Sade Lamidi's Research Blog
The aim of this research is to develop theoreticl knowledge on photosensitizer binding to amyloids and to apply the outcome in prospective experimental analysis.
The overarching goal is to develop a hybrid drug delivery system containing photosensitizer binding to amyloids inside liquid condensates. The photosensitizer to be used has Aggregation induced emission (AIE) characteristics such that it becomes a photoactive sensitizer or triplet oxygen on binding to amyloid domain.
The theoretical analysis centers around, looking at conformational changes in the pi-conjugated photosensitizers upon binding to amyloidal domains. The idea is to look for pi-ring planarization that would increase aromatization in the molecule and hence make it photoactive (Fig. 1). This change can be experimentally observed in emission spectroscopy. During the study various photosensitizers and amyloid combinations will be studied for optimized results.
Detailing further, we will synthesize dyes and mix them into condensates containing fibers. We will observe binding properties of the dyes and correlate those properties with the strength of interactions with the amyloid fibers. Fluorescent light emission will be the guiding parameter along with the production of reactive oxygen species. Computationally, structural modifications will be made to the dye based on binding efficiency and conformation of the dyes when interacting with the fibers. The new dye candidates will be tested. The targeted fibers are hydrophobic, so we will be increasing the conjugation of the dye which in return will increase the hydrophobicity allowing for better interaction between the dye and fibers. This experimental analysis and computational results will be interchangeable, allowing for the application of computational lessons to experiments. Our end goal will be to produce reactive oxygen species by generating novel high efficiency photosensitizers, which will be killing cancer cells and activating the prodrug.
Fig 1. Generalized molecular framework of the D-π-A (Electron Donor-Pi conjugated rings- Electron Acceptor) photosensitizers. Angles 1 and 2 are torsional angles between indicated benzene ring planes.
Fig 2. Variation of torsional angle 1 in PS1 as observed in simulation frames. Inset shows the molecular structure with relevant benzene planes marked.
Fig 3. Variation of torsional angle 2 in PS1 as observed in simulation frames. Inset shows the molecular structure with relevant benzene planes marked.
Fig 4. Variation of torsional angle 1 vs angle 2 in PS1 as observed in simulation frames. Inset shows the molecular structure with relevant benzene planes marked.
Computational Methods
Appropriate tutorials were completed through Maestro Schrödinger to gain familiarity with computation modeling.
Molecular dynamics, Conformational dynamics to prepare three- dimensional dye and amyloid
Amyloid structure obtained from the Protein Data Bank was utilized until a more comprehensive understanding of dye binding is achieved
Desmond system builder used to simulate PS1 in water, using SPC water model containing fixed partial charges, bond lengths, and three interaction sites. High aggregation, linked to higher emission intensity and efficient binding is observed when dye interacts with water versus when bound to previously tested solvent THF 1. It is predicted that dye will better bind to the LVFFAR9 beta sheet than it does water.
Using the SPC simulation, we then observed and calculated the geometric parameters of the two aromatic moieties on PS1 by measuring the dihedral angles upon aggregation. One dimensional histogram was created for each given angle (l and r) on PS1.
To calculate probability of planarity of molecules, a dimensional statistical analysis was done (Fig. 2-4).
5/30/2024
After evaluation of angles 1 and 2, it is important to include angle three binding the two
phenyl rings, out of plane with the remainder of the molecule. Because this portion of the
molecule is out of plane it requires a relaxed torsional scan.
To do so a third histogram is to be calculated. Because angle three does not have as significant of a correlation with the remainder of the molecule
Due to symmetry, there is low correlation between angles 1 and 2, and
changing them doesn't require the neighboring hydrogen atoms to move so rigid
scans will be performed, first.
On angle 3 a relaxed scan will be performed.
Questions:
1. When considering the third angle, what is the energy of each trajectory possible?
Summer 2024- SYNTHESIS
start of TTPM syntehsis
we started by synthesizing 4-pyradilne-iodo-benzene. To do so a suzuki recation is done under heated conditions of 72 degrees celcius.
7/15- Suzuki reaction/sample TLC
Appropriate glassware was warmed in the RV for 5 days, to avoid water disturbing the reaction. 1-bromo-iodo-benzene (0.552 g), pyridine baronic acid (0.200g), and Pottasium phosphate (1.04g) was added into a double neck round bottom flask, one end sealed and one end containing a gas adapter. 16mL of DMF, sealed and dried by molecular seeds was nirtogentaed and added to A, B, and C inside of the double headed round bottom flask. Distilation glassware, warmed for 5 days in RV was set up. The solution was degassed for thirty minutes. The method of nitrogenation we chose for the DMF solution was piercing a needle attatche to a nitrogen filled ballon throuygh the sealed piercable bottle lid. To degas the reaction, a needle connected to a syringe and tube was pierced into the sealed end of the flask. Nitrogen flowed through the tube at a pressure of 10 psi. On the other head of the round bottom, a gas adapter was switched on allowing for Oxygen to exit the recation as Nitrogen flowed in. Bubbling of the solution signified successful degassing. After degassing the catalyst was added
9/20/2023
-Start of SL03 synthesis
Oct 10, 2024
We will proceed in binding amyloids and photosensitizer via glide extension on Maestro Schrodinger.
