ItemMechanism of RNA-independent cleavage of CRISPR-Cas9(2020) Liu, Jin; Chen, Xiongping; Hayatshahi, Sayyed; Wang, Duen-ShianPurpose: CRISPR(Clustered-Regularly-interspaced-short-palindromic-repeats)-Cas9 (CRISPR-associated-protein-9) uncovered a new path toward gene therapy. However, non-specific cleavage of Cas9 raises concerns on human therapeutic applications so that it is critical to understand and minimize those non-specifics cuttings. Recent in vitro studies showed that Cas9 cleavage occurred even without the guidance from the guide RNA (gRNA) in the presence of Mn2+, implying the serious issue of off-target effect of Cas9. The purpose of this study is to elucidate the mechanism of the RNA-independent cleavage of CRISPR-Cas9, which may provide insights for the improvement of Cas9 specificity. Method: Based on our previously captured structure of catalytically-active Cas9-gRNA-dsDNA complex, we performed molecular dynamic (MD) simulations on Cas9 complexed with and without gRNA, respectively. We also compared the simulations in the presence of Mn2+vs. Mg2+. All MD simulations were performed using AMBER package with GPU acceleration. Result: We expect our MD simulations to demonstrate the different coordination environments of Mn2+ and Mg2+ in the presence or absence of gRNA, elucidating a novel mechanism for Cas9 off-target effects. Conclusion: In this study, we expect to identify the mechanism of RNA-independent cleavage of CRISPR-Cas9, shedding light on the development of new Cas9 variants to reduce off-target effects. ItemLipoprotein Based Targeted Therapy for Acute Myelogenous Leukemia(2020) Sabnis, Nirupama; Lacko, Andras; Saranya Conjeevaram Nagarajan, Bhavani; Dossou, Akpedie; Raut, Sangram; Ahmed, Nadia; Kvinta, RyanPurpose: The purpose of this project was to initiate studies to develop nano-formulations for anti-cancer agents to treat Acute Myeloid Leukemia (AML) via enhancing their targeting efficiency, cytotoxicity and reducing off-target toxicities. Methods: A computer optimized formulation of rHDL-Valrubicin was characterized for its physical and chemical properties. The diameter of the nanoparticles and the zeta potential were measured by Malvern Zetasizer. The anisotropy was measured by Cary eclipse spectrofluometer. The drug entrapment efficiency was measured by absorbance of valrubicin at 490nm. The cytotoxicity of the formulation was tested in 2 Leukemia cell lines, Kasumi-1 and AML-193, using cell counting kit 8, Dojindo Molecular Technologies. The cardioprotective effect of the formulation was investigated in Rat cardiomyocytes cells, (H9C2). Results: rHDL-Valrubicin formulations were successfully prepared with drug entrapment efficiency of 31.8%. The formulation was non leaky and homogeneous with average particle diameter of the 13.2 ± 2.2 nm and the average zeta potential was found to be -2.39 ± 9.81 mV. The rHDL-Valrubicin formulation exhibited 50 times more cardioprotective effect as compared to free valrubicin. In the Leukemia cell lines Kasumi-1 and AML-193, the inhibitory concentration required to kill 50% of cells (IC50) using the rHDL-Valrubicin formulation was observed to be 1.5 and 1.9 times more effective than the free valrubicin respectively. Conclusion: These proof of concept studies indicate the potential of rHDL as a drug delivery platform to mitigate the cardiotoxicity associated with anthracycline use in the treatment of AML. ItemDiscovery of Small Molecule Slack Inhibitors for the Treatment of MMPSI: SAR Development in the Western and Central Region of Hit Compound VU0606170(2020) Qunies, Alshaima'a; Acuna, Valeria; Acebo, Jonathan; Spitznagel, Brittany; Weaver, C.; Emmitte, Kyle; Mishra, NigamIntroduction: Malignant Migrating Partial Seizures of Infancy (MMPSI) is a severe and pharmacoresistant form of epilepsy. Slack channels are sodium-activated potassium channels that regulate essential electrical activity in the CNS. Gain-of-function mutations in Slack channels have been linked to MMPSI. Objective: To develop small molecule selective inhibitors of Slack employing a library synthesis based iterative hit optimization approach to discover leads for development into MMPSI therapeutics. Methods: Classical and state-of-the-art synthetic chemistry techniques including microwave assisted organic synthesis and flow chemistry were employed. Purification was by automated liquid chromatography. Bruker Fourier 300HD and Agilent 6230 time-of-flight LC/MS were utilized to obtain NMR and HRMS, respectively. Inhibitory activity of Slack was measured utilizing a Thallium flux assay in HEK293 