bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Dynamics and Distance‐from‐Average‐Structure Characteristics of Human Cytochrome CYP2D6
Authors: Colwell Quinton E , Furge Laura 
Published: November 2023

CYP2D6 is a major enzyme involved in metabolism of at least 10% of all chemicals including ~12% of pharmaceutical drugs. Understanding of how CYP2D6, and CYPs in general, are able to metabolize such a wide‐range of chemicals despite having a buried active site is partly achieved through understanding the motion of structural regions of the protein relative to one another. However, these conformational dynamics of CYP2D6 are poorly defined in part because structure‐determining techniques (e.g. X‐ray crystallography) are limited to a single conformation per protein crystal structure. The current study uses molecular dynamics in conjunction with analytical methods to overcome this limitation. In particular, distance‐from‐average‐structure measurements were made as a means to understanding the conformational dynamics of ligand unbound structures of CYP2D6 in a dilute, aqueous, simulated in vitro environment. Our results show that the protein conformation is constantly changing in statistically significant ways that support regional movements and an overall “breathing” motion of the protein that may allow for ligand movement to and from the active site. Also, specific secondary structural elements were found to have greater ranges in motion than others. These differences followed the commonsensical trend of loop regions being more dynamic than helical regions, although there was significant variability between similar structural elements. Furthermore, significant intra‐regional variability in the ranges of motion existed for all structural elements. Though more analyses will need to be done to understand the significance of these dynamic differences, overall the conclusions from the analysis support the validity of our molecular dynamic simulations approach.Support or Funding InformationSupport: NIH 1R15‐GM086767‐02 and the F. W. and Elsie L. Heyl, Roger F. and Harried G. Varney, and Hutchcroft Endowments of Kalamazoo College.
Amino Acid Changes in Allelic Variants of CYP2D6 Alter Kinetics and Susceptibility to Inactivation
Authors: Cho Christi W. , Glass Sarah M. , Osorio Victoria M. , Sunden Kyle F. , Fujiwara Rina , Furge Laura Lowe 
Published: November 2023

Cytochrome P450s (CYPs) are a superfamily of heme‐containing enzymes that are responsible for the metabolism of many xenobiotics and other small endogenous substrates. CYP2D6 is a major P450 enzyme studied during drug development due to its ability to metabolize ~20% of all pharmaceuticals. Moreover, there are over a 100 allelic variants of CYP2D6 resulting in a wide‐range of drug responses among individuals including cases of adverse drug events. The kinetics and susceptibility to inactivation of four allelic variants ‐ three with a series of distal mutations (*34, *17‐2, *17‐3) and one ultra‐metabolizer (*53), and one active‐site mutant (CYP2D6‐T309A), were investigated in the current study. The variants were expressed in E. coli and characterized with the prototypical substrates bufuralol and dextromethorphan. Decreased activity was observed for *34, *17‐2, and *17‐3 while the increased activity of *53 confirmed it as an ultra‐metabolizer. CYP2D6‐T309A displayed similar kinetic values as reference CYP2D6*1. A new abundant metabolite of dextromethorphan formed by *53 was identified using mass spectrometry. The inactivation of 2D6 variants by a known‐mechanism based inactivator, SCH66712, was investigated in a time‐ and concentration‐dependent manner for *1, *53, and T309A. Though *1 was inactivated by SCH66712, *53 and T309A were not susceptible to inactivation. These findings support the designation of T309A as the nucleophilic target for inactivation of CYP2D6 by SCH66712. The greatly decreased rate of inactivation of *53 suggests that inactivation may be dependent on time and positioning of inactivator in the active site. Computational methods using Molecular Dynamics and AMBER tools were used to gain insights into how individual amino acid changes cause alterations in structural stability and plasticity, and may lead to observed differences in reaction rates and susceptibilities to inactivation.Support or Funding InformationSupport: NIH 1R15‐GM086767‐02 and the F.W. and Elsie L. Heyl, Hutchroft, and Roger F. and Harriet G. Varney Endowments of Kalamazoo College.
Deciphering the Resistance Profile of Cancerous Thymidylate Synthase
Authors: Islam Zahidul , Gurevic Ilya , Saeed Muhammad , Rasool Sobia , Kohen Amnon 
Published: November 2023

Thymidylate Synthase (TSase) is an enzyme that converts 2′‐deoxyuridinemonophosphate (dUMP) to thymidylate (dTMP), a base for DNA. Since there is no other de novo route to dTMP in cells, TSase is critical to cell replication and survival. 5F‐dUMP – produced intracellularly from the chemotherapeutic pro‐drug 5‐fluorouracil (5‐FU) – inhibits TSase, thus halting the supply of the DNA base thymidylate, causing cessation of DNA synthesis and leading to thymine‐less cell death. While it is very common for pathogenic organisms to become resistant to certain antibiotic drugs, cancerous cells have also developed some mechanisms to cope with the cytotoxic effect of chemotherapeutic drugs. 1A variant of human (hs) TSase (Y33H) derived from a primary culture of human colonic tumor was reportedly found to confer resistance to 5‐FU.2 Crystal structures of hs TSase indicate Y33 is a remote residue that does not make direct contact either with the inhibitor or the residues interacting with it. Nevertheless, mutation at Y33 affects the affinity of the inhibitor for the enzyme. To resolve this enigma, we aim to investigate the molecular origin behind the resistance due to this remote mutation. Recombinant WT hs TSase and the mutant Y33H were expressed and purified in E. coli host systems. We structurally and kinetically interrogated WT and the mutant. Our studies revealed that the mutation at Y33 causes subtle changes across the protein that affect the chemical transformations at the active site. This change in protein could account for the low affinity of the mutant for the drug 5‐FdUMP.
Identification of Molecular Interactions within Histone Deacetylase Isozymes for the Development of Selective HDAC2 Inhibitors for the Design of Targeted Cancer Therapies
Authors: May Xavier , Watkins Davita , Stoddard Shana V. 
Published: November 2023

Histone deacetylase inhibitors (HDACi) are necessary for the therapeutic intervention of various cancers. HDACi decrease the function of a class of isozymes called histone deacetylases (HDAC). HDAC's are involved in the regulation of gene expression. Various members of this HDAC family are being targeted for therapeutic design. HDAC2 is seen to have elevated levels of expression with colorectal cancer. Our two part docking study compared our nine potential synthetic targets and known HDACi's Belinostat, Mocetinostat, Vorinostat, and Panobinostat within 11 HDAC isozymes to determine which candidates would be the best for synthesis. Additionally, we looked to determine if there are any molecular interactions that can be taken advantage of for the design of a more selective HDACi. ETS4 shows promising signs of in silico selectivity. HDAC2 and HDAC11 both experience binding score values that are 100 times higher than the other receptors. We observed pi‐pi stacking interaction with F155 and F 210 within the core of the receptor, as well as metal ion coordination and multiple hydrogen bonds throughout the receptor for HDAC2. Interestingly, HDAC11 does not have this pi‐pi stacking interaction occur, yet still has a high binding score. The work from this