Alzheimer's Disease Immunotherapy and Mimetic Peptide Design for Drug Development: Mutation Screening, Molecular Dynamics, and a Quantum Biochemistry Approach Focusing on Aducanumab::Aß2-7 Binding Affinity.

Seven treatments are approved for Alzheimer's disease, but five of them only relieve symptoms and do not alter the course of the disease. Aducanumab (Adu) and lecanemab are novel disease-modifying antiamyloid-ß (Aß) human monoclonal antibodies that specifically target the pathophysiology of Alzheimer's disease (AD) and were recently approved for its treatment. However, their administration is associated with serious side effects, and their use is limited to early stages of the disease. Therefore, drug discovery remains of great importance in AD research. To gain new insights into the development of novel drugs for Alzheimer's disease, a combination of techniques was employed, including mutation screening, molecular dynamics, and quantum biochemistry. These were used to outline the interfacial interactions of the Aducanumab::Aß2-7 complex. Our analysis identified critical stabilizing contacts, revealing up to 40% variation in the affinity of the Adu chains for Aß2-7 depending on the conformation outlined. Remarkably, two complementarity determining regions (CDRs) of the Adu heavy chain (HCDR3 and HCDR2) and one CDR of the Adu light chain (LCDR3) accounted for approximately 77% of the affinity of Adu for Aß2-7, confirming their critical role in epitope recognition. A single mutation, originally reported to have the potential to increase the affinity of Adu for Aß2-7, was shown to decrease its structural stability without increasing the overall binding affinity. Mimetic peptides that have the potential to inhibit Aß aggregation were designed by using computational outcomes. Our results support the use of these peptides as promising drugs with great potential as inhibitors of Aß aggregation.

Tofacitinib and peficitinib inhibitors of Janus kinase for autoimmune disease treatment: a quantum biochemistry approach.

Autoimmune inflammatory diseases, such as rheumatoid arthritis (RA) and ulcerative colitis, are associated with an uncontrolled production of cytokines leading to the pronounced inflammatory response of these disorders. Their therapy is currently focused on the inhibition of cytokine receptors, such as the Janus kinase (JAK) protein family. Tofacitinib and peficitinib are JAK inhibitors that have been recently approved to treat rheumatoid arthritis. In this study, an in-depth analysis was carried out through quantum biochemistry to understand the interactions involved in the complexes formed by JAK1 and tofacitinib or peficitinib. Computational analyses provided new insights into the binding mechanisms between tofacitinib or peficitinib and JAK1. The essential amino acid residues that support the complex are also identified and reported. Additionally, we report new interactions, such as van der Waals; hydrogen bonds; and alkyl, pi-alkyl, and pi-sulfur forces, that stabilize the complexes. The computational results revealed that peficitinib presents a similar affinity to JAK1 compared to tofacitinib based on their interaction energies.

Unveiling fructose and glucose binding to human serum albumin: fluorescence measurements and docking, molecular dynamics and quantum biochemistry computations.

This research examines the interaction between human serum albumin (HSA) and various sugar forms (ß-D-fructofuranose (FRC), α-D-glucopyranose (GLC), Keto-D-fructose (FRO), Aldehydo-D-glucose (GLO), and modified Aldehydo-D-glucose (GLOm)) using fluorescent spectroscopy, molecular docking simulations, molecular dynamics, protein conformational clusters (EnGens), molecular fractionation with conjugate caps (MFCC) and quantum biochemistry analysis. We analyze molecular and quantum aspects, uncovering interaction energies between sugar atoms and amino acids. Total interaction energy considers protein fragmentation, energetic decomposition, and interaction energy from a bottom-up perspective. Molecular dynamics reveal that unmodified Aldehydo-D-glucose (GLO) escapes HSA binding sites, explaining gradual glycation. We pioneer studying HSA's binding mechanism with glucose and fructose in a 1:1 ratio using long molecular dynamics simulations. Results suggest the transitional GLOm form has a higher Sudlow I site propensity than unmodified glucose, crucial for K195 glycation. FRO and GLOm interaction tendencies move toward a deeper FA7 cavity, near its center. This approach effectively elucidates small molecule binding mechanisms, consistent with previous experimental results.Communicated by Ramaswamy H. Sarma.