cells stably expressing either WT or Slack mutants. Results: SAR studies around hit compound VU0606170 identified a chiral-methyl analog in piperizine core as optimal for potency. Other new core analogs were less potent than their piperazine counterparts. Several western urea and amide analogs were prepared, and a few moderately potent compounds were identified. Replacement of sulfamide linkers with a sulfonamide gave encouraging results. Lastly, invitro DMPK studies with selected compounds revealed high clearance, high protein binding, and good permeability. Conclusion: SAR was identified for Slack activity, mutant selectivity, and DMPK properties around the western and central region of VU0606170. Presently, synthesis of analogs that combine optimal functional groups from the entire chemotype are underway with a goal of improving potency and DMPK properties. ItemThe effect of phenylthiophene as an allosteric pharmacophore on the affinity of phenylpiperazine derivatives for dopamine receptors(2020) Liu, Jin; Hayatshahi, Sayyed; Amani, ArmaghanPurpose: Dopamine receptors are important therapeutic targets in treatment of many neurological diseases such as schizophrenia and Parkinson's disease. Compounds like phenylpiperazine derivatives that selectively target D3R subtype have shown significant clinical benefits. The selectivity of these compounds for D3R over D2R is shown to increase when a phenylthiophene is added as a second pharmacophores. Here, we used a computational approach to see how the second pharmacophore affects the affinity of these ligands. Method: In an effort to evaluate the difference of these compounds, docking on the crystal structure of D2R and D3R was completed using AutoDock vina after the ligands were prepared for docking with Autodock tools. Results: The binding energies from the docking poses followed expected trend that the addition of phenylthiophene compounds would increase the affinity for D3R. We further studied the binding of the more selective ligands that have 3,5-dicholoro and 3CN substitution on the phenylpiperazine to analyze the important interactions that stabilize the D3R binding. Conclusion: Studying compounds that have higher selectivity towards D3R shed light on important interactions that the phenylthiophene can have with D3R. The added phenylthiophene seems to potentially interact with Tyr7.35 and the ECL2 region in D3R. Additional investigation is needed to determine the neurological applicability of these compounds. ItemDetermining the binding site of Carisoprodol on GABAA receptor(2020) Liu, Jin; Huang, Renqi; Claudio, Maria; Hayatshahi, SayyedPurpose: Carisoprodol (CSP) is prescribed to treat musculoskeletal pain. CSP exerts inhibitory action on GABAA receptors (GABAA Rs) in certain concentrations. However, its binding sites remain elusive. The purpose of this study is to determine the binding site for CSP's inhibitory action on GABAA Rs. Our electrophysiological studies have shown that CSP inhibitory action is diminished by alpha1 T261F mutation of the picrotoxin (PTX) binding site. Therefore, we hypothesize that CSP shares PTX's binding site at GABAA Rs. Methods: We docked CSP on wild type alpha1beta2gamma2 and mutant alpha1(T261F)beta2gamma2 GABAA Rs using Glide program. We further performed molecular dynamics (MD) simulations of wild type and mutant GABAA Rs in unbound forms and in complex with PTX and CSP. Results: The docking reproduced the experimental pose of PTX and the effect of mutations on its binding, but could not predict the effect of the mutations on the CSP binding. However, the MD simulations showed that the local channel conformation is changed upon the mutations, and consequently, the binding of both ligands is significantly deteriorated. We further used the observed receptor-ligand interactions of CSP to predict molecular changes that would improve its binding. Conclusions: We demonstrate that the consideration of the pocket dynamics is necessary to capture the changes mutations potentially cause in GABAA Rs. The similar trend for CSP and PTX in MD simulation results validate our hypothesis that the two molecules share the same binding pocket. These data provide further information on how CSP may interact with the receptors. ItemUsing Artificial Intelligence Algorithms to Develop Target-specific Ligands(2020) Emmitte, Kyle; Hayatshahi, Sayyed; Liu, Jin; Escobedo, Daniel; Li, Leo; Yang, YanmingPurpose: Pharmaceutical research has recently taken advantage of the rapid advancement in artificial intelligence (AI). The purpose of this study is to use AI to facilitate