Development of HDAC inhibitors for treatment of pancreatic cancer in mouse models
Muhammad Murtaza Hassan, University of Toronto Mississauga
Abstract: Histone Deacetylases are a class of ‘eraser’ enzymes in epigenetics that regulate transcription by the removal of an acetyl group of histone lysine residues. As such, their aberrant expression has been demonstrated in various cancers such as neuroblastoma, leukemia, breast, colorectal and pancreatic cancer. Pancreatic cancer specifically is known to be an incurable and aggressive cancer with a 20% 1 year survival rate. Prior in vitroand in vivostudies of clinically approved HDAC inhibitor, belinostat, have demonstrated inhibition of pancreatic cancer growth. There are currently four FDA approved HDAC inhibitors, all of which show pan-inhibition of HDACs resulting in a wide range of side effects and host-toxicity. We have developed AES135, an HDAC inhibitor that prolongs the survival of mouse models of pancreatic cancer. It exhibits nanomolar HDAC IC50 for HDAC3, 6, and 11, kills patient-derived spheroids selectively over cancer-associated fibroblasts, and demonstrates great in vivopharmacokinetic properties. Furthermore, this inhibitor has led to the discovery of a variety of promising HDAC selective inhibitors by our group for the treatment of cancers such as leukemia, lymphoma and neuroblastoma.
The origins of life: Bridging the gap between nucleotides and protocells
Renée-Claude Bider, McMaster University
Abstract: The hydrothermal field hypothesis for the origin of life suggests that cyclical wetting and drying of the edge of volcanic ponds, due to tides, seasons, or day night cycles, coupled with extreme temperature promoted the synthesis of the first RNA and the formation of first protocells . Compounds found in those ponds such as clay, inorganic salts and amphiphiles are important to align nucleotides into pre-polymers . While RNA has been polymerized from nucleotides in experiments using rudimentary hydration-dehydration cycles (HD cycles), these experiments were limited by possible number of cycles and precise environmental control. To mimic the contents of these volcanic ponds, lipids, clays and inorganic salts were mixed with adenosine 3'-phosphate(AMP) and uridine 3’-phosphate (UMP). Solutions were dried on silicon wafers and fast HD cycles run in the Planet Simulator in McMaster’s newly constructed Origins of Life Lab. Gel electrophoresis was used measure the length and volume of synthesized RNA. Conjointly, the samples were analyzed through microscopic imaging and with X-ray diffraction, which allowed us analyze changes in the structure of the free nucleotides in different environments and compare them to uncycled samples. Protocells become visible in the microscopy image of a warm little pond samples containing lipids and nucleotides that was cycled for about 3 months. I will present first results of a pathway for the formation of first RNA and protocells under prebiotic conditions.
Quantifying Multilayer Regions in the Expansion of Twitching Bacterial Colonies
Erin Shelton, University of Guelph
Abstract: Type IV pili (T4P) are very thin (5-8 nm in diameter) protein filaments that can be extended and retracted by certain classes of Gram-negative bacteria including P. aeruginosa . These bacteria use T4P to move across viscous interfaces, referred to as twitching motility. Twitching can occur for isolated cells or in a collective manner . We have used optical microscopy, together with a custom-built, temperature- and humidity-controlled environmental chamber, to study the expansion of twitching colonies across an agar-glass interface for a range of agar concentrations 1.0 % w/v < C< 1.9% w/v. The advancing front consists of finger-like protrusions (fingers) consisting of many bacteria, with the cells within the expanding colony arranged in a lattice-like pattern. The fingers consist of aligned bacteria 5 to 30 cells across, which move radially outward across the agar-glass interface. We observed a significant decrease in edge speed and a corresponding transition from monolayer to multilayer coverage within the fingers at an agar concentration of 1.6% w/v. We have studied this transition by characterizing multilayer formation and dissolution, and transient and stable multilayer regions within fingers. We observed that a minimum finger width is required for multilayer stability, and we have developed a simple nucleation model that describes the dependence of multilayer lifetime on finger width.
TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry
Jay Bassan, University of Toronto
Abstract: Protein synthesis is a fundamental process in biology but no ‘gold standard’ probe exists for its quantification in living organisms. We took advantage of the synthetic accessibility and biological stability of tellurophenes to design TePhe, which mimics phenylalanine and is incorporated into proteins in human cell culture and living mice. The tellurium atom in TePhe is measurable by mass cytometry and provides excellent signal-to-noise ratio due to the zero biological background of tellurium. Using imaging mass cytometry, we showed that TePhe is incorporated into healthy and cancerous tissues in mice, and imaged the spatial variation of protein synthesis in the intestine and the brain. We multiplexed TePhe with another tellurium-containing probe for hypoxia, Telox 2, to image for the first time the hypoxic suppression of protein synthesis in a PANC-1 xenograft tumour. In this presentation I will detail our current work with TePhe and suggest future directions for the measurement of protein synthesis with imaging mass cytometry.
How Reactive are Druggable Cysteines in Protein Kinases? — A Computational Study
Ernest Awoonor-Williams, Memorial University of Newfoundland
Abstract: The protein kinase family of signalling enzymes is a popular target for enzyme inhibition by therapeutic drugs, particularly for cancer treatments. Traditionally, most drugs bind to their targets through non-covalent interactions like hydrogen bonding. Recently, there has been renewed interest among drug developers and medicinal chemists to design drugs that bind covalently to their targets, since these drugs tend to be more therapeutically potent than conventional non-covalent binding drugs. Cysteine-targeting covalent inhibitors are a promising branch of kinase drug development that have the potential to increase potency and residence time. Cysteines reactivity towards a drug molecule depends on its acidity, a property quantified by a pKa value. Experimentally identifying a targetable cysteine residue and determining its pKais a difficult task because of the need to express and purify the protein, and the presence of many ionizable residues. We have developed a computational method to predict the reactivity of cysteine residues in proteins based on their pKa's. We have used this method together with other rigorous computational approaches to predict the reactivity of druggable cysteines across the protein kinase family of popular drug targets.
Do Soft Anions Promote Protein Denaturation Through Binding Interactions? A Case Study Using Ribonuclease A
Mazdak Khajepour, University of Manitoba
Abstract: It has long been known that large soft anions like bromide, iodide and thiocyanate are protein denaturing agents, but their mechanism of action is still unclear. In this work we have investigated the protein denaturing properties of these anions using Ribonuclease A (RNase A) as a model protein system. Salt-induced perturbations to the protein folding free energy were determined using differential scanning calorimetry and the results demonstrate that the addition of sodium iodide and sodium thiocyanate significantly decreases the melting temperature of the protein. In order to account for this reduction in protein stability, we show that the introduction of salts that contain soft anions to the aqueous solvent perturbs the protein unfolding free energy through three mechanisms: a) screening Coulomb interactions that exist between charged protein residues, b) Hofmeister effects, and c) specific anion binding to CH and CH2 moieties in the protein polypeptide backbone. Using the micellization of 1,2-hexanediol as a ruler for hydrophobicity, we have devised a practical methodology that separates the Coulomb and Hofmeister contributions of salts to the protein unfolding free energy. This allowing us to isolate the contribution of soft anion binding interactions to the unfolding process. The analysis shows that binding contributions have the largest magnitude, confirming that it is the binding of soft anions to the polypeptide backbone that is the main promoter of protein unfolding.
Development of DNA Aptamers to Salivary Exosomes for Oral-pharyngeal Cancer Diagnostics and Application
Vanessa Susevski, University of Ottawa
Abstract: Exosomes are a heterogeneous group of extracellular vesicles secreted from most cells into body fluids such as urine and saliva. Exosomes are attractive candidate as biomarker for the non-invasive diagnostic of various cancer, such as oropharyngeal cancer. Human salivary exosomes have been isolated from healthy patients and characterized by NTA, TEM and by validating the presence of several common exosome markers in parallel. Aptamers were then developed to bind to salivary exosomes by Systematic Exponential Enrichment of Ligands (SELEX) and validated by FACS. The sequences were recovered by NGS and aptamer clones were chosen after bioinformatic analysis. Aptamer-Faciliated Biomarker Discovery (AptaBiD) protocol will be followed to identify the binding partner of the aptamers for the purpose of a potential novel biomarker that can be used for screening or diagnostics.
