Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

A study of Pt(II)-phenanthroline complex interactions with double-stranded and G-quadruplex DNA by ESI-MS, circular dichroism, and computational docking.

The binding interactions of a series of square-planar platinum(II)-phenanthroline complexes of the type [Pt(PL)(AL)]2+ [where PL = variously methyl-substituted 1,10-phenanthroline (phen) and AL = ethane-1,2-diamine (en)] were assessed with a G-quadruplex DNA (5'-TTG GGG GT-3', G4DNA) and a double-stranded DNA (5'-CGC GAA TTC GCG-3', dsDNA) sequence by ESI-MS. The results indicate a strong correlation between G4DNA affinity and increasing phenanthroline methyl substitution. Circular dichroism (CD) spectroscopy and molecular docking studies also support the finding that increased substitution of the phenanthroline ligand increased selectivity for G4DNA. ESI-MS was used to probe the interaction of a range of square-planar Pt(II)-phenanthroline complexes with double-stranded and G-quadruplex DNA.

Quadruplex DNA-Stabilising Dinuclear Platinum(II) Terpyridine Complexes with Flexible Linkers.

Four dinuclear terpyridineplatinum(II) (Pt-terpy) complexes were investigated for interactions with G-quadruplex DNA (QDNA) and duplex DNA (dsDNA) by synchrotron radiation circular dichroism (SRCD), fluorescent intercalator displacement (FID) assays and fluorescence resonance energy transfer (FRET) melting studies. Additionally, computational docking studies were undertaken to provide insight into potential binding modes for these complexes. The complexes demonstrated the ability to increase the melting temperature of various QDNA motifs by up to 17 °C and maintain this in up to a 600-fold excess of dsDNA. This study demonstrates that dinuclear Pt-terpy complexes stabilise QDNA and have a high degree of selectivity for QDNA over dsDNA.

Multifaceted Studies of the DNA Interactions and In Vitro Cytotoxicity of Anticancer Polyaromatic Platinum(II) Complexes.

This study reports a detailed biophysical analysis of the DNA binding and cytotoxicity of six platinum complexes (PCs). They are of the type [Pt(PL )(SS-dach)]Cl2 , where PL is a polyaromatic ligand and SS-dach is 1S,2S-diaminocyclohexane. The DNA binding of these complexes was investigated using six techniques including ultraviolet and fluorescence spectroscopy, linear dichroism, synchrotron radiation circular dichroism, isothermal titration calorimetry and mass spectrometry. This portfolio of techniques has not been extensively used to study the interactions of such complexes previously; each assay provided unique insight. The in vitro cytotoxicity of these compounds was studied in ten cell lines and compared to the effects of their R,R enantiomers; activity was very high in Du145 and SJ-G2 cells, with some submicromolar IC50 values. In terms of both DNA affinity and cytotoxicity, complexes of 5,6-dimethyl-1,10-phenanthroline and 2,2'-bipyridine exhibited the greatest and least activity, respectively, suggesting that there is some correlation between DNA binding and cytotoxicity.

Linear dichroism as a probe of molecular structure and interactions.

Linear dichroism (LD) spectroscopy involves measuring the wavelength (or energy) dependence of the difference in absorption of light parallel and perpendicular to an orientation direction. It requires samples to have a net orientation. The aim of this review is to summarise some UV-visible linear dichroism (LD) methods that can be usefully applied to increase our understanding of biomacromolecules and their complexes that have a high aspect ratio. LD shares the advantages of most spectroscopic techniques including the fact that data collection is fairly straightforward and many sample types can be investigated. Conversely, LD shares the disadvantage that the measured signal is an average over all species in the sample on which the light beam is incident. LD mitigates this disadvantage somewhat in that only species which are oriented give a net signal. How the data can be analysed to give structural information about small molecules in stretched films and membrane systems or bound to biomacromolecules and directly about biomacromolecules such as DNA and protein fibres forms part of this review. In the UV-visible region LD often suffers noticeably from light scattering since the samples tend to be large relative to the wavelength of the incident light, so consideration is also given to data analysis challenges including removal of scattering contributions to an observed signal. Brief mention is made of fluorescence detected LD.

Transition Metal Intercalators as Anticancer Agents-Recent Advances.

The diverse anticancer utility of cisplatin has stimulated significant interest in the development of additional platinum-based therapies, resulting in several analogues receiving clinical approval worldwide. However, due to structural and mechanistic similarities, the effectiveness of platinum-based therapies is countered by severe side-effects, narrow spectrum of activity and the development of resistance. Nonetheless, metal complexes offer unique characteristics and exceptional versatility, with the ability to alter their pharmacology through facile modifications of geometry and coordination number. This has prompted the search for metal-based complexes with distinctly different structural motifs and non-covalent modes of binding with a primary aim of circumventing current clinical limitations. This review discusses recent advances in platinum and other transition metal-based complexes with mechanisms of action involving intercalation. This mode of DNA binding is distinct from cisplatin and its derivatives. The metals focused on in this review include Pt, Ru and Cu along with examples of Au, Ni, Zn and Fe complexes; these complexes are capable of DNA intercalation and are highly biologically active.