Noncovalent binding of carbofuran to acetylcholinesterase from Homo sapiens, Danio rerio, Apis mellifera and Caenorhabditis elegans: Homology modelling, molecular docking and dynamics, and quantum biochemistry description.

Although various regulatory agencies have banned or severely restricted the use of carbofuran (CAR), recent reports indicate the presence of CAR residues in both cultivated and wild areas. This pesticide is a potent inhibitor of acetylcholinesterase (AChE), which acts by preventing the hydrolysis of acetylcholine (ACh). Given the critical role of AChE::ACh in the proper functioning of the nervous system, we thought it appropriate to investigate the binding of CAR to AChEs from Homo sapiens, Danio rerio, Apis mellifera, and Caenorhabditis elegans using homology modelling, molecular docking, molecular dynamics, and quantum biochemistry. Molecular docking and dynamics results indicated peculiar structural behavior in each AChE::CAR system. Quantum biochemistry results showed similar affinities for all complexes, confirming the description of carbofuran as a broad-spectrum pesticide, and have a limited correlation with IC50 values. We found the following decreasing affinity order of AChE species: H. sapiens > A. mellifera > C. elegans > D. rerio. The computational results suggest that CAR occupies different pockets in the AChEs studied. In addition, our results showed that CAR binds to hsAChE and ceAChE in a very similar manner: it has high affinities for the same subsites in both species and forms hydrogen bonds with residues (hsTYR124 and ceTRP107) occupying homologous positions in the peripheral site. This suggests that this nematode is a potential model to evaluate the toxicity of carbamates, even though the sequence identity between them is only 41 %. Interestingly, we also observed that the catalytic histidines of drAChE and amAChE exhibited favorable contacts with carbofuran, suggesting that the non-covalent binding of carbofuran to these proteins may promote faster carbamylation rates than the binding modes to human and worm acetylcholinesterases. Our computational results provide a better understanding of the binding mechanisms in these complexes, as well as new insights into the mechanism of carbamylation.

In silico description of the adsorption of cell signaling pathway proteins ovalbumin, glutathione, LC3, TLR4, ASC PYCARD, PI3K and NF-Kß on 7.0 nm gold nanoparticles: obtaining their Lennard-Jones-like potentials through docking and molecular mechanics.

The impact of vaccination on the world's population is difficult to calculate. For developing different types of vaccines, adjuvants are substances added to vaccines to increase the magnitude and durability of the immune response and the effectiveness of the vaccine. This work explores the potential use of spherical gold nanoparticles (AuNPs) as adjuvants. Thus, we employed docking techniques and molecular mechanics to describe how a AuNP 7.0 nm in diameter interacts with cell signaling pathway proteins. Initially, we used X-ray crystallization data of the proteins ovalbumin, glutathione, LC3, TLR4, ASC PYCARD, PI3K, and NF-Kß to study the adsorption with an AuNP through molecular docking. Therefore, interaction energies were obtained for the AuNP complexes and individual proteins, as well as the AuNP and OVA complex (AuNP@OVA) with each cellular protein, respectively. Results showed that AuNPs had the highest affinity for OVA individually, followed by glutathione, ASC PYCARD domain, LC3, PI3K, NF-Kß, and TLR4. Furthermore, when evaluating the AuNP@OVA complex, glutathione showed a greater affinity with more potent interaction energy when compared to the other studied systems.

Quantum biochemistry description of PI3Kα enzyme bound to selective inhibitors.

The PI3K class I is composed of four PI3K isoforms that serve as regulatory enzymes governing cellular metabolism, proliferation, and survival. The hyperactivation of PI3Kα is observed in various types of cancer and is linked to poor prognosis. Unfortunately, the development inhibitors selectively targeting one of the isoforms remains challenging, with only few agents in clinical use. The main difficulty arises from the high conservation among residues at the ATP-binding pocket across isoforms, which also serves as target pocket for inhibitors. In this work, molecular dynamics and quantum calculations were performed to investigate the molecular features guiding the binding of selective inhibitors, alpelisib and GDC-0326, into the ATP-binding pocket of PI3Kα. While molecular dynamics allowed crystallographic coordinates to relax, the interaction eergy between each amino acid residues and inhibitors was obtained by combining the Molecular Fractionation with Conjugated Caps scheme with Density Functional Theory calculations. In addition, the atomic charge of ligands in the bound and unbound (free) was calculated. Results indicated that the most relevant residues for the binding of alpelisib are Ile932, Glu859, Val851, Val850, Tyr836, Met922, Ile800, and Ile848, while the most important residues for the binding of GDC-0326 are Ile848, Ile800, Ile932, Gln859, Glu849, and Met922. In addition, residues Trp780, Ile800, Tyr836, Ile848, Gln859 Val850, Val851, Ile932 and Met922 are common hotspots for both inhibitors. Overall, the results from this work contribute to improving the understanding of the molecular mechanisms controlling selectivity and highlight important interactions to be considered during the rational design of new agents.Communicated by Ramaswamy H. Sarma.

