Pharmaceutical Sciences
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30821
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Item Design of man-made miniature CRISPR-Cas systems using computational technologies(2022) Arachchige, Vindi Mahesha Jayasinghe; Liu, JinPurpose: An RNA-guided targeted genome engineering platform, CRISPR/Cas system is one of the breakthroughs of the twenty-first century. Despite the wealth of its advancement, there are some associated limitations that need to be overcome for the betterment of this revolutionized technology. Among them, the larger size of the available Cas proteins that are essential for the functioning of these tools limits their in vivo administration due to the low delivery efficiency. To address this issue, we have used computational chemistry tools to design smaller versions or compact size Cas proteins that can be used as an alternative. Methods: The available crystal structures of CRISPR-Cas systems were utilized and the reduction was done preserving the regions that are essential for the DNA binding and cleavage functions using Chimera, Yasara, and the Swiss Model software. Molecular Dynamics (MD) simulations were performed to obtain stable conformations of the reduced structures. The minimized sequences were used to generate their structures by the Swiss Model. Results/Conclusions: Four stable man-made miniature Cas proteins were generated that are less than half the size of the currently used CRISPR systems such as Cas9 or Cas12a. The sequence-based modeling studies using the Swiss model have shown the similar folding of these reduced proteins compared to their original counterparts. Further experimental validation of their ds-DNA cleavage activities remains to be determined at this point of the study.Item Meta-Analysis and Systematic Review of Post-Transplant Infection Rates Following Prophylactic and Preemptive Strategies in Solid Organ Transplant Recipients(2022) Te Poele, Nicholas; Ho, Jason; McLeroy-Te Poele, Ashley; Darden, Chase; Meyer, Kollin; Howell, CrystalPurpose : Standard of care to prevent infectious complications post solid organ transplant (SOT) generally involves prophylactic antimicrobial or preemptive monitoring strategies. This systematic review and meta analysis aims to compare rates of common viral and fungal post-SOT infections following either preemptive or prophylactic therapy strategies in kidney, liver, pancreas, lung, and heart SOTs. Methods : Data sources included PubMed, Embase, Medline Complete, Scopus, Web of Science, US National Institutes of Health's ongoing trials registry, Cochrane Library, email listservs, and references of included articles. Studies were considered for inclusion if they involved SOT patients who received an abdominal or thoracic organ, aged over 18 years, and recipients of either prophylaxis or preemptive prevention strategies for a qualifying pathogen within the first year of transplantation. Qualifying organisms included Candida species, Aspergillus species, Zygomycosis (multiple species), Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), and Varicella Zoster Virus (VZV). Data extracted from eligible studies included study methods, prevention strategy used, and development of fungal and/or viral infections. The primary outcome was the odds ratio of opportunistic infections post-SOT when comparing preemptive and prophylactic strategies. Secondary outcomes included time to infection, rates of transplant rejection, rates of antimicrobial adverse events, and mortality rate. Results : 107 studies were extracted and pooled for analysis. Of the 14,464 patients included, 12,194 patients reportedly received prophylactic therapy, while 1,257 patients reportedly received preemptive therapy. Two of the most common infections were more prevalent in patients receiving preemptive therapy, CMV (OR 6.47; 95% CI, 5.72-7.32) and Aspergillus species (OR 2.92; 95% CI 1.99-4.27). Of patients who received preemptive therapy, 59.45% reported an infection with one of the identified study pathogens (n=739) and of patients who received prophylactic therapy, 18.84% reported an infection (n=2,297) (OR 6.32; 95% CI, 5.59-7.14). Patients who received preemptive therapy consistently had an increased odds of infection across all categories, heart transplants (OR 12.73; 95% CI, 8.86-18.30), lung transplants (OR 10.95; 95% CI, 5.35-22.43), liver transplants (OR 4.35; 95% CI, 3.25-5.83), and kidney transplants (OR 2.94; 95% CI, 2.57-3.36). The mortality rate was not significantly different