Improved DNA equilibrium binding affinity determinations of platinum(II) complexes using synchrotron radiation circular dichroism.

The binding affinity of a series of square planar platinum(II) compounds of the type [Pt(A(L))(I(L))](2+), where A(L) is 1,2-diaminoethane and I(L) are 1,10-phenanthroline (phen), 4-methyl-1,10-phenanthroline (4Mephen), 5-methyl-1,10-phenanthroline (5Mephen), 4,7-dimethyl-1,10-phenanthroline (47Me2phen), 5,6-dimethyl-1,10-phenanthroline (56Me2phen) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3478Me4phen) has been reinvestigated using Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The additional peaks exhibited considerably greater intensity than those observed between 200 and 400 nm affording additional binding affinity determinations. In addition, the authors have reviewed the various mathematical approaches used to estimate equilibrium binding constants and thereby demonstrate that their mathematical approach, implemented with Wolfram Mathematica, has merit over other methods.

Exposing "Bright" Metals: Promising Advances in Photoactivated Anticancer Transition Metal Complexes.

BACKGROUND: Photodynamic therapy (PDT) is an increasingly prominent field in anticancer research. PDT agents are typically nontoxic in the absence of light and can be stimulated with nonionising irradiation to "activate" their cytotoxic effect. Photosensitzers are not classified as chemotherapy drugs although it is advantageous to control the toxicity of a drug through localised irradiation allowing for selective treatment. Transition metals are an extremely versatile class of compounds with various unique properties such as oxidation state, coordination number, redox potential and molecular geometry that can be tailored for specific uses. This makes them excellent PDT candidates as their properties can be manipulated to absorb a specific range of light wavelengths, cross cellular membranes or target specific sites in vitro. This article reviews recent advances in transition metal PDT agents, with a focus on structural scaffolds from which several metal complexes in a series are synthesised, as well as their in vitro cytotoxicity in the presence or absence of irradiation. CONCLUSION: The success of clinical photoactive agents such as Photofrin® has inspired the development of thousands of potential PDT agents. Transition metal complexes in particular have demonstrated excellent versatility and diversity when it comes to PDT for treatment of invasive cancers. This review has highlighted some of the many recent advances of transition metal PDT agents with high in vitro and in vivo phototoxic activity. Photoactive transition metal complexes have proven their potential due to their inherent physicochemical variety, allowing them to fill a niche in the PDT world.

Metal complex interactions with DNA.

Increasing numbers of DNA structures are being revealed using biophysical, spectroscopic and genomic methods. The diversity of transition metal complexes is also growing, as the unique contributions that transition metals bring to the overall structure of metal complexes depend on the various coordination numbers, geometries, physiologically relevant redox potentials, as well as kinetic and thermodynamic characteristics. The vast range of ligands that can be utilised must also be considered. Given this diversity, a variety of biological interactions is not unexpected. Specifically, interactions with negatively-charged DNA can arise due to covalent/coordinate or subtle non-coordinate interactions such as electrostatic attraction, groove binding and intercalation as well as combinations of all of these modes. The potential of metal complexes as therapeutic agents is but one aspect of their utility. Complexes, both new and old, are currently being utilised in conjunction with spectroscopic and biological techniques to probe the interactions of DNA and its many structural forms. Here we present a review of metal complex-DNA interactions in which several binding modes and DNA structural forms are explored.

Synthesis and analysis of the anticancer activity of platinum(II) complexes incorporating dipyridoquinoxaline variants.

Eight platinum(II) complexes with anticancer potential have been synthesised and characterised. These complexes are of the type [Pt(I(L))(A(L))](2+), where I(L) is either dipyrido[3,2-f:2',3'-h]quinoxaline (dpq) or 2,3-dimethyl-dpq (23Me2dpq) and A(L) is one of the R,R or S,S isomers of either 1,2-diaminocyclohexane (SS-dach or RR-dach) or 1,2-diaminocyclopentane (SS-dacp or RR-dacp). The CT-DNA binding of these complexes and a series of other complexes were assessed using fluorescent intercalator displacement assays, resulting in unexpected trends in DNA binding affinity. The cytotoxicity of the eight synthesised compounds was determined in the L1210 cell line; the most cytotoxic of these were [Pt(dpq)(SS-dach)]Cl2 and [Pt(dpq)(RR-dach)]Cl2, with IC50 values of 0.19 and 0.80 µM, respectively. The X-ray crystal structure of the complex [Pt(dpq)(SS-dach)](ClO4)2·1.75H2O is also reported.