Pharmacokinetic Profile Evaluation of Novel Combretastatin Derivative, LASSBio-1920, as a Promising Colorectal Anticancer Agent.

LASSBio-1920 was synthesized due to the poor solubility of its natural precursor, combretastatin A4 (CA4). The cytotoxic potential of the compound against human colorectal cancer cells (HCT-116) and non-small cell lung cancer cells (PC-9) was evaluated, yielding IC50 values of 0.06 and 0.07 µM, respectively. Its mechanism of action was analyzed by microscopy and flow cytometry, where LASSBio-1920 was found to induce apoptosis. Molecular docking simulations and the enzymatic inhibition study with wild-type (wt) EGFR indicated enzyme-substrate interactions similar to other tyrosine kinase inhibitors. We suggest that LASSBio-1920 is metabolized by O-demethylation and NADPH generation. LASSBio-1920 demonstrated excellent absorption in the gastrointestinal tract and high central nervous system (CNS) permeability. The pharmacokinetic parameters obtained by predictions indicated that the compound presents zero-order kinetics and, in a human module simulation, accumulates in the liver, heart, gut, and spleen. The pharmacokinetic parameters obtained will serve as the basis to initiate in vivo studies regarding LASSBio-1920's antitumor potential.

In silico analysis of ACE2 from different animal species provides new insights into SARS-CoV-2 species spillover.

Aim: This study aimed to analyze the phylogenetic relationships between the ACE2 of humans and other animals and investigate the potential interaction between SARS-CoV-2 RBD and ACE2 of different species. Materials & methods: The phylogenetic construction and molecular interactions were assessed using computational models. Results & conclusion: Despite the evolutionary distance, 11 species had a perfect fit for the interaction between their ACE2 and SARS-CoV-2 RBD (Chinchilla lanigera, Neovison vison, Rhinolophus sinicus, Emballonura alecto, Saccopteryx bilineata, Numida meleagris). Among them, the avian N. meleagris was reported for the first time in this study as a probable SARS-CoV-2 host due to the strong molecular interactions. Therefore, predicting potential hosts for SARS-CoV-2 for understanding the epidemiological cycle and proposal of surveillance strategies.

Here, computational analysis was employed to predict the interaction between the Spike protein from SARS-COV-2 with the ACE2 receptor with animals that could serve as a reservoir for SARS-CoV-2 spillover. Our results reported for the first time that N. meleagris could act as a possible host for SARS-CoV-2.

Losartan as an ACE inhibitor: a description of the mechanism of action through quantum biochemistry.

Losartan (LST) is a potent and selective angiotensin II (Ang II) type 1 (AT1) receptor antagonist widely used in the treatment of hypertension. The formation of Ang II is catalyzed by the angiotensin I-converting enzyme (ACE) through proteolytic cleavage of angiotensin I (Ang I), which is involved in the control of blood pressure. Despite the vast literature on the relationship of losartan with the renin-angiotensin system (RAS), the actions of losartan on the sACE enzyme are so far poorly understood. In view of this, we investigated how losartan can interact with the sACE enzyme to block its activity and intracellular signaling. After performing docking assays following quantum biochemistry calculations using losartan and sACE crystallographic data, we report that their interaction results reveal a new mechanism of action with important implications for understanding its effects on hypertension.

Quantum Biochemistry and MM-PBSA Description of the ZIKV NS2B-NS3 Protease: Insights into the Binding Interactions beyond the Catalytic Triad Pocket.

