the enzyme in contact with the nanomaterial. Experimental conditions, such as pH, and material/LA ratio, contact time were optimized. LA activity was quantified by Nessler reaction (2). The first results reveal a total adsorption of LA by the CNTs. Depending on the CNT functionalization/ treatment, different values of recovered activity of LA were obtained. The modified CNTs are shown to be very promising nanomaterials for the purification of LA. The LA was easily attached to CNTs by adsorption under mild conditions. CNTs supports can be a real alternative for a single step immobilization/purification of LA.publishe

Magnetoliposomes based on manganese ferrite nanoparticles for guided transport of antitumor drugs

Publicado em "RICI6 abstract book"In this work, manganese ferrite nanoparticles with size distribution of 46 ± 17 nm and superparamagnetic behavior were synthesized by coprecipitation method. These magnetic nanoparticles were either entrapped in liposomes, originating aqueous magnetoliposomes (AMLs), or covered with a lipid bilayer, forming solid magnetoliposomes (SMLs).MAP-Fis PhD Programme, FEDER, COMPETE/QREN/EU for financial support to CFUM (PEst-C/FIS/UI0607/2013) and FCT and POPH/QREN for PhD grant (SFRH/BD/90949/2012)

Purification of antileukemic drugs through silica-based supported ionic liquids

L-asparaginase (LA) is an enzyme used as a biopharmaceutical for the treatment of acute lymphoblastic leukemia. LA can be produced via fermentation and its purification usually comprises several steps including precipitation, liquid-liquid extraction and chromatography techniques. Among these, ion exchange chromatography, which is often preceded by precipitation with salts as a first pre-chromatographic step, is the most used. However, theses common strategies for protein purification result in low yields and purity, requiring long processing times, while leading to a consequent increase of the process costs. Therefore, the demand for new cost-effective production/purification processes play now a priority role. This work aims the development of cost-effective technologies to purify LA from the complex fermentation medium from Bacillus Subtillis. Silica-based supported ionic liquids (SILs) are investigated as cost-effective purification materials for the target enzyme. The concentration of the extract from the fermentation, material/ extract from fermentation ratio and contact time effects in the purity and yield of LA were optimized. With this strategy, process costs, energy consumed, and waste generated, may be significantly decreased, which may lead to this biopharmaceutical price decrease and wider application.publishe