LH1-RC Light-Harvesting Photocycle under Realistic Light-Matter Interaction Scheme
Chern Chuang, University of Toronto
Abstract: We simulate the photocycle of the light-harvesting complex 1 (LH1) and the reaction center (RC) pigment-protein complex in purple bacteria using quantum master equations. We take into account the influences of the radiation and the protein environments and the full photocycle of the complexes including the charge separation and recovery processes of the RC afterward. Particular emphasis was placed on the steady state excitation energy transfer rate between the LH1 and the RC, the pertinent limit of these devices under natural conditions, and the steady state dependence on the light intensity. The transfer rate scales linearly with the light intensity, nearing the value of the natural habitat and is capped by the rate-determining step of the photocycle, the RC turnover rate, at higher light intensities. Transient dynamics, however, shows rates higher than the steady state value and continues to scale linearly with the intensity. Results show the correlation between the transfer rate and the manner in which the donor state is prepared in the donor-acceptor context. In addition, the transition from the transient to the steady state results can be understood as a cascade of ever slower rate-determining steps and quasi-stationary states inherent to multi-scale sequential processes. This transition is relevant in most light-induced biological machineries.
Investigating the Structure of a Dendritic Nanoparticle using Coarse-Grained Simulations
Nicole Drossis, University of Ontario Institute of Technology
Abstract: Phytoglycogen is a naturally occurring dendritic nanoparticle found in sweet corn. It is composed of repeatedly branching units of glucose. Despite glucose being a common form of energy storage in plants, many questions still remain about the structure of this naturally occurring particle. In this work pytoglycogen was modelled with a coarse-grained approach, simplifying each glucose unit to a single particle. A hydrophobic attraction between chains was observed in atomistic simulations and this was modelled in the coarse-grained simulations with a simple attraction between glucose particles. The strength of this attraction was used as a free parameter to investigate how changing it changes the structure of the particle. The simulated particles with radii similar to those seen in experiment were observed to have a hairy colloid shape, with a dense core and hairs extending out from the core.
Surface Modification of NaLnF4 NPs using Lipid-coating for High-Sensitivity Mass Cytometry
Loryn Arnett, University of Toronto
Abstract: Detection and quantification of cellular markers present in very low quantities (<103 copies per cell) is an enduring challenge in cell biology. One method that is currently used for cellular analysis is mass cytometry. Mass cytometry uses Abs labeled with a heavy metal “mass tag” for targeting and detection, respectively, where the sensitivity of detection is proportional to the number of metal atoms per mass tag. Current mass tags based on lanthanide-chelating polymers contain ~200 metal atoms each, and are insufficient for detection of low copy number markers. We hypothesize that using lanthanide (Ln) containing nanoparticles (NPs) as mass tags, which contain ~10^4 Ln atoms per NP, will provide enhanced signal sensitivity to mass cytometry. The LnNPs should be uniform in size, colloidally stable in physiological media, and be functional for Ab conjugation. I have successfully encapsulated 35 nm NaYF4: Yb, Er NPs with a lipid mixture based on DEC221 (2 Dioleoylphosphatidylcholine : 2 Egg sphingomyelin : 1 Cholesterol), where the cholesterol molecules are conjugated to polyethylene glycol (PEG-600) to reduce non-specific interactions of the LnNPs with cells. Biotin-PEG-conjugated 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2k-biotin) lipids were added to this lipid mixture to provide a functional handle for Ab conjugation. The resulting lipid-coated LnNPs are colloidally stable in phosphate buffer for a minimum of two months by dynamic light scattering (dh~90 nm), and show minimal non-specific binding to cells (< 0.2%) by mass cytometry. The next step will be to conjugate the lipid-coated LnNPs to Abs and test for specific binding.
The Bending Rigidity of Red Blood Cell Membranes Determined from Solid-Supported Multi-lamellar Membranes
Sebastian Himbert, McMaster University
Abstract: The preparation of Red Blood Cell (RBC) Ghosts is a well-known protocol in biological and medical research and describes the extraction of the membrane from RBCs. Another well-known protocol is the preparation of highly ordered stacks of artificial lipid bilayers on silicon wafers. There are various attempts to adapt this protocol to a native cell membrane. For the first time we were able to combine both techniques and prepare highly ordered stacks of RBC membranes on silicon wafers . This assay can now be used as inexpensive and safe platform for testing the effect of drugs and bacteria on RBC membranes in-vitro using biophysical techniques, such as X-ray and Neutron diffraction. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. This patchy nature of the red blood cell membrane has a significant effect on the bending rigidity of the membranes. We present a novel method to determine the bending modulus and membrane interaction modulus of a biological membrane on the molecular scale from 2-dimensinoal X-ray diffraction measurements. These measurements are complemented by Molecular Dynamics simulations and Neutron Spin-Echo measurement which allow a direct determination of the membrane undulations.