The Zika virus protease NS2B-NS3 has a binding site formed with the participation of a H51-D75-S135 triad presenting two forms, active and inactive. Studies suggest that the inactive conformation is a good target for the design of inhibitors. In this paper, we evaluated the co-crystallized structures of the protease with the inhibitors benzoic acid (5YOD) and benzimidazole-1-ylmethanol (5H4I). We applied a protocol consisting of two steps: first, classical molecular mechanics energy minimization followed by classical molecular dynamics were performed, obtaining stabilized molecular geometries; second, the optimized/relaxed geometries were used in quantum biochemistry and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations to estimate the ligand interactions with each amino acid residue of the binding pocket. We show that the quantum-level results identified essential residues for the stabilization of the 5YOD and 5H4I complexes after classical energy minimization, matching previously published experimental data. The same success, however, was not observed for the MM-PBSA simulations. The application of quantum biochemistry methods seems to be more promising for the design of novel inhibitors acting on NS2B-NS3.

Characterization of the binding interaction between atrazine and human serum albumin: Fluorescence spectroscopy, molecular dynamics and quantum biochemistry.

Atrazine (ATR), one of the most used herbicides worldwide, causes persistent contamination of water and soil due to its high resistance to degradation. ATR is associated with low fertility and increased risk of prostate cancer in humans, as well as birth defects, low birth weight and premature delivery. Describing ATR binding to human serum albumin (HSA) is clinically relevant to future studies about pharmacokinetics, pharmacodynamics and toxicity of ATR, as albumin is the most abundant carrier protein in plasma and binds important small biological molecules. In this work we characterize, for the first time, the binding of ATR to HSA by using fluorescence spectroscopy and performing simulations using molecular docking, classical molecular dynamics and quantum biochemistry based on density functional theory (DFT). We determine the most likely binding sites of ATR to HSA, highlighting the fatty acid binding site FA8 (located between subdomains IA-IB-IIA and IIB-IIIA-IIIB) as the most important one, and evaluate each nearby amino acid residue contribution to the binding interactions explaining the fluorescence quenching due to ATR complexation with HSA. The stabilization of the ATR/FA8 complex was also aided by the interaction between the atrazine ring and SER454 (hydrogen bond) and LEU481(alkyl interaction).

Gallic acid leads to cell death of Candida albicans by the apoptosis mechanism.

Aim: To evaluate the antifungal activity of gallic acid (GA) against the strains of Candida spp. resistant to fluconazole and to determine its mechanism of action. Materials & methods: Antifungal activity was evaluated using the broth microdilution and flow cytometry techniques. Results: GA presented minimum inhibitory concentrations ranging from 16 to 72 µg/ml, causing alterations of the membrane integrity and mitochondrial transmembrane potential, production of reactive oxygen species and externalization of phosphatidylserine. Conclusion: GA has potential antifungal activity against Candida spp.

ACE2-derived peptides interact with the RBD domain of SARS-CoV-2 spike glycoprotein, disrupting the interaction with the human ACE2 receptor.

Vaccines could be the solution to the current SARS-CoV-2 outbreak. However, some studies have shown that the immunological memory only lasts three months. Thus, it is imperative to develop pharmacological treatments to cope with COVID-19. Here, the in silico approach by molecular docking, dynamic simulations and quantum biochemistry revealed that ACE2-derived peptides strongly interact with the SARS-CoV-2 RBD domain of spike glycoprotein (S-RBD). ACE2-Dev-PepI, ACE2-Dev-PepII, ACE2-Dev-PepIII and ACE2-Dev-PepIV complexed with S-RBD provoked alterations in the 3D structure of S-RBD, leading to disruption of the correct interaction with the ACE2 receptor, a pivotal step for SARS-CoV-2 infection. This wrong interaction between S-RBD and ACE2 could inhibit the entry of SARS-CoV-2 in cells, and thus virus replication and the establishment of COVID-19 disease. Therefore, we suggest that ACE2-derived peptides can interfere with recognition of ACE2 in human cells by SARS-CoV-2 in vivo. Bioinformatic prediction showed that these peptides have no toxicity or allergenic potential. By using ACE2-derived peptides against SARS-CoV-2, this study points to opportunities for further in vivo research on these peptides, seeking to discover new drugs and entirely new perspectives to treat COVID-19.Communicated by Ramaswamy H. Sarma.

Quantum biochemistry, molecular docking, and dynamics simulation revealed synthetic peptides induced conformational changes affecting the topology of the catalytic site of SARS-CoV-2 main protease.