target-specific ligands development in drug discovery. Here, we used a machine learning algorithm to identify target-specific features of compounds to the metabotropic G-protein coupled receptors 2 and 3 (mGlu2 and mGlu3), which have been targeted for treatment of CNS disorders. Methods: A convolutional neural network (CNN) with 3 hidden layers was made using Tensorflow. We obtained data sets of 315 mGlu2 and 118 mGlu3 compounds and split them into testing and training sets. 75% of each data set was used for training and the remaining was used for testing. We fed the data sets into the CNN and ran the program over 100 iterations with each data set. Results: The neural network was able to differentiate between mGlu2 and mGlu3 compounds in the testing data set with up to a 99.1% accuracy. Visualization of the hidden layers revealed areas in the 2D images that the CNN viewed as important to distinguish the compounds. These identified chemical features can be considered as the target-specific features of the compounds. Conclusions: The neural network is able to differentiate mGlu2 and mGlu3 compounds by 2D representation alone and provide insight to distinguish target-specific features of the compounds through hidden layer visualization. Further testing through cross validation and introduction of a control data set with compounds that bind to neither receptor is needed. ItemEffect of increased blood glucose on anti-cancer nanoparticle interactions with tissue(2020) Kastellorizios, Michail; Mishra, Ina; Nguyen, AlexandraPurpose: Little research has been conducted on liposomal drug delivery in pediatric diabetics and non-diabetics with cancer. Preliminary data demonstrates that increased liposomal interactions with collagen in the tumor's extracellular matrix affect tumor targeting. The goal of the present study was to understand the effect that glucose has on liposomes' interaction with collagen and its interference with the anti-cancer action of liposomal drugs in diabetic pediatric patients. Methods: Liposomes were formulated according to the Doxil molar ratio. Varying amounts of glucose, glycated albumin (GA), albumin, and liposomes were dissolved into phosphate-buffered saline (PBS) to make solutions representative of the mean pediatric hypoglycemic, normoglycemic, and hyperglycemic diabetic and non-diabetic patient. An Optical Contact Angle instrument quantified the interactions between liposomes and collagen by advancing and receding a drop of each solution onto human and rat collagen-coated slides. Results: The contact angle measured was lower in human collagen than rat collagen in the PBS and Liposomes samples. In samples with added glucose and albumin, the contact angle was lower in rat collagen than human collagen. Conclusions: Liposomes by themselves in PBS have a greater interaction with human collagen than rat collagen, but the addition of glucose, GA, and albumin reverses this trend so that liposomes have a greater interaction with rat collagen than human collagen. The protein's interactions also overpower the liposomes' interaction with collagen. Further investigation is necessary to determine if the presence of elevated glucose has a significant effect on how liposomes interact with collagen. ItemPseudo-ternary Phase Diagrams for Lipid Formulation(2020) Dong, Xiaowei; Dulie Kom Nzia, JeannePurpose: Lipid-based formulations are frequently studied as they improve oral bioavailability of water-insoluble drugs that constitute 40% of new drugs. A pseudo-ternary diagram is a thesaurus tool used to define a specific physical composition of a thermodynamically stable solution. The aim of this study was to construct phase diagrams of lipid-based formulations that could be used for identifying optimum compositions for drug delivery. Method: The aqueous titration experimental method was used. A lipid, including oleic acid and Miglyol 812, and a surfactant, including Cremophor EL, Tween 80 and TPGS, were mixed in a fixed ratio. No co-surfactant was used. The mixture was heated at 45°C. Water was used as a titer and was added in increment of 5%-10% to up to 95% of the total mixture. After equilibrium, the mixture was observed for characterization. Data were gathered on a table, showing each increment of water, the amount of each component, and the percentage of each component in the mixture. The pseudo-ternary phase diagrams were constructed. Result: All phase diagrams have various areas of emulsion and microemulsion. Comparing sets of phase diagrams, Miglyol and TPGS produced the largest areas of one continuous phase. Oleic