9 research outputs found
Strained Ruthenium Complexes Are Potent Light-Activated Anticancer Agents
Strained ruthenium (Ru) complexes have been synthesized
and characterized
as novel agents for photodynamic therapy (PDT). The complexes are
inert until triggered by visible light, which induces ligand loss
and covalent modification of DNA. An increase in cytotoxicity of 2
orders of magnitude is observed with light activation in cancer cells,
and the compounds display potencies superior to cisplatin against
3D tumor spheroids. The use of intramolecular strain may be applied
as a general paradigm to develop light-activated ruthenium complexes
for PDT applications
Design of Cytochrome P450 1B1 Inhibitors <i>via</i> a Scaffold-Hopping Approach
Cytochrome
P450 1B1 (CYP1B1) is a potential drug target in cancer
research that is overexpressed in several solid tumors but is present
only at low levels in healthy tissues. Its expression is associated
with resistance to common chemotherapeutics, while inhibitors restore
efficacy to these drugs in model systems. The majority of CYP1B1 inhibitors
are derived from a limited number of scaffolds, and few have achieved
outstanding selectivity against other human CYPs, which could impede
clinical development. This study explores a new chemical space for
CYP1B1 inhibitors using a scaffold-hopping approach and establishes
2,4-diarylthiazoles as a promising framework for further development.
From a small library, compound 15 emerged as the lead,
with picomolar CYP1B1 inhibition, and over 19,000-fold selectivity
against its relative, CYP1A1. To investigate the activity of 15, molecular dynamics, optical spectroscopy, point mutations,
and traditional structure–activity relationships were employed
and revealed key interactions important for the development of CYP1B1
inhibitors
Photoactive Ru(II) Complexes With Dioxinophenanthroline Ligands Are Potent Cytotoxic Agents
Two
novel strained rutheniumÂ(II) polypyridyl complexes containing a 2,3-dihydro-1,4-dioxinoÂ[2,3-<i>f</i>]-1,10-phenanthroline (dop) ligand selectively ejected
a methylated ligand when irradiated with >400 nm light. The best
compound exhibited a 1880-fold increase in cytotoxicity in human cancer
cells upon light-activation and was 19-fold more potent than the well-known
chemotherapeutic, cisplatin
Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes
Target
identification and mechanistic studies of cytotoxic agents
are challenging processes that are both time-consuming and costly.
Here we describe an approach to mechanism of action studies for potential
anticancer compounds by utilizing the simple prokaryotic system, <i>E. coli,</i> and we demonstrate its utility with the characterization
of a ruthenium polypyridyl complex [RuÂ(bpy)<sub>2</sub>dmbpy<sup>2+</sup>]. Expression of the photoconvertible fluorescent protein Dendra2
facilitated both high throughput studies and single-cell imaging.
This allowed for simultaneous ratiometric analysis of inhibition of
protein production and phenotypic investigations. The profile of protein
production, filament size and population, and nucleoid morphology
revealed important differences between inorganic agents that damage
DNA vs more selective inhibitors of transcription and translation.
Trace metal analysis demonstrated that DNA is the preferred nucleic
acid target of the ruthenium complex, but further studies in human
cancer cells revealed altered cell signaling pathways compared to
the commonly administrated anticancer agent cisplatin. This study
demonstrates <i>E. coli</i> can be used to rapidly distinguish
between compounds with disparate mechanisms of action and also for
more subtle distinctions within in studies in mammalian cells
Direct Measurement of Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator to the Cell Surface and Binding to a Chemical Chaperone
Mutations
in the cystic fibrosis transmembrane conductance regulator
(CFTR) result in the disease cystic fibrosis. Deletion of Phe508,
the most prevalent mutation associated with this disease, disrupts
trafficking of the protein. Small molecule correctors yield moderate
improvements in the trafficking of ΔF508-CFTR to the plasma
membrane. It is currently not known if correctors increase the level
of trafficking through improved cargo loading of transport vesicles
or through direct binding to CFTR. Real-time measurements of trafficking
were utilized to identify the mechanistic details of chemical, biochemical,
and thermal factors that impact CFTR correction, using the corrector
molecule VX-809, a secondary mutation (I539T), and low-temperature
conditions. Each individually improved trafficking of ΔF508-CFTR
to approximately 10% of wild-type levels. The combination of VX-809
with either low temperature or the I539T mutation increased the amount
of CFTR on the plasma membrane to nearly 40%, indicating synergistic
activity. The number of vesicles reaching the surface was significantly
altered; however, the amount of channel in each vesicle remained the