The recent outbreak caused by SARS-CoV-2 continues to threat and take many lives all over the world. The lack of an efficient pharmacological treatments are serious problems to be faced by scientists and medical staffs worldwide. In this work, an in silico approach based on the combination of molecular docking, dynamics simulations, and quantum biochemistry revealed that the synthetic peptides RcAlb-PepI, PepGAT, and PepKAA, strongly interact with the main protease (Mpro) a pivotal protein for SARS-CoV-2 replication. Although not binding to the proteolytic site of SARS-CoV-2 Mpro, RcAlb-PepI, PepGAT, and PepKAA interact with other protein domain and allosterically altered the protease topology. Indeed, such peptide-SARS-CoV-2 Mpro complexes provoked dramatic alterations in the three-dimensional structure of Mpro leading to area and volume shrinkage of the proteolytic site, which could affect the protease activity and thus the virus replication. Based on these findings, it is suggested that RcAlb-PepI, PepGAT, and PepKAA could interfere with SARS-CoV-2 Mpro role in vivo. Also, unlike other antiviral drugs, these peptides have no toxicity to human cells. This pioneering in silico investigation opens up opportunity for further in vivo research on these peptides, towards discovering new drugs and entirely new perspectives to treat COVID-19.Communicated by Ramaswamy H. Sarma.

Insulin degludec and glutamine dipeptide modify glucose homeostasis and liver metabolism in diabetic mice undergoing insulin-induced hypoglycemia.

This study investigated whether a 30-day co-treatment with 1 g/kg glutamine dipeptide (GdiP) and 1 U/kg regular (rapid acting) or 5 U/kg degludec (long acting) insulins modifies glucose homeostasis and liver metabolism of alloxan-induced type 1 diabetic (T1D) male Swiss mice undergoing insulin-induced hypoglycemia (IIH). Glycemic curves were measured in fasted mice after IIH with 1 U/kg regular insulin. One hour after IIH, the lipid profile and AST and ALT activities were assayed in the serum. Morphometric analysis was assessed in the liver sections stained with hematoxylin-eosin and glycolysis, glycogenolysis, gluconeogenesis and ureagenesis were evaluated in perfused livers. T1D mice receiving GdiP or the insulins had a smaller blood glucose drop at 60 minutes after IIH, which was not sustained during the subsequent period up to 300 minutes. The 30-day treatment of T1D mice with insulin degludec, but not with regular insulin, improved fasting glycemia, body weight gain and serum activity of AST and ALT. Treatments with insulin degludec, GdiP and insulin degludec + GdiP decreased the liver capacity in synthesizing glucose from alanine. GdiP, in combination with both insulins, was associated with increases in the serum triglycerides and, in addition, regular insulin and GdiP increased AST and ALT activities, which could be the consequence of hepatic glycogen overload. GdiP and the insulins improved the IIH, although to a small extent. Caution is recommended, however, with respect to the use of GdiP because of its increasing effects on serum triglycerides and AST plus ALT activities.

Computational approach, scanning electron and fluorescence microscopies revealed insights into the action mechanisms of anticandidal peptide Mo-CBP3-PepIII.

AIMS: The Candida genus is composed of opportunistic pathogens that threaten public health. Given the increase in resistance to current drugs, it is necessary to develop new drugs to treat infections by these pathogens. Antimicrobial peptides are promising alternative molecules with low cost, broad action spectrum and low resistance induction. This study aimed to clarify the action mechanisms of synthetic peptides against Candida albicans. MAIN METHODS: The mode of action of the anticandidal peptides Mo-CBP3-PepIII were analyzed through molecular dynamics and quantum biochemistry methods against Exo-ß-1,3-glucanase (EXG), vital to cell wall metabolism. Furthermore, scanning electron (SEM) and fluorescence (FM) microscopies were employed to corroborate the in silico data. KEY FINDINGS: Mo-CBP3-PepIII strongly interacted with EXG (-122.2 kcal mol-1) at the active site, higher than the commercial inhibitor pepstatin. Also, molecular dynamics revealed the insertion of Mo-CBP3-PepIII into the yeast membrane. SEM analyses revealed that Mo-CBP3-PepIII induced cracks and scars of the cell wall and FM analyses confirmed the pore formation on the Candida membrane. SIGNIFICANCE: Mo-CBP3-PepIII has strong potential as a new drug with a broad spectrum of action, given its different mode of action compared to conventional drugs.

Molecular insight on the binding of stevia glycosides to bovine serum albumin.