acid and TPGS produced a large area of emulsion. Conclusion: The pseudo-ternary phase diagrams of one lipid and one surfactant were constructed by using a titration method. Miglyol and TPGS form stable binary systems that could be used for further formulation development. ItemEvaluation of Lipoprotein Mimetic Anthracycline Formulation for the Treatment of Ewing Sarcoma(2020) Kvinta, Ryan; Dossou, Akpedie; Saranya Conjeevaram Nagarajan, Bhavani; Sabnis, Nirupama; Raut, Sangram; Lacko, Andras; Ahmed, NadiaPurpose: Anthracyclines are effective in treating many types of cancer, including pediatric cancers such as Ewing Sarcoma. Currently, their use is limited due to cardiotoxicity, but a novel drug delivery method using lipoprotein-based technologies could mitigate this via selective delivery of its payload to cancer cells using a receptor-mediated mechanism. Methods: A soluble formulation of HDL-mimetic peptide (Myr-5A)-Valrubicin was fabricated using Nanoassemblr. Payload efficiency was characterized using spectrophotometric measurements. Size and zeta potential were measured with Zetasizer. Homogeneity was measured using FPLC. Hemolysis assays were performed by incubating Rabbit Red Blood Cells with Myr-5A-Valrubicin at different time intervals and measuring the absorbance of the supernatant at 550nm after centrifugation for 5 minutes. Cytotoxicity assays were performed on Ewing sarcoma cell lines TC205, TC32, CHLA10 and rat cardiomyocytes H9C2 using Cell counting kit (CCK 8). Results: The Myr-5A-Valrubicin nanoparticle formulation was observed to be stable, non-leaky and homogeneous with payload efficiency of 65%, size 87.75 ± 28.4nm, zeta potential -2.89 ± 9.81mV. The percentage hemolysis for Myr-5A-Valrubicin formulation was 5 and 2 times less compared to the free valrubicin formulation at 3.25 and 32.5�g/mL Valrubicin concentration, respectively. The cytotoxicity assays revealed Myr-5A-Valrubicin is effective in protecting cardiomyocytes. Conclusion: These studies indicate the potential of this novel drug delivery platform as an alternative or adjuvant therapy for combating Ewing Sarcoma. More experiments with tumor bearing model are needed to evaluate the efficacy of this formulation before reaching clinic. ItemCharacterize the Pre-catalytic State of CRISPR/Cas9(2020) Liu, Jin; Chen, XiongpingPurpose: CRISPR-Cas9 has been widely used as a gene-editing tool, but its catalytic mechanism remains elusive. Cas9 has two catalytic domains, HNH domain and RuvC domain. In our previous study, we have successfully identified the pre-catalytic state of the HNH domain. The purpose of this study is to identify the pre-catalytic state of the RuvC domain, which may provide an understanding of the Cas9 catalytic mechanism and strategies for Cas9 engineering. Methods: Here we use molecular dynamic simulations to discover the pre-catalytic state of Cas9. The initial structure was obtained from our previous simulations, where the HNH domain was in the pre-catalytic state. We placed two Mg2+ ions and non-target DNA strand at the RuvC domain and performed molecular dynamics simulations to capture the pre-catalytic state of the RuvC domain. Results: Our molecular dynamics simulations revealed a pre-catalytic state of Cas9 that both target DNA strand and non-target DNA strand are posed to be catalyzed by Cas9. Conclusions: In this study, we identified the first atomic-level structure of CRISPR/Cas9 with the pre-catalytic state in both catalytic domains. ItemMicrobial Natural Product Drug Discovery Through Systematic Sampling of Diverse Texas Soils(2020) Cheng, Eric; Patel, Rinkal; Currens, GrantPurpose: To prove a concept of discovering microbial natural products through systematic sampling of diverse Texas soils by constructing and screening a pilot library of secondary metabolites produced by Texas soil-derived microbes for cytotoxicity against three human cancer cell lines. Methods: Secondary metabolites were extracted from microbial isolates using MeOH and EtOAc. These metabolites were fractionated by flash chromatography and screened for cytotoxicity against MIA PaCa-2, SH-SY5Y, and COLO 829 using an ATP-luciferase assay. A short-list of active fractions was compiled and further activity-guided purification through HPLC was performed to purify the active compounds. From this, isolated active compounds were identified through methods including TOF-MS, MS/MS, NMR, and X-ray crystallography. Results: Malformin, a bicyclic pentapeptide which has been shown to elicit potent anti-cancer effects was purified and helped to validate our