A Multipurpose Metallophore and Its Copper Complexes with Diverse Catalytic Antioxidant Properties to Deal with Metal and Oxidative Stress Disorders: A Combined Experimental, Theoretical, and In Vitro Study.

We report the discovery that the molecule 1-(pyridin-2-ylmethylamino)propan-2-ol (HL) can reduce oxidative stress in neuronal C6 glioma cells exposed to reactive oxygen species (O2-•, H2O2, and •OH) and metal (Cu+) stress conditions. Furthermore, its association with Cu2+ generates [Cu(HL)Cl2] (1) and [Cu(HL)2](ClO4)2 (2) complexes that also exhibit antioxidant properties. Potentiometric titration data show that HL can coordinate to Cu2+ in 1:1 and 1:2 Cu2+:ligand ratios, which was confirmed by monocrystal X-ray studies. The subsequent ultraviolet-visible, electrospray ionization mass spectrometry, and electron paramagnetic resonance experiments show that they can decompose a variety of reactive oxygen species (ROS). Kinetic studies revealed that 1 and 2 mimic the superoxide dismutase and catalase activities. Complex 1 promotes the fastest decomposition of H2O2 (kobs = 2.32 × 107 M-1 s-1), efficiently dismutases the superoxide anion (kcat = 3.08 × 107 M-1 s-1), and scavenges the hydroxyl radical (RSA50 = 25.7 × 10-6 M). Density functional theory calculations support the formation of dinuclear Cu-peroxide and mononuclear Cu-superoxide species in the reactions of [Cu(HL)Cl2] with H2O2 and O2•-, respectively. Furthermore, both 1 and 2 also reduce the oxidative stress of neuronal glioma C6 cells exposed to different ROS, including O2•- and •OH.

Tuneable redox-responsive albumin-hitchhiking drug delivery to tumours for cancer treatment.

This paper outlines a novel drug delivery system for highly cytotoxic mertansine (DM1) by conjugating to an albumin-binding Evans blue (EB) moiety through a tuneable responsive disulfide linker, providing valuable insights for the development of effective drug delivery systems toward cancer therapy.

Structural studies of catalytic peptides using molecular dynamics simulations.

Many self-assembling peptides can form amyloid like structures with different sizes and morphologies. Driven by non-covalent interactions, their aggregation can occur through distinct pathways. Additionally, they can bind metal ions to create enzyme like active sites that allow them to catalyze diverse reactions. Due to the non-crystalline nature of amyloids, it is quite challenging to elucidate their structures using experimental spectroscopic techniques. In this aspect, molecular dynamics (MD) simulations provide a useful tool to derive structures of these macromolecules in solution. They can be further validated by comparing with experimentally measured structural parameters. However, these simulations require a multi-step process starting from the selection of the initial structure to the analysis of MD trajectories. There are multiple force fields, parametrization protocols, equilibration processes, software and analysis tools available for this process. Therefore, it is complicated for non-experts to select the most relevant tools and perform these simulations effectively. In this chapter, a systematic methodology that covers all major aspects of modeling of catalytic peptides is provided in a user-friendly manner. It will be helpful for researchers in this critical area of research.

Application of molecular dynamic simulations in modeling the excited state behavior of confined molecules.

Relative to isotropic organic solvent medium, the structure and conformation of a reactant molecule in an organized and confining medium are often different. In addition, because of the rigidity of the immediate environment, the reacting molecule have a little freedom to undergo large changes even upon gaining energy or modifications in the electronic structure. These alterations give rise to differences in the photochemistry of a molecular and supramolecular species. In this study, one such example is presented. α-Alkyl dibenzylketones upon excitation in isotropic solvents give products via Norrish type I and type II reactions that are independent of the chain length of the alkyl substituent. On the other hand, when these molecules are enclosed within an organic capsule of volume ~ 550 Å3, they give products that are strikingly dependent on the length of the α-alkyl substitution. These previously reported experimental observations are rationalized based on the structures generated by molecular modeling (docking and molecular dynamics (MD) simulations). It is shown that MD simulations that are utilized extensively in biologically important macromolecules can also be useful to understand the excited state behavior of reactive molecules that are part of supramolecular assemblies. These simulations can provide structural information of the reactant molecule and the surroundings complementing that with the one obtained from 1 and 2D NMR experiments. MD simulated structures of seven α-alkyl dibenzylketones encapsulated within the octa acid capsule provide a clear understanding of their unique behavior in this restricted medium. Because of the rigidity of the medium, these structures although generated in the ground state can rationalize the photochemical behavior of the molecules in the excited state.