The interaction of the steviol and its glycosides (SG), steviolbioside, and rebaudioside A, with bovine serum albumin (BSA) was studied by absorption and fluorescence spectroscopy techniques alongside molecular docking. The stevia derivatives quenched the fluorescence of BSA by a dynamic quenching mechanism, indicating the interaction between the stevia derivatives and BSA. The binding constant (Kb) of steviol was 100-1000-fold higher than those of SG. The stevia derivative/BSA binding reaction was spontaneous and involved the formation of hydrogen bonds and van der Waals interactions between steviol and steviolbioside with BSA, and water reorganization around the rebaudioside A/BSA complex. Molecular docking pointed out the FA1 and FA9 binding sites of BSA as the probable binding sites of steviol and SG, respectively. In conclusion, steviol enhanced hydrophobicity and small size compared to SG may favor its binding to BSA. As steviol and its glycosides share binding sites on BSA with free fatty acids and drugs, they may be competitively displaced from plasma albumin under various physiological states or disease conditions. These findings are clinically relevant and provide an insight into the pharmacokinetics and pharmacodynamics of the stevia glycosides.

Graphene: Insights on Biological, Radiochemical and Ecotoxicological Aspects.

Graphene, including graphene quantum dots, its oxide and unoxidized forms (pure graphene) have several properties, like fluorescence, electrical conductivity, theoretical surface area, low toxicity, and high biocompatibility. In this study, we evaluated genotoxicity (in silico analysis using the functional density theory-FDT), cytotoxicity (human glioblastoma cell line), in vivo pharmacokinetics, in vivo impact on microcirculation and cell internalization assay. It was also radiolabeled with lutetium 177 (177Lu), a beta emitter radioisotope to explore its therapeutic use as nanodrug. Finally, the impact of its disposal in the environment was analyzed using ecotoxicological evaluation. FDT analysis demonstrated that graphene can construct covalent and non-covalent bonds with different nucleobases, and graphene oxide is responsible for generation of reactive oxygen species (ROS), corroborating its genotoxicity. On the other hand, non-cytotoxic effect on glioblastoma cells could be demonstrated. The pharmacokinetics analysis showed high plasmatic concentration and clearance. Topical application of 0.1 and 1 mg/kg of graphene nanoparticles on the hamster skinfold preparation did not show inflammatory effect. The cell internalization assay showed that 1-hour post contact with cells, graphene can cross the plasmatic membrane and accumulate in the cytoplasm. Radio labeling with 177Lu is possible and its use as therapeutic nanosystem is viable. Finally, the ecotoxicity analysis showed that A. silina exposed to graphene showed pronounced uptake and absorption in the nauplii gut and formation of ROS. The data obtained showed that although being formed exclusively of carbon and carbon-oxygen, graphene and graphene oxide respectively generate somewhat contradictory results and more studies should be performed to certify the safety use of this nanoplatform.

Quantum biochemistry in cancer immunotherapy: New insights about CTLA-4/ipilimumab and design of ipilimumab-derived peptides with high potential in cancer treatment.

Cancer is a group of diseases involving disordered growth of abnormal cells with the potential to invade and spread to other parts of the body. Today, immunotherapy is the most efficient treatment, with fewer side effects. Notably, the employment of monoclonal antibodies to inhibit checkpoint proteins, such as CTLA-4, has caused much excitement among cancer immunotherapy researchers. Thus, in-depth analysis through quantum biochemistry and molecular dynamics simulations was performed to understand the complex formed by ipilimumab and its target CTLA-4. Our computational results provide a better understanding of the binding mechanisms and new insights about the CTLA-4: ipilimumab interaction, identifying essential amino acid residues to support the complex. Additionally, we report new interactions such as aromatic-aromatic, aromatic-sulfur, and cation-pi interactions to stabilize the CTLA-4:ipilimumab complex. Finally, quantum biochemistry analyses reveal the most important amino acid residues involved in the CTLA-4:ipilimumab interface, which were used to design synthetic peptides to inhibit CTLA-4. The computational results presented here provide a better understanding of the CTLA-4:ipilimumab binding mechanisms, and can support the development of alternative antibody-based drugs with high relevance in cancer immunotherapy.

Crystal structure and specific location of a germin-like protein with proteolytic activity from Thevetia peruviana.

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical ß-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.