methodology. Subsequently, two related compounds, Aspergillin PZ and Trichoderone B, exhibiting anti-cancer effects were purified from a fungal isolate. Though previously identified, these compounds have not been extensively studied for their general cytotoxicity or differential potency, and have thus been enrolled in the US National Cancer Institute - 60 human tumor cell lines screen (NCI60). Conclusions: While a previously unidentified compound has not yet been discovered through this pilot study, bioactive secondary metabolites have been re-discovered which not only validates our methodology but also provides promising opportunity to address gaps in scientific understanding of previously reported compounds. Theoretically, enlarging our library size should afford new and active natural products. ItemComputer-Assisted Design and In Silico Screening of Brain-Targeting Peptide Prodrugs(2020) Prokai, Laszlo; Prokai, Katalin; Nguyen, Vien; De La Cruz, DanielPurpose: Prodrug approaches promise overcoming obstacles for the brain delivery of therapeutically useful small neuropeptides. However, their rational design and large-scale computational screening have been elusive. Recently, we have introduced a method that not only enhances a prodrug's access to the brain through lipoamino acid residues (LAAs), but also ensures site-specific bioactivation by prolyl oligopepdidase (POP) selectively expressed in neuronal milieu. Using thyrotropin-releasing hormone (TRH) as a model, we show a computer-aided strategy to assist the design and optimization of brain-targeting prodrugs for small neuropeptides. Methods: Using SCIGRESS molecular modeling and AutoDock Vina docking, lipophilicity (indicated by calculated logarithm of octanol/water partition coefficient, clogP) and binding affinity to POP (expressed as free energy change, ΔG) were assessed in silico for a virtual library of prodrugs. A computational model of human TRH receptor (hTRHr-1) was also adapted to provide proof of concept for the prodrug principle. Results: Prodrugs of a virtual library docked computationally to POP's active site displayed ΔG values comparable to that of a co-crystallized POP ligand, but with subtle differences based on the configuration of the LAAs and the POP-sensitive linker. Expectedly, they showed no affinity for binding to hTRHr-1's active site. Co-optimization based on clogP has allowed for the selection of top TRH prodrug candidates for further in vitro and in vivo evaluations. Conclusion: We have created a comprehensive in silico workflow to aid the rational design and large-scale virtual screening of brain-targeting neuropeptide prodrugs. ItemDiscovery of Small Molecule Slack Inhibitors for the Treatment of MMPSI: SAR Development in the Eastern Region of Hit Compound VU0606170(2020) Mishra, Nigam; Spitznagel, Brittany; Weaver, C.; Emmitte, Kyle; Tran, Elizabeth; Qunies, Alshaima'aIntroduction: Malignant Migrating Partial Seizures of Infancy (MMPSI) is a severe form of epilepsy without effective treatments. Slack channels are sodium-activated potassium channels, which are critical regulators of electrical activity in the CNS. MMPSI has been linked to gain-of-function mutations of Slack channels. Objective: To develop small molecule selective Slack inhibitors through an iterative hit optimization library synthesis approach to identify lead compounds for development into MMPSI therapeutics. Methods: Classical and state-of-the-art synthetic chemistry techniques including microwave assisted organic synthesis and flow chemistry were employed. Purification was by automated liquid chromatography. Bruker Fourier 300HD and Agilent 6230 time-of-flight LC/MS were utilized to obtain NMR and HRMS, respectively. Inhibitory activity of Slack was measured utilizing a Thallium flux assay in HEK293 cells stably expressing either WT or Slack mutants. Results: SAR studies developed around hit compound VU0606170 revealed that a 2,5-di-substitution pattern on the eastern phenyl ring was optimal for Slack activity. Compounds were identified that are selective for the A934T Slack variant versus WT. Modifications to the linker portion led to a loss of Slack activity. Lastly, in vitro DMPK studies with selected compounds revealed high clearance, high protein binding, and good permeability. Conclusion: SAR was identified for Slack activity, mutant selectivity, and DMPK properties around the eastern portion of VU0606170. These findings are presently being combined with SAR obtained from other regions of the molecule in search of compounds with improved potency and a more favorable DMPK profile.