between preemptive and prophylactic therapies (OR 1.57; 95% CI, 0.98-2.53) nor was the rate of adverse effects (OR 1.04; 95% CI, 0.86-1.27). Meta-analysis of infection rates in 14 studies showed that the odds of infection in the preemptive group were 3.84 times that of the prophylactic group (OR 3.84; 95% CI, 2.03-7.26; p < 0.05). Analysis of rejection rates in 8 studies and of mortality rates in 4 studies showed no statistically significant differences. Conclusion : The incidence of infection within the first year of SOT is lower in recipients that receive prophylaxis compared to those that receive preemptive therapy but the rates of mortality and adverse effects were similar.Item Allosteric Modulation of Small-Molecule Drugs on ACE2 Conformational Change upon Binding to SARS-CoV-2 Spike Protein(2022) Wang, Duen-Shian; Hayatshahi, Hamed; Arachchige, Vindi Mahesha JayasingheSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused Coronavirus disease (COVID-19) pandemic. Drug repurposing studies, including drugs such as dexamethasone (DEX), chloroquine (CQ), and telmisartan(TLS), have been performed in COVID-19 clinical trials. DEX and CQ have been demonstrated in vitro to bind angiotensin-converting enzyme 2 (ACE2), a cellular entry receptor utilized by SARS-CoV-2. However, how DEX/CQ bind to ACE2 and their mechanisms of action are still unknown. Here we demonstrated that DEX, CQ, and TLS disrupt the interactions between SARS-CoV-2 spike protein and human ACE2 via binding to an allosteric site close to the viral spike protein binding region at the peptidase domain of ACE2, causing a conformational change of the ACE2. We defined four conformational states of ACE2 based on the two helices distances. Our molecular dynamics simulations suggested that binding to the viral spike protein shifted ACE2 conformation populations away from "Open" conformation. Such conformation population shift is further enhanced by the Delta variant. The binding of the drugs to ACE2 rescues this conformation population shift allosterically to keep ACE2 in "Open" conformation mostly. Our findings provide a potential insight that modulating the conformation of ACE2 may prevent SARS-CoV-2 invasion due to unfavored poses for spike protein binding.Item An Investigation of the Allosteric Effects of Agonist and Antagonist Ligands on Sigma-1 Receptor using MD Simulation and Machine Learning Methods(2022) Kumari, Pratibha; Liu, JinPurpose: Allosteric regulation is the control of the activity of a protein or protein complex by the binding of a ligand or effector molecule, at a site topographically distinct from the active site of the protein. The sigma-1 receptor (Sig1R), a small-ligand operated transmembrane protein, has been implicated in various neural processes such as calcium signalling, cell survival and function, inflammation, and synaptogenesis. Many small molecules act as agonist or antagonist ligands to Sig1R based on their ability to recapitulate the phenotype of receptor overexpression or knockdown, respectively. Sig1R exists in multiple oligomeric states, and agonist and antagonist are found to have a different impact on the oligomeric form of the receptor. The crystal structure of human Sig1R reveals that both agonist and antagonist ligands share the same binding pocket. However, why agonists and antagonists have distinct activities while binding to the same pocket remains unknown. It is also not clear why binding to a pocket not at the oligomer interface could allosterically affect oligomer formation of Sig1R. Our objective is to gain a molecular-level understanding of how agonist and antagonist ligands allosterically modulate the oligomer interactions differently. Method: An atomistic molecular dynamics (MD) simulation study was employed to investigate how the interface of homotrimer human Sig1R bound to agonist ((+)-pentazocine) and antagonist (PD 144418) ligands are allosterically affected. Machine learning algorithms developed by our lab were used to identify the residues that are impacted allosterically. Results: A significant decrease in the interactions between the interface residues of protomer units in agonist bound Sig1R has been found. MM/GBSA and PCA analysis reveal lowered stability of agonist-bound trimer in simulations compared to an antagonist-bound structure. The coordinated actions between the pocket and interface residues depend substantially on the type of ligands present in the binding pocket. The residue response map obtained using machine learning algorithms reflects