Discovery of SARS-CoV-2 Inhibitors Featuring Novel Histidine α-Nitrile Motif.

As COVID-19 infection caused severe public health concerns recently, the development of novel antivirals has become the need of the hour. Main protease (Mpro ) has been an attractive target for antiviral drugs since it plays a vital role in polyprotein processing and virus maturation. Herein we report the discovery of a novel class of inhibitors against the SARS-CoV-2, bearing histidine α-nitrile motif embedded on a simple dipeptide framework. In-vitro and in-silico studies revealed that the histidine α-nitrile motif envisioned to target the Mpro contributes to the inhibitory activity. Among a series of dipeptides synthesized featuring this novel structural motif, some dipeptides displayed strong viral reduction (EC50 =0.48 µM) with a high selectivity index, SI>454.54. These compounds also exhibit strong binding energies in the range of -28.7 to -34.2 Kcal/mol. The simple dipeptide structural framework, amenable to quick structural variations, coupled with ease of synthesis from readily available commercial starting materials are the major attractive features of this novel class of SARS-CoV-2 inhibitors. The histidine α-nitrile dipeptides raise the hope of discovering potent drug candidates based on this motif to fight the dreaded SARS-CoV-2.

Distinct chemical factors in hydrolytic reactions catalyzed by metalloenzymes and metal complexes.

The selective hydrolysis of the extremely stable phosphoester, peptide and ester bonds of molecules by bio-inspired metal-based catalysts (metallohydrolases) is required in a wide range of biological, biotechnological and industrial applications. Despite the impressive advances made in the field, the ultimate goal of designing efficient enzyme mimics for these reactions is still elusive. Its realization will require a deeper understanding of the diverse chemical factors that influence the activities of both natural and synthetic catalysts. They include catalyst-substrate complexation, non-covalent interactions and the electronic nature of the metal ion, ligand environment and nucleophile. Based on our computational studies, their roles are discussed for several mono- and binuclear metallohydrolases and their synthetic analogues. Hydrolysis by natural metallohydrolases is found to be promoted by a ligand environment with low basicity, a metal bound water and a heterobinuclear metal center (in binuclear enzymes). Additionally, peptide and phosphoester hydrolysis is dominated by two competing effects, i.e. nucleophilicity and Lewis acid activation, respectively. In synthetic analogues, hydrolysis is facilitated by the inclusion of a second metal center, hydrophobic effects, a biological metal (Zn, Cu and Co) and a terminal hydroxyl nucleophile. Due to the absence of the protein environment, hydrolysis by these small molecules is exclusively influenced by nucleophile activation. The results gleaned from these studies will enhance the understanding of fundamental principles of multiple hydrolytic reactions. They will also advance the development of computational methods as a predictive tool to design more efficient catalysts for hydrolysis, Diels-Alder reaction, Michael addition, epoxide opening and aldol condensation.

Deconvoluting binding sites in amyloid nanofibrils using time-resolved spectroscopy.

Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-ß fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-ß, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 µM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-ß fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-ß in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-ß also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.

In vitro anti-Leishmania activity of new isomeric cobalt(II)complexes and in silico insights: Mitochondria impairment and apoptosis-like cell death of the parasite.

