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.

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).

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.

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.

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.

Antitumor Potential of the Isoflavonoids (+)- and (-)-2,3,9-Trimethoxypterocarpan: Mechanism-of-Action Studies.

Synthetically derived samples of (+)-(6aS,11aS)-2,3,9-trimethoxypterocarpan [(+)-1] and its enantiomer [(-)-1], both of which are examples of naturally occurring isoflavonoids, were evaluated, together with the corresponding racemate, as cytotoxic agents against the HL-60, HCT-116, OVCAR-8, and SF-295 tumor cell lines. As a result it was established that compound (+)-1 was particularly active with OVCAR-8 cells being the most sensitive and responding in a dose-dependent manner. A study of cell viability and drug-induced morphological changes revealed the compound causes cell death through a mechanism characteristic of apoptosis. Finally, a computational study of the interactions of compound (+)-1 and (S)-monastrol, an established, synthetically derived, potent, and cell-permeant inhibitor of mitosis, with the kinesin-type protein Eg5 revealed that both bind to this receptor in a similar manner. Significantly, compound (+)-1 binds with greater affinity, an effect attributed to the presence of the associated methoxy groups.

Explaining urokinase type plasminogen activator inhibition by amino-5-hydroxybenzimidazole and two naphthamidine-based compounds through quantum biochemistry.

Urokinase plasminogen activator (uPA) is a biomarker and therapeutic target for several cancer types whose inhibition has been shown to slow tumor growth and metastasis. In this work, crystallographic data of uPA complexed with distinct ligands (PDB id: 1SQA, 1SQO, and 1FV9) were used to perform quantum biochemistry calculations based on the framework of density functional theory (DFT) and within the molecular fractionation with conjugated caps (MFCC) scheme. Our calculations revealed a total energy interaction of -107.30, -99.5, and -35.30 kcal mol-1 for two naphthamidine-based compounds (Ul1 and UI2) and 2-amino-5-hydroxybenzimidazole (172), respectively, which are in good agreement with known inhibitory experiments. Residues Asp189, Ser190, Cys191-Cys220, Gln192, Trp 215, Gly216, and Gly219 were identified as the main interacting amino acid residues with interaction energy contributions lower than -4.0 kcal mol-1 for uPA/UI1 and UPA/UI2 complexes. In the case of compound 172, our calculations have shown that the most important interactions occur with residues Asp189, Cys191-Cys220, and Ser190. Our results highlight the relevance of the protonation state of ligands and residues and that the naphthamidine scaffold of UI1 and UI2 is the main determinant of their potency, followed by their aminopyrimidine substitution. Altogether, the results of this work contribute to the understanding of the uPA binding mechanisms of the inhibitory compounds Ul1 and 172, stimulating the use of quantum biochemistry theoretical approaches for the development of new uPA inhibitors as new medicines for cancer treatment.

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

Encapsulation of nor-ß-lapachone into poly(d,l)-lactide-co-glycolide (PLGA) microcapsules: full characterization, computational details and cytotoxic activity against human cancer cell lines.

In this work, we characterize nor-ß-lapachone-loaded (NßL-loaded) microcapsules prepared using an emulsification/solvent extraction technique. Features such as surface morphology, particle size distribution, zeta potential, optical absorption, Raman and Fourier transform infrared spectra, thermal analysis data, drug encapsulation efficiency, drug release kinetics and in vitro cytotoxicity were studied. Spherical microcapsules with a size of 1.03 ± 0.46 µm were produced with an encapsulation efficiency of approximately 19%. Quantum DFT calculations were also performed to estimate typical interaction energies between a single nor-ß-lapachone molecule and the surface of the microparticles. The NßL-loaded PLGA microcapsules exhibited a pronounced initial burst release. After the in vitro treatment with NßL-loaded microcapsules, a clear phagocytosis of the spheres was observed in a few minutes. The cytotoxic activity against a set of cancer cell lines was investigated.

Controlled Release of Nor-ß-lapachone by PLGA Microparticles: A Strategy for Improving Cytotoxicity against Prostate Cancer Cells.

Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-ß-lapachone (NßL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 µm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NßL on PLGA. Finally, the cytotoxic activity of NßL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.

Structural basis of ConM binding with resveratrol, an anti-inflammatory and antioxidant polyphenol.

Resveratrol can also inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5mM for 2h of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an Rfactor of 16.0% and an Rfree of 25.5%. Resveratrol binds in the rigid ß-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth ß-strands of the rigid ß-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.