same. Direct binding measurements of VX-809 in native membranes using
backscattering interferometry indicate tight binding to CFTR, which
occurred in a manner independent of mutation. The similar values obtained
for all forms of the channel indicate that the binding site is not
compromised or enhanced by these mutations
Photoactive Ru(II) Complexes With Dioxinophenanthroline Ligands Are Potent Cytotoxic Agents
Two
novel strained rutheniumÂ(II) polypyridyl complexes containing a 2,3-dihydro-1,4-dioxinoÂ[2,3-<i>f</i>]-1,10-phenanthroline (dop) ligand selectively ejected
a methylated ligand when irradiated with >400 nm light. The best
compound exhibited a 1880-fold increase in cytotoxicity in human cancer
cells upon light-activation and was 19-fold more potent than the well-known
chemotherapeutic, cisplatin
Photoactive Ru(II) Complexes With Dioxinophenanthroline Ligands Are Potent Cytotoxic Agents
Two
novel strained rutheniumÂ(II) polypyridyl complexes containing a 2,3-dihydro-1,4-dioxinoÂ[2,3-<i>f</i>]-1,10-phenanthroline (dop) ligand selectively ejected
a methylated ligand when irradiated with >400 nm light. The best
compound exhibited a 1880-fold increase in cytotoxicity in human cancer
cells upon light-activation and was 19-fold more potent than the well-known
chemotherapeutic, cisplatin
Photochemical and Photobiological Activity of Ru(II) Homoleptic and Heteroleptic Complexes Containing Methylated Bipyridyl-type Ligands
Light-activated
compounds are powerful tools and potential agents for medical applications,
as biological effects can be controlled in space and time. Ruthenium
polypyridyl complexes can induce cytotoxic effects through multiple
mechanisms, including acting as photosensitizers for singlet oxygen
(<sup>1</sup>O<sub>2</sub>) production, generating other reactive
oxygen species (ROS), releasing biologically active ligands, and creating
reactive intermediates that form covalent bonds to biological molecules.
A structure–activity relationship (SAR) study was performed
on a series of RuÂ(II) complexes containing isomeric tetramethyl-substituted
bipyridyl-type ligands. Three of the ligand systems studied contained
strain-inducing methyl groups and created photolabile metal complexes,
which can form covalent bonds to biomolecules upon light activation,
while the fourth was unstrained and resulted in photostable complexes,
which can generate <sup>1</sup>O<sub>2</sub>. The compounds studied
included both bis-heteroleptic complexes containing two bipyridine
ligands and a third, substituted ligand and tris-homoleptic complexes
containing only the substituted ligand. The photophysics, electrochemistry,
photochemistry, and photobiology were assessed. Strained heteroleptic
complexes were found to be more photoactive and cytotoxic then tris-homoleptic
complexes, and bipyridine ligands were superior to bipyrimidine. However,
the homoleptic complexes exhibited an enhanced ability to inhibit
protein production in live cells. Specific methylation patterns were
associated with improved activation with red light, and photolabile
complexes were generally more potent cytotoxic agents than the photostable <sup>1</sup>O<sub>2</sub>-generating compounds
Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450<sub>BM3</sub> Variants
Cytochrome
P450<sub>BM3</sub> is a heme-containing enzyme from <i>Bacillus
megaterium</i> that exhibits high monooxygenase activity
and has a self-sufficient electron transfer system in the full-length
enzyme. Its potential synthetic applications drive protein engineering
efforts to produce variants capable of oxidizing nonnative substrates
such as pharmaceuticals and aromatic pollutants. However, promiscuous
P450<sub>BM3</sub> mutants often exhibit lower stability, thereby
hindering their industrial application. This study demonstrated that
the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is
destabilized because of the disruption of hydrophobic contacts and
salt bridge interactions. This was directly observed from crystal
structures of PM in the presence and absence of ligands (palmitic
acid and metyrapone). The instability of the tertiary structure and
heme environment of substrate-free PM was confirmed by pulse proteolysis
and circular dichroism, respectively. Binding of the inhibitor, metyrapone,
significantly stabilized PM, but the presence of the native substrate,
palmitic acid, had no effect. On the basis of high-temperature molecular
dynamics simulations, the lid domain, β-sheet 1, and Cys ligand
loop (a β-bulge segment connected to the heme) are the most
labile regions and, thus, potential sites for stabilizing mutations.
Possible approaches to stabilization include improvement of hydrophobic
packing interactions in the lid domain and introduction of new salt
bridges into β-sheet 1 and the heme region. An understanding
of the molecular factors behind the loss of stability of P450<sub>BM3</sub> variants therefore expedites site-directed mutagenesis
studies aimed at developing thermostability