Light exposure of tetra-cationic porphyrins containing peripheral Pd(II)-bipyridyl complexes and the induced effects on purified DNA molecule, fibroblast and melanoma cell lines.

Photodynamic therapy (PDT) combines a light source, oxygen, and a photosensitizer (PS) to generate reactive oxygen species (ROS) for treating diseases. In this study, we evaluated two meso-tetra-pyridyl porphyrins with [Pd(bpy)Cl]+, namely 3-PdTPyP and 4-PdTPyP, as PS for PDT application. DNA interaction was assessed by spectroscopic measurements (UV-Vis and fluorescence emission), viscosity analysis, and molecular docking simulations. The results indicate that Pd(II)-porphyrins do not intercalate into DNA, suggesting that the minor groove is the primary interaction site, mainly through van der Waals forces. These metalloporphyrins effectively induced nitrogenous bases oxidation, particularly in purines, after white light irradiation. The induced DNA lesions were able to inactivate plasmid DNA metabolism (DNA replication and transcription) in a bacterial model. 3-PdTPyP and 4-PdTPyP significantly decreased the viability of treated melanoma cell lines (A375 and B16-F10), demonstrating that melanoma cell lines were more sensitive to these Pd(II)-porphyrins than the fibroblast cell line (L929). Moreover, 3-PdTPyP was more photototoxic to A375 cells (IC50 = 0.43 µM), whereas 4-PdTPyP was more photototoxic to B16-F10 cells (IC50 = 0.51 µM). These findings suggest that these porphyrins are promising PS for future PDT research focused on skin cancer.

Revisiting and Updating the Interaction between Human Serum Albumin and the Non-Steroidal Anti-Inflammatory Drugs Ketoprofen and Ketorolac.

Ketoprofen (KTF) and ketorolac (KTL) are among the most primarily used non-steroidal anti-inflammatory drugs (NSAIDs) in humans to alleviate moderate pain and to treat inflammation. Their binding affinity with albumin (the main globular protein responsible for the biodistribution of drugs in the bloodstream) was previously determined by spectroscopy without considering some conventional pitfalls. Thus, the present work updates the biophysical characterization of the interactions of HSA:KTF and HSA:KTL by 1H saturation-transfer difference nuclear magnetic resonance (1H STD-NMR), ultraviolet (UV) absorption, circular dichroism (CD), steady-state, and time-resolved fluorescence spectroscopies combined with in silico calculations. The binding of HSA:NSAIDs is spontaneous, endothermic, and entropically driven, leading to a conformational rearrangement of HSA with a slight decrease in the α-helix content (7.1% to 7.6%). The predominance of the static quenching mechanism (ground-state association) was identified. Thus, both Stern-Volmer quenching constant (KSV) and binding constant (Kb) values enabled the determination of the binding affinity. In this sense, the KSV and Kb values were found in the order of 104 M-1 at human body temperature, indicating moderate binding affinity with differences in the range of 0.7- and 3.4-fold between KTF and KTL, which agree with the previously reported experimental pharmacokinetic profile. According to 1H STD-NMR data combined with in silico calculations, the aromatic groups in relation to the aliphatic moiety of the drugs interact preferentially with HSA into subdomain IIIA (site II) and are stabilized by interactions via hydrogen bonding and hydrophobic forces. In general, the data obtained in this study have been revised and updated in comparison to those previously reported by other authors who did not account for inner filter corrections, spectral backgrounds, or the identification of the primary mathematical approach for determining the binding affinity of HSA:KTF and HSA:KTL.

Folding Kinetics and Volume Variation of the ß-Hairpin Peptide Chignolin upon Ultrafast pH-Jumps.