Generate and locate binding pocket on amyloid, and create receptor grid. Binding site is typically in location of the ligand, since our amyloud does not contain a ligand we will lcoate one at random or use "site map"
next step:manually dock ligand then perform glide.
induced fit docking changes side chains of both amyloid and dye to fit.
Previous studies of THT binding to KLVFFA are studied and refrenced. Glide docking was tested. for our binding for TTPM and TPPM to LVFFAR.
11/04/24
Confocal microscopy was done to observe AlE-1 binding to amyloidal condensates: 20% LVFFAR, + 80% Rg + ATP + 0.5% AlE-1. LVFFAR, shows visible microscopic structure at concentrations above CAC with AIE-1 staining the fibers specifically. Proceed with induced fit.
12/15/2024
Our initial efforts were concentrated on Glide, a commercially available docking software, meant for rigid receptor docking. The Glide program is generally useful in identifying high-affinity ligand binding poses by assuming a static receptor conformation, an assumption that may not adequately describe the flexibility of amyloid fibrils.
Given the dynamic nature of amyloids and their binding sites, we shifted to using the Induced Fit Docking protocol. The Induced Fit allows for simultaneous flexibility of both the ligand and the receptor, hence allowing for a more realistic simulation of ligand-amyloid interactions.
Challenges and Observations-
Failed Docking Jobs: Consecutive job failures during the process of Induced Fit runs called for reassessment of system preparation. My initial hypothesis included the following as the probable cause: improper preparation of proteins and ligands. Indeed, using Protein Prep and LigPrep tools did result in partial resolution of some structural issues, which resulted in improved runtime before failures occurred.
Manual Ligand Placement: Ligands were manually placed into the amyloid workspace based on key binding regions identified in recent literature:
"The Binding of Thioflavin T and Its Neutral Analog BTA-1 to Protofibrils" by Wu et al. (2008): Grooves on the surface of a β-sheet were highlighted as primary binding regions with both micromolar and nanomolar affinities. This paper suggests that targeting the FF (phenylalanine) region yields optimal binding.
"Mechanism of Thioflavin T Binding to Amyloid Fibrils" Khurana et al., 2005: Points to the outer face of the molecule and proposes micelle-driven fluorescence enhancement mechanisms. This suggests that the binding efficiency of the ligand depends on its correct positioning.
Proposed Directions
Larger Ligand Construction: You had mentioned constructing a larger ligand to enhance the binding affinity. I have two proposals in this regard:
Manual Ligand Extension: Adding the R9 sequence to the existing ligand structure.
Using PDB Molecule: Using PDB entry 6O4J for the molecule, due to relevance in structural aspects.
Winter Break Goals:
Further tune the docking protocols with the goal of resolving job failures.
Apply suggested ligand modifications and run binding affinity calculations.
Use the β-sheet grooves and FF region previously identified further in more docking experiments.
References:
Zhuang, W., Yang, L., Ma, B., Kong, Q., Li, G., Wang, Y., & Ben Zhong Tang. (2019). Multifunctional Two-Photon AIE Luminogens for Highly Mitochondria-Specific Bioimaging and Efficient Photodynamic Therapy. ACS Applied Materials & Interfaces, 11(23), 20715–20724. https://doi.org/10.1021/acsami.9b04813
Khurana, R., Coleman, C., Ionescu-Zanetti, C., Carter, S. A., Krishna, V., Grover, R. K., Roy, R., & Singh, S. (2005). Mechanism of thioflavin T binding to amyloid fibrils. Journal of Structural Biology, 151(3), 229–238. https://doi.org/10.1016/j.jsb.2005.06.006
O. Ousman, T. (2017). Catalytic diversity in self-propagating peptide assemblies. NatureChemistry.
Rodríguez-Rodríguez, C., Rimola, A., Rodríguez-Santiago, L., Ugliengo, P., Álvarez-Larena, Á., Gutiérrez-de-Terán, H., Sodupe, M., & González-Duarte, P. (2010). Crystal structure of thioflavin-T and its binding to amyloid fibrils: insights at the molecular level. Chemical Communications, 46(7), 1156. https://doi.org/10.1039/b912396b
Wu, C., et al. (2008). The Binding of Thioflavin T and Its Neutral Analog BTA-1 to Protofibrils. Journal of Molecular Biology, 384, 718-729.
Khurana, R., et al. (2005). Mechanism of Thioflavin T Binding to Amyloid Fibrils. Journal of Structural Biology, 151, 229-238.