that the properties of most of the interface residues (T141, H54, H55, G87, L111, H116, R119, A183, D188, S192, Q194, D195, and T198) are affected in different manners. Conclusion: It is shown that even though agonist and antagonist ligands bound at the same pocket, their ability to allosterically impact the interface residues is significantly different which may lead to lesser stability of high molecular weight oligomers in the agonist bound Sig1R. Our research presents a potential to collaborate MD and machine learning methods to identify the allosteric response of different ligands binding at the same pocket in protein.Item Development and Validation of a Novel RP-HPLC Analytical Method for Quantification of Amphotericin B(2022) Mans, Jaylen C.Amphotericin B (AmB) is an antifungal and antiparasitic natural product. AmB is biosynthesized through bacterial fermentation of Streptomyces nodosus. For decades researchers have been investigating new methods to improve the drug formulation. However, drug development research requires validated quantitative analysis techniques to accurately measure drug concentrations. Previously, AmB has been quantified by one of several reverse phase-high performance liquid chromatography (RP-HPLC) that have been reported in literature. However, these methods rely on relatively high disodium EDTA concentration in the mobile phase to separate AmB from its impurities. As a chelating agent, EDTA can bind to metal surfaces within HPLC instrument. Over time this effect can be detrimental, as excess EDTA can precipitate out of solution and negatively impact the integrity of internal instrument components or column performance. Further, reported RP-HPLC methods have relatively high sample run time (> 35 minutes) due to their reliance on isocratic separation. Shorter Run times (< 20 minutes) are desirable for HPLC analytical methods due to the reduced cost mobile phase, as well as to prolong lifetime of detector lamp and stationary phase column. The purpose of this work is to develop and validate a new alternative HPLC method with shorter run times that avoid high EDTA usage. Our developed method achieves a shorter run time by utilizing a gradient HPLC method and avoids the use of EDTA by replacing the agent with 0.2% Acetic Acid.Item Design and discovery of new tool compounds for studying the role of Slack potassium channels in malignant migrating partial seizure of infancy(2022) Qunies, Alshaima'a; Spitznagel, Brittany; Du, Yu; Weaver, C.; Emmitte, KylePurpose: Slack channels are sodium-activated potassium channels encoded by the KCNT1 gene, and are key regulators of electrical activity in the central nervous system. Slack channels belong to the Slo family of potassium channels (Slo2.2). The development of malignant migrating partial seizure of infancy (MMPSI), a type of severe infantile epilepsy, has been linked to KCNT1 gain of function mutations. The aim of this project is to design and synthesize Slack channel inhibitors for use as in vivo probes via an iterative hit optimization approach. Methods: 110K-member library was screened in a cell-based assay against wild-type Slack and three MMPSI-associated KCNT1 mutants. The hit compound VU0531245 (VU245) was selected for the development of structure-activity relationship (SAR) studies in order to optimize its potency and drug metabolism and pharmacokinetic (DMPK) properties. A thallium flux assay in HEK-293 cell lines stably expressing Slack channels was utilized to evaluate Slack inhibitory activity of the resulting compounds. Results: Compound libraries were designed around VU245 through the systematic scanning of the chemical space and incorporating various bioisosteric replacements. Our data suggest that modifications at the phenyl ring A lead to mode switching from inhibition to activation. However, fluorinated, alicyclic, and deuterated alkoxy groups at 2-position of the phenyl ring maintained Slack inhibitory activity; moreover, they improved some DMPK properties. We have demonstrated that piperidine replacement at ring B was tolerated. In addition, an ethylene linker in place of the 1,2,4-oxadiazole ring at position C maintained the Slack activity. A 4-fluorophenyl ring at position D was tolerated, improved the metabolic stability, and was used as the basis for further SAR exploration. Finally, a sulfonamide linker was optimal for Slack inhibitory activity. Collectively, the tested analogs within this series demonstrated good passive permeability with no evidence of P-gp-mediated efflux; however, high