In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex biomacromolecules. We have previously reported on the folding/unfolding kinetics of a model α-helix. Here, we study folding transitions in chignolin (GYDPETGTWG), a short ß-hairpin peptide previously used as a model to study conformational changes in ß-sheet proteins. Although previously suggested, until now, the role of the Tyr2-Trp9 interaction in the folding mechanism of chignolin was not clear. In the present work, pH-dependent conformational changes of chignolin were characterized by circular dichroism (CD), nuclear magnetic resonance (NMR), ultrafast pH-jump coupled with time-resolved photoacoustic calorimetry (TR-PAC), and molecular dynamics (MD) simulations. Taken together, our results present a comprehensive view of chignolin's folding kinetics upon local pH changes and the role of the Tyr2-Trp9 interaction in the folding process. CD data show that chignolin's ß-hairpin formation displays a pH-dependent skew bell-shaped curve, with a maximum close to pH 6, and a large decrease in ß-sheet content at alkaline pH. The ß-hairpin structure is mainly stabilized by aromatic interactions between Tyr2 and Trp9 and CH-π interactions between Tyr2 and Pro4. Unfolding of chignolin at high pH demonstrates that protonation of Tyr2 is essential for the stability of the ß-hairpin. Refolding studies were triggered by laser-induced pH-jumps and detected by TR-PAC. The refolding of chignolin from high pH, mainly due to the protonation of Tyr2, is characterized by a volume expansion (10.4 mL mol-1), independent of peptide concentration, in the microsecond time range (lifetime of 1.15 µs). At high pH, the presence of the deprotonated hydroxyl (tyrosinate) hinders the formation of the aromatic interaction between Tyr2 and Trp9 resulting in a more disorganized and dynamic tridimensional structure of the peptide. This was also confirmed by comparing MD simulations of chignolin under conditions mimicking neutral and high pH.

Profiling the Interaction between Human Serum Albumin and Clinically Relevant HIV Reverse Transcriptase Inhibitors.

Tenofovir (TFV) is the active form of the prodrugs tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), both clinically prescribed as HIV reverse transcriptase inhibitors. The biophysical interactions between these compounds and human serum albumin (HSA), the primary carrier of exogenous compounds in the human bloodstream, have not yet been thoroughly characterized. Thus, the present study reports the interaction profile between HSA and TFV, TDF, and TAF via UV-Vis, steady-state, and time-resolved fluorescence techniques combined with isothermal titration calorimetry (ITC) and in silico calculations. A spontaneous interaction in the ground state, which does not perturb the microenvironment close to the Trp-214 residue, is classified as weak. In the case of HSA/TFV and HSA/TDF, the binding is both enthalpically and entropically driven, while for HSA/TAF, the binding is only entropically dominated. The binding constant (Ka) and thermodynamic parameters obtained via ITC assays agree with those obtained using steady-state fluorescence quenching measurements, reinforcing the reliability of the data. The small internal cavity known as site I is probably the main binding pocket for TFV due to the low steric volume of the drug. In contrast, most external sites (II and III) can better accommodate TAF due to the high steric volume of this prodrug. The cross-docking approach corroborated experimental drug-displacement assays, indicating that the binding affinity of TFV and TAF might be impacted by the presence of different compounds bound to albumin. Overall, the weak binding capacity of albumin to TFV, TDF, and TAF is one of the main factors for the low residence time of these antiretrovirals in the human bloodstream; however, positive cooperativity for TAF and TDF was detected in the presence of some drugs, which might improve their residence time (pharmacokinetic profile).

Increasing the polarity of ß-lapachone does not affect its binding capacity with bovine plasma protein.

Despite ortho-quinones showing several biological and pharmacological activities, there is still a lack of biophysical characterization of their interaction with albumin - the main carrier of different endogenous and exogenous compounds in the bloodstream. Thus, the interactive profile between bovine serum albumin (BSA) with ß-lapachone (1) and its corresponding synthetic 3-sulfonic acid (2, under physiological pH in the sulphonate form) was performed. There is one main binding site of albumin for both ß-lapachones (n ≈ 1) and a static fluorescence quenching mechanism was proposed. The Stern-Volmer constant (KSV) values are 104 M-1, indicating a moderate binding affinity. The enthalpy (-3.41 ± 0.45 and - 8.47 ± 0.37 kJ mol-1, for BSA:1 and BSA:2, respectively) and the corresponding entropy (0.0707 ± 0.0015 and 0.0542 ± 0.0012 kJ mol-1 K-1) values indicate an enthalpically and entropically binding driven. Hydrophobic interactions and hydrogen bonding are the main binding forces. The differences in the polarity of 1 and 2 did not change significantly the affinity to albumin. In addition, the 1,2-naphthoquinones showed a similar binding trend compared with 1,4-naphthoquinones.

Interaction between a water-soluble anionic porphyrin and human serum albumin unexpectedly stimulates the aggregation of the photosensitizer at the surface of the albumin.