The synthesis, physico-chemical characterization and in vitro antiproliferative activity against the promastigote form of Leishmania amazonensis of two new cobalt(II) coordination compounds (i.e. [Co(HL1)Cl2]0.4,2H2O (1) and [Co(HL2)(Cl)(CH3OH)](ClO4).2H2O (2)) are reported, where HL1 = 4-{3-[bis(pyridin-2-ylmethyl)amino]-2-hydroxypropoxy}-2H-chromen-2-one and HL2 = 7-{3-[bis(pyridin-2-ylmethyl)amino]-2-hydroxypropoxy}-2H-chromen-2-one. X-ray diffraction studies were performed for complex (2) and the structure of complex (1) was built through Density Functional Theory (DFT) calculations. Complex (1) presented no cytotoxicity to LLC-MK2, but complex (2) was toxic. IC50 against promastigotes of L. amazonensis for complex (1) were 4.90 (24 h), 3.50 (48 h) and 3. 80 µmol L-1 (72 h), and for complex (2) were 2.09, 4.20 and 2.80 µmol L-1, respectively. Due to the high toxicity presented by complex (2) against LLC-MK2 host cells, mechanistic studies, to shed light on the probable mode of leishmanicidal activity, were carried out only for the non-cytotoxic complex. Complex (1) was able to elevate mitochondrial membrane potential of the parasites after treatment. Transmission electron microscopy revealed typical apoptotic condensation of chromatin, altered kinetoplast and mitochondria structures, suggesting that apoptosis-like cell death of the protozoa is probably mediated by an apoptotic mechanism associated with mitochondrial dysfunction (intrinsic pathway). Molecular docking studies with complex (1) upon protein tyrosine phosphatase (LmPRL-1) suggests a plausible positive complex anchoring mainly by hydrophobic and hydrogen bond forces close to the enzyme's catalytic site. These promising results for complex 1 will prompt future investigations against amastigote form of L. amazonensis.

Structure-Activity Relationship of Carbon Nitride Dots in Inhibiting Tau Aggregation.

Due to the numerous failed clinical trials of anti-amyloid drugs, microtubule associated protein tau (MAPT) now stands out as one of the most promising targets for AD therapy. In this study, we report for the first time the structure-dependent MAPT aggregation inhibition of carbon nitride dots (CNDs). CNDs have exhibited great promise as a potential treatment of Alzheimer's disease (AD) by inhibiting the aggregation of MAPT. In order to elucidate its structure-activity relationship, CNDs were separated via column chromatography and five fractions with different structures were obtained that were characterized by multiple spectroscopy methods. The increase of surface hydrophilic functional groups is consistent with the increase of polarity from fraction 1 to 5. Particle sizes (1-2 nm) and zeta potentials (~-20 mV) are similar among five fractions. With the increase of polarity from fraction 1 to 5, their MAPT aggregation inhibition capacity was weakened. This suggests hydrophobic interactions between CNDs and MAPT, validated via molecular dynamics simulations. With a zebrafish blood-brain barrier (BBB) model, CNDs were observed to cross the BBB through passive diffusion. CNDs were also found to inhibit the generation of multiple reactive oxygen species, which is an important contributor to AD pathogenesis.

Promiscuous Catalytic Activity of a Binuclear Metallohydrolase: Peptide and Phosphoester Hydrolyses.