protein binding (fu ~ 0.01 - 0.04) was observed. Conclusions: SAR for Slack activity and DMPK properties was identified around VU245. Further optimization is required to develop suitable Slack in vivo probes with improved potency and DMPK properties.Item Molecular Docking Study of Positive Allosteric Modulators of the Sigma-1 Receptor for the COVID treatment of elderly patients(2022) Contractor, Sareena; Kumari, Pratibha; Liu, JinPurpose: Our goal with this project is to determine conformations of ligands that have strong affinities to allosteric sites of the Sigma-1 Receptor (Sig1R). The SARS-CoV-2 strain virus is part of a group of viruses known as RNA Positive Sense Coronaviruses. The virus enters the cell through endocytosis and replicates in a cellular compartment derived for the Endoplasmic Reticulum (ER). Viral replication causes ER stress and forces the cell to adapt. The Sig1R found within the membranes of the nucleus, ER, and mitochondria. The receptor protein can change conformation to help the cell cope with ER stress. Positive Allosteric Modulators (PAMs) bind to the Sig1R and cause conformational changes that can alter its response to natural ligand binding. By targeting the Sig1R we can create therapeutic responses to the SARS-CoV-2 virus by modulating ER stress response signaling pathways for elderly patients. Method: We performed molecular docking for the Nonselective, Selective and Putative ligands of Sig1R.With molecular docking, we were able to determine the binding affinities and conformations of three types of PAMs in relation to the Sig1R. Results: The study showed that nonselective allosteric modulators have the strongest binding affinities for the Sig1R. Through molecular docking and 3D visualization, the data showed that most PAMs bind to the monomeric Sig1R at an orthosteric binding site. The only ligand to bind at a site other than the orthosteric site was SCH23390. The rest of the ligands bound to the receptor at the orthosteric site, which would indicate that they would need to compete with the receptor's natural ligands for binding. To avoid the competition, the PAMs need to bind to the receptor at a site different from their first pose, or most preferred configuration. Conclusion: This project showed us that non-selective PAMs have a higher binding affinity. We were also able to identify a need to explore other binding configurations, besides the first pose, to find allosteric binding of the PAMs to the Sig-1R.Item A sensitive LC-MS/MS method to quantitate nitrite in human plasma(2022) Wang, Jianmei; Hemingway, Holden; Coyle, Donna; Romero, StevenPurpose: Numerous methods are available to quantitate nitrite in biological samples, including fluorescence, chemi-luminescence, capillary electrophoresis, colorimetric and ultraviolet (UV) spectrophotometry and gas chromatography-mass spectrometry (GC-MS). However, limitations associated with these techniques (e.g., lack of specificity, low sensitivity, etc.) prompted us to develop a LC-MS/MS method to quantitate nitrite in biological samples. Methods: We compared two derivatization methods which are used to convert the endogenous nitrite to a more stable organic compound that is able to be analyzed by LC-MS/MS. The first S-nitrosoglutathione (GSNO)-based method was performed by selected-reaction monitoring specific mass transition m/z 337 ([M+H]+)→m/z 307 ([M+H-14NO]+•) for GS14NO and GS15NO as internal standard (IS) by reaction of nitrite and glutathione (GSH). The second 2,3-naphthotriazole (NAT)-based method was performed by measuring NAT m/z 170 ([M+H]+)→m/z 115 ([C9H7+] and NAT-N15 as IS following the reaction of 2,3-diaminonaphthalene (DAN) with nitrite to produce NAT. We found that the latter NAT-based derivatization method is reproducible, stable, and 100 times more sensitive than the GSNO method. In addition, we validated the NAT-based method for precision and accuracy, recovery, stability and derivatization time and temperature. We then utilized the optimized method to quantitate nitrite in human plasma. Results: We found that nitrite stability in human plasma filtrate can be affected by freeze-thaw cycles and storage temperature which were evidenced by a decrease in nitrite levels over 50% after 24 hours at -20°C. However, the derivatized and extracted samples were stable for 24 hours at room temperature. When derivatization is fully complete and performed at 370C and 45 minute incubation, the recovery is 108%. The precision and accuracy are 15%, and 106%, respectively. The