The 5,10,15,20-tetrakis(2,6-difluoro-3-sulfophenyl)porphyrin (TDFPPS4) was reported as a potential photosensitizer for photodynamic therapy. The capacity of the photosensitizers to be carried in the human bloodstream is predominantly determined by its extension of binding, binding location, and binding mechanism to human serum albumin (HSA), influencing its biodistribution and ultimately its photodynamic therapy efficacy in vivo. Thus, the present work reports a biophysical characterization on the interaction between the anionic porphyrin TDFPPS4 and HSA by UV-visible absorption, circular dichroism, steady-state, time-resolved, and synchronous fluorescence techniques under physiological conditions, combined with molecular docking calculations and molecular dynamics simulations. The interaction HSA:TDFPPS4 is spontaneous (ΔG° < 0), strong, and enthalpically driven (ΔH° = -70.1 ± 3.3 kJ mol-1) into subdomain IIA (site I). Curiously, despite the porphyrin binding into an internal pocket, about 50 % of TDFPPS4 structure is still accessible to the solvent, making aggregation in the bloodstream possible. In silico calculations were reinforced by spectroscopic data indicating porphyrin aggregation between bound and unbound porphyrins. This results in an adverse scenario for anionic porphyrins to achieve their therapeutical potential as photosensitizers and control of effective dosages. Finally, a trend of anionic porphyrins to have a combination of quenching mechanisms (static and dynamic) was noticed.

Photophysical, photobiological, and biomolecule-binding properties of new tri-cationic meso-tri(2-thienyl)corroles with Pt(II) and Pd(II) polypyridyl derivatives.

We report the synthesis and characterization of new tri-cationic corrole derivatives, containing Pt(II) or Pd(II) complexes attached at the peripheral position of thienyl moieties. Corrole derivatives were characterized through microanalysis, electrochemical, spectrometry and spectroscopy analysis. Singlet and triplet excited-states are investigated by photophysical/theoretical calculation methods and photobiological parameters were also evaluated spectroscopic techniques (UV-Vis and EPR). Also, the binding capacity of each corrole derivative with nucleic acids (DNA) and human serum albumin (HSA) was determined by UV-Vis, steady-state, and time-resolved fluorescence spectroscopy, combined with molecular docking analysis. Moreover, the new corroles containing peripheral complexes improve their interactions with biomacromolecules, generate reactive oxygen species under light source irradiation studied and has potential for application in photodynamic therapeutic processes.

Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Phytochemical profile of Cespedesia spathulata leaves (Ochnaceae) and its effect on tyrosinase enzyme.

Phytochemical studies of Cespedesia spathulata (Ochnaceae) leaves using 1H, 13C NMR, and GC-MS have led to the isolation of some metabolites identified for the first time in these species such as cathechin, epicatechin, vitexin, orientin, 6''-O-acetyl-vitexin, sitosterol, stigmasterol, phytol, 4,5-dihydrovomifoliol and a mixture of aliphatic methyl esters, together with ochnaflavone, which was previously isolated from this plant. The modulating activity of some fractions and compounds from Cespedesia spathulata towards tyrosinase enzyme was assayed by spectroscopic and theoretical means/experiments. The dichloromethane fraction (133 µg mL-1) and ochnaflavone (333 µM) inhibited tyrosinase activity by 20 % and 2.0 %, respectively, whereas the ethyl acetate fraction (666 µg mL-1) and ±catechins (catechin and epicatechin - 800 µM) activated it by 104 % and 384 %, respectively. Quantum chemical calculations suggested that catechin and epicatechin are better activators than L-DOPA by interacting with Cu (II) ions. Molecular docking results suggested that hydrogen bonding and hydrophobic interactions are the main binding forces between each tyrosinase activator and the amino acid residues inside the active protein binding pocket.

Monosaccharides interact weakly with human serum albumin. Insights for the functional perturbations on the binding capacity of albumin.

Monosaccharides, e.g. fructose, glucose, and arabinose are present in most foods consumed daily, whether, in natural or industrialized forms, and their concentration in the human bloodstream can impact the formation of advanced glycation end-products (AGEs, prevalent in people with diabetes) impacting the profile of Human Serum Albumin (HSA) in biodistribution of endogenous and exogenous compounds. Multiple spectroscopic techniques (UV-vis, circular dichroism, steady-state, and time-resolved fluorescence) combined with molecular docking showed that carbohydrates interact weakly and spontaneously via a ground-state association with HSA. The binding is enthalpically and entropically driven in the subdomain IIA (site I) and perturb weakly the secondary structure of the albumin. Hydrogen bonding and van der Waals forces are the main intermolecular interactions involved in the ligand binding, as well as hydrophobic effects related to the release of hydration shell upon ligand binding. Overall, the results indicated that an increase in glucose, fructose or arabinose level in the human bloodstream may cause functional perturbation on the binding capacity of albumin. Therefore, there is the necessity of carbohydrate level control in the bloodstream to not compromise the interaction and distribution of exogenous and endogenous compounds by HSA.