In this study, chemical promiscuity of a binuclear metallohydrolase Streptomyces griseus aminopeptidase (SgAP) has been investigated using DFT calculations. SgAP catalyzes two diverse reactions, peptide and phosphoester hydrolyses, using its binuclear (Zn-Zn) core. On the basis of the experimental information, mechanisms of these reactions have been investigated utilizing leucine p-nitro aniline (Leu-pNA) and bis(4-nitrophenyl) phosphate (BNPP) as the substrates. The computed barriers of 16.5 and 16.8 kcal/mol for the most plausible mechanisms proposed by the DFT calculations are in good agreement with the measured values of 13.9 and 18.3 kcal/mol for the Leu-pNA and BNPP hydrolyses, respectively. The former was found to occur through the transfer of two protons, while the latter with only one proton transfer. They are in line with the experimental observations. The cleavage of the peptide bond was the rate-determining process for the Leu-pNA hydrolysis. However, the creation of the nucleophile and its attack on the electrophile phosphorus atom was the rate-determining step for the BNPP hydrolysis. These calculations showed that the chemical nature of the substrate and its binding mode influence the nucleophilicity of the metal bound hydroxyl nucleophile. Additionally, the nucleophilicity was found to be critical for the Leu-pNA hydrolysis, whereas double Lewis acid activation was needed for the BNPP hydrolysis. That could be one of the reasons why peptide hydrolysis can be catalyzed by both mononuclear and binuclear metal cofactors containing hydrolases, while phosphoester hydrolysis is almost exclusively by binuclear metallohydrolases. These results will be helpful in the development of versatile catalysts for chemically distinct hydrolytic reactions.

Peptide hydrogel with self-healing and redox-responsive properties.

We have rationally designed a peptide that assembles into a redox-responsive, antimicrobial metallohydrogel. The resulting self-healing material can be rapidly reduced by ascorbate under physiological conditions and demonstrates a remarkable 160-fold change in hydrogel stiffness upon reduction. We provide a computational model of the hydrogel, explaining why position of nitrogen in non-natural amino acid pyridyl-alanine results in drastically different gelation properties of peptides with metal ions. Given its antimicrobial and rheological properties, the newly designed hydrogel can be used for removable wound dressing application, addressing a major unmet need in clinical care.

Organic Host Encapsulation Effects on Nitrosobenzene Monomer-Dimer Distribution and C-NO Bond Rotation in an Aqueous Solution.

The intermolecular (monomer-dimer equilibrium) and intramolecular (C-NO and C-NMe2 rotations) dynamics of 4-nitrosocumene (1a) and 4-(N,N-dimethylamino)nitrosobenzene (1b), respectively, were found to be controlled by the medium (water) and the host environment (organic capsules and cavitands). The ability of water to shift the equilibrium toward the dimer appears to result from dipolar stabilization of the polar dimer structure and has a resemblance to water's known ability to favor organic cycloaddition reactions. In an aqueous medium, a range of organic hosts selectively include only the nitrosocumene monomer 1a. Encapsulation in the octa acid duplex (OA2) selects two 1a monomers rather than a dimer structure. Octa acid encapsulation also results in more restricted intramolecular C-N rotations of the guest 1b.

Interactions of Urea-Based Inhibitors with Prostate-Specific Membrane Antigen for Boron Neutron Capture Therapy.

In this study, molecular interactions of prostate-specific membrane antigen (PSMA) with five chemically distinct urea-based boron-containing inhibitors have been investigated at the atomic level using molecular docking and molecular dynamics simulations. The PSMA-inhibitor complexations have been analyzed by comparing their binding modes, secondary structures, root-mean-square deviations, noncovalent interactions, principal components, and binding free energies. PSMA is a cell surface glycoprotein upregulated in cancerous cells and can be targeted by boron-labeled inhibitors for boron neutron capture therapy (BNCT). The effective BNCT requires the selective boron delivery to the tumor area and highly specific PSMA-mediated cellular uptake by tumor. Thus, a potent inhibitor must exhibit both high binding affinity and high boron density. The computational results suggest that the chemical nature of inhibitors affects the binding mode and their association with PSMA is primarily dominated by hydrogen bonding, salt bridge, electrostatic, and π-π interactions. The binding free energies (-28.0, -15.2, -43.9, -23.2, and -38.2 kcal/mol) calculated using λ-dynamics for all inhibitors (In1-5) predict preferential binding that is in accordance with experimental data. Among all inhibitors, In5 was found to be the best candidate for BNCT. The binding of this inhibitor to PSMA preserved its overall secondary structure. These results provide computational insights into the coordination flexibility of PSMA and its interaction with various inhibitors. They can be used for the design and synthesis of efficient BNCT agents with improved drug selectivity and high boron percentage.