linearity range is 0.13-16 µM with linear regression, 1/x weighing correlation coefficients = (r), R2>0.9990. The same amount of labeled isotope as IS was added to each sample to keep track of signal fluctuation between study samples, In doing so, it was confirmed that the quantitation of endogenous nitrite was indeed affected by individual complex sample content. And the use of isotope-labeled nitrite reduced the impact of high background levels of nitrite in biological matrices, which other methods cannot achieve. Conclusion: We have developed and validated a reproducible and highly sensitive LC-MS/MS method for quantitation of nitrite in biological samples. In addition, we anticipate that our method can be utilized in other fluidic human biological samples.Item Library synthesis of Slack potassium channel activators based on a high-throughput screening hit(2022) Nguyen, Dalena; Qunies, Alshaima'a; Du, Yu; Weaver, C.; Emmitte, KyleIntroduction: Fragile X syndrome (FXS) is an X-linked disorder that is associated with cognitive disabilities. Previous studies have shown an association between a mutation in the FMR1 gene and FXS. The mutation is an overexpansion of the promoter region, resulting in hypermethylation and silencing of fragile X mental retardation protein 1 (FMRP).1 FMRRP is necessary for activating Slack proteins, which are important for normal neuronal activity.2, 3 Objective: To synthesize a library of small molecules in two distinct regions of an HTS hit chemotype made of sulfonamides and heteroaryl amines for functional testing versus Slack channels. Methods: Solution-phase and microwave-assisted organic chemistry were utilized to synthesis small molecules. Purification of compounds involved using automated liquid chromatography. Final compounds were characterized through NMR and HRMS data obtained from a Bruker Fourier 300HD and Agilent 6230 time-of-flight LC/MS, respectively. Activity of new compounds versus Slack was measured utilizing a Thallium flux assay in HEK293 cells stably expressing WT Slack channels. Results: SAR studies conducted around hit compound VU0521448 have thus far discovered analogs showing only weak activity against Slack proteins. Data for SAR studies developed around hit compound VU0521398 are in the process of being collected. Conclusion: The sulfonamide library prepared using different sulfonyl chloride substituents gave some analogs with weak activity in activating Slack proteins. Additional SAR studies will be carried out to examine other aspects of the chemotype, such as modifying the piperidine ring to pyrroline or pyrrolidine. FUNDING STATEMENT Research reported in this publication was supported by the National Heart, Lung, and Blood Institute (R25HL125447) and the National Institute of Mental Health (R21MH125257), both of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health.Item Identification of Potential Positive Allosteric Modulators of Sigma-1 Receptor using Computational Molecular Docking and Virtual Screening(2022) Olson, Zachary Gunnar; Kumari, Pratibha; Liu, JinPurpose: Coronaviruses (such as SARS-COV-2) can achieve replication in host cells by activating pathways in the endoplasmic reticulum (ER), which causes ER stress. As it is known that the mortality rate of elderly populations in COVID-19 infection is dramatically high, indicating a vital role in the timely response of cell stress response signaling pathways in the management of the treatment of COVID-19. The sigma-1 receptor (Sig1R) is an important upstream modulator of ER stress, which regulates folding/degradation of proteins, Ca+2 homeostasis, ER stress responses, and cellular survival. Therefore, ligands enhancing Sig1R activities may improve the treatment of COVID-19 of the elderly patients. Positive Allosteric Modulators (PAM) can enhance protein activities by binding at an allosteric site. Several PAMs of Sig1R have been reported. However, the molecular basis of interactions of PAMs in Sig1R is poorly understood. Further, we do not have much information about the allosteric binding sites in Sig1R yet. Our purpose in this research is to identify possible chemical scaffolds/compounds that can bind at the allosteric sites of Sig1R and selectively elicit the activity of Sig1R. Method: In this study, we have assessed several known PAMs of Sig1R to investigate their binding affinity, the molecular basis of their interactions at three possible allosteric binding sites in Sig1R using the efficient docking suite, Glide. In addition to this, we explored ZINC and DRUG bank databases to search for compounds/chemical scaffolds that are similar to PAMs, which can be docked and engineered further to get a highly efficient drug target/PAM of Sig1R. Results: We have found that methylphenylpiracetam, SKF38393, and SCH23390 show high affinity for allosteric pockets. Further, by virtual screening of small drug-like compounds of the ZINC database in Auto Dock Vina, we obtained a list of 1000 compounds for each allosteric pocket of Sig1R. In the next step, we plan to continually refine our search by performing docking of these compounds and the compounds we obtained through ligand-based search in Glide to identify the promising set of compounds that bind efficiently at an allosteric site in Sig1R. Conclusion: Using molecular docking, we have found three compounds methylphenylpiracetam, SKF38393, and SCH23390 that bind to Sig1R at the allosteric pockets with high binding affinities and identified a list of 1000 compounds for each potential allosteric sites, shedding light on the further development of selective PAMs of Sig1R.Item Contact Angle as Characterization Method for Vitreous Humor and Vitreous Humor Substitute Gels(2022) Jacobsen, DylanPurpose: In clinical ophthalmology, intraocular injections and long-acting implants are currently utilized to facilitate controlled drug release into the eye. However, in vivo pharmacokinetic testing not possible due to the inaccessibility of intraocular tissue. This has created a need for bio-relevant in vitro dissolution methods that reflect in vivo pharmacokinetic studies. To achieve that, tissue properties need to be assessed for relevancy to drug release and, if necessary, incorporated into the in vitro dissolution method design and media selection. Currently, synthetic gel substitutes are utilized in ocular drug release studies and release medium viscosity is the primary property that is adjusted to biorelevant ranges to mimic human vitreous humor. Here, surface characterization of vitreous humor is studied as a potential marker to develop biorelevant in vitro release media. To achieve this, contact angle measurements of human vitreous humor, as well as synthetic vitreous humor substitutes, were obtained using an optical goniometer (DataPhysics Instruments USACorp.). It is hypothesized that obtaining contact angle values of these substances allows for more accurate predictability of drug release. Water contact angle is a simple and efficient method to obtain characterization of the hydrophobicity or hydrophilicity of surfaces and this technique has yet to be utilized in vitreous humor studies. Methods: An analysis of eight different samples of human vitreous humor (donated by Dr. Abe Clark, North Texas Eye Research Institute) and a vitreous humor substitute gel formulation made of agar and hyaluronic acid was completed. The synthetic gel formulation has been reported in the literature as a vitreous humor substitute. Both vitreous humor samples and gel samples were deposited on glass microscope slides and allowed to air dry at room temperature for 24 hours. Water contact angle was then measured at multiple points on each slide and the average values of each run were compiled. Human vitreous humor demonstrated two different phases: a viscous, gel-like portion and a thin, watery portion. These phases were separated in each sample and were tested independently. Contact angles were measured at different hyaluronic acid content of the synthetic gel formulation. Results: Water contact angle measurements were reproducible in both the vitreous humor and synthetic gel samples. The gel-like, more viscous phase of vitreous humor had an average contact angle of 77.46±25°. In contrast, the less viscous, watery phase produced an average contact angle of 38.31±25°. The synthetic gel formulation demonstrated a contact angle of 66.79±10°. Additionally, when altering the concentration of hyaluronic acid in the gel formulation, contact angles were reduced by 8±2° with every .05% reduction in concentration of hyaluronic acid. Conclusions: Water contact angle shows promising capabilities as a reliable method of surface characterization of vitreous humor and vitreous humor substitute gels. Our data showed that vitreous humor-mimicking gels can still have contact angles that are different than vitreous humor, but they can be corrected by simple formulation adjustments. Future studies will utilize the method developed here to study the effect of contact angle on controlled drug release studies of intraocular formulations.