Modeling membrane selectivity of antimicrobial peptides
Shokoofeh Nourbakhsh, University of Waterloo
Abstract: Antimicrobial peptides (AMPs) are naturally-occurring peptide antibiotics. The way they work has inspired a vigorous search for optimized peptide antibiotics for fighting resistant bacteria. Cationic AMPs cleverly utilize their electrostatic interactions with the bacterial membrane to selectively attack bacteria. Here, we present a coarse-grained model of membrane selectivity of these peptides, which includes several competing effects (e.g., lipid demixing and peptide-peptide interactions on the membrane surface). Using the model, we relate peptide’s intrinsic (cell-density-independent) selectivity to an apparent, cell-density-dependent one, and clarify the relative roles of peptide parameters and cell densities in determining their selectivity. A natural consequence of this relationship is that the selectivity is more sensitive to peptide parameters at low cell densities; as a result, the optimal peptide charge, at which the selectivity is maximized, increases with the cell density such that this notion becomes less meaningful at high cell densities.
Evaluating the structure and function of tau protein
Sanela Martic, Trent University
Abstract: A neuronal tau protein plays an important cellular role, but it has been linked to neurodegeneration. Hence, tau protein is a viable drug target. At the biochemical level, tau protein interacts with microtubules, is a substrate for post-translational modifications, and is prone to misfolding and aggregation. However, the intricate details of tau protein biochemistry are still not known. In addition, the therapeutic inhibition of tau pathogenesis remains elusive. We have demonstrated that antitau antibodies modulate the interactions of tau with microtubules, tau phosphorylation by protein kinases and tau aggregation in vitro. These various aspects of tau biochemistry and its inhibition have been evaluated by fluorescence spectroscopy and transmission electron microscopy.
Coarse-grained simulations of disordered protein condensates: Effect of interaction potentials and charge pattern parameters
Suman Das, University of Toronto
Abstract: Recent experimental studies have revealed that membranelessorganelles are multi-component viscous liquiddroplets, assembled through weak, dynamic, multivalent interactions among biomolecules, such as proteins and RNAs. These organelles are involved in many important biological functions and also been linked to amyloid diseases. Understanding their formation and physio-chemical properties is challenging due to the intrinsic complexity of cellular environment and heteropolymeric nature of the biomolecules. Extensive Langevin dynamic simulations have been performed with coarse-grain model to understand how phase separation propensity depends on the charge pattern of the amino acid sequences. Our studies indicate that liquid-liquid phase separation propensity for a previously-studied set of sequences correlates well with two well-known charge pattern parameters, the “blockiness measure” κ andthe “sequence charge decoration” SCD. Moreover, quantitative analysis suggests that overall phase separation propensity is enhanced by background residue-residue attraction. However, for a novel set of sequences we designed to exhibit an anti-correlation between κ and −SCD, the simulated T*cr’s are quite insensitive to κ or SCD. In general, our results reveal both the utility and limitations of analytical theory as well as the charge pattern parameters,and point to several fruitful future directions in the development of theory and simulation for the phase behaviors of disordered proteins.
Physical bases of forming a smooth boundary between two rough-edged materials: an expanding Arp2/3 actin network and a contractile actomyosin network
Medha Sharma, University of Toronto
Abstract: Smooth boundaries commonly form for cell and tissue morphogenesis. One example is the cytokinetic ring which drives cell division, and another is the leading-edge purse string for wound healing. In addition, smooth boundaries form between growing Arp2/3 actin networks and encircling actomyosin networks to bend the plasma membrane and form nuclear compartments during syncytial Drosophilaembryogenesis. To understand how these initially rough-edged materials can interact to form a smooth boundary, we utilize mathematical modeling. The contractile actomyosin material is constructed with hundreds of myosin nodes that interconnect randomly and pull on each other with myosin activity and passive elasticity. Our model mimics reported in vitrobehaviors of reconstituted actomyosin networks, including shape changes following patterned activation. The Arp2/3 network is modelled based on the properties of these branched networks. The simulated networks also mimic reported in vitrobehavior, such as a non-elastic, local density increase in response to physical restriction. By reconstituting a single Arp2/3 network encircled by a myosin network, we found that Arp2/3 network growth can disrupt an initially smooth myosin border. However, such growth can increase the smoothness of a myosin border with a more natural rough edge, indicating the importance of the network-network interaction. In both cases, increased myosin activity also promotes smoothness of the boundary. To determine if such heightened myosin activity is compatible with in vivonetwork organization, and to further probe the properties of this organization, we are currently testing a natural configuration of an actomyosin network embedded with an array of individual Arp2/3 networks.