Degradation of a Main Plastic Pollutant Polyethylene Terephthalate by Two Distinct Proteases (Neprilysin and Cutinase-like Enzyme).

In this DFT study, hydrolysis of polyethylene terephthalate (PET), a major cause of plastic pollution, by two distinct enzymes, neprilysin (NEP, a mononuclear metalloprotease) and cutinase-like enzyme (CLE, a serine protease), has been investigated. These enzymes utilize different mechanisms for the degradation of PET. NEP uses either the metal-bound hydroxide attack (MH) mechanism or reverse protonation (RP) mechanism, while CLE utilizes a general acid/base mechanism that includes acylation and deacylation processes. Additionally, the RP mechanism of NEP can proceed through three pathways, RP0, RP1, and RP2. The DFT calculations predict that, among all these mechanisms, the MH mechanism is the energetically most favorable one for the NEP enzyme. In comparison, CLE catalyzes this reaction with a significantly higher barrier. These results suggest that the Lewis acid and nucleophile activations provided by the Zn metal center of NEP are more effective than the hydrogen bonding interactions afforded by the catalytic Ser85-His180-Asp165 triad of CLE. They have provided intrinsic details regarding PET degradation and will pave the way for the design of efficient metal-based catalysts for this critical reaction.

Dynamics of Anthracene Excimer Formation within a Water-Soluble Nanocavity at Room Temperature.

Excited anthracene is well-known to photodimerize and not to exhibit excimer emission in isotropic organic solvents. Anthracene (AN) forms two types of supramolecular host-guest complexes (2:1 and 2:2, H:G) with the synthetic host octa acid in aqueous medium. Excitation of the 2:2 complex results in intense excimer emission, as reported previously, while the 2:1 complex, as expected, yields only monomer emission. This study includes confirming of host-guest complexation by NMR, probing the host-guest structure by molecular dynamics simulation, following the dynamics AN molecules in the excited state by ultrafast time-resolved experiments, and mapping of the excited surface through quantum chemical calculations (QM/MM-TDDFT method). Importantly, time-resolved emission experiments revealed the excimer emission maximum to be time dependent. This observation is unique and is not in line with the textbook examples of time-independent monomer-excimer emission maxima of aromatics in solution. The presence of at least one intermediate between the monomer and the excimer is inferred from time-resolved area normalized emission spectra. Potential energy curves calculated for the ground and excited states of two adjacent anthracene molecules via the QM/MM-TDDFT method support the model proposed on the basis of time-resolved experiments. The results presented here on the excited-state behavior of a well-investigated aromatic molecule, namely the parent anthracene, establish that the behavior of a molecule drastically changes under confinement. The results presented here have implications on the behavior of molecules in biological systems.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)3 template mediated strategy.

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(II)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(II)A2B heterotrimers, while d sites provide pure Pb(II)C2D or Pb(II)CD2 scaffolds. Altering the metal from Pb(II) to Hg(II) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(II)(histidine)3(H2O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.

Room-Temperature Phosphorescence from Encapsulated Pyrene Induced by Xenon.

Phosphorescence from pyrene especially at room temperature is uncommon. This emission was recorded utilizing a supramolecular organic host and the effect due to the heavy atom. Poor intersystem crossing from S1 to T1, small radiative rate constant from T1, and large rate constant for oxygen quenching hinder the phosphorescence of aromatic molecules at room temperature in solution. In this study, these limitations are overcome by encapsulating a pyrene molecule within a water-soluble capsule (octa acid, OA) and purging with xenon. While OA suppressed oxygen quenching, xenon enabled the intersystem crossing from S1 to T1 and radiative process from T1 to S0 through the well-known heavy atom effect. The close interaction facilitated between the pyrene and the heavy atom perturber xenon in the three-component supramolecular assembly (OA, pyrene, and xenon) resulted in phosphorescence from pyrene. Computational modeling and NMR studies supported the postulate that pyrene and more than one molecule of xenon are present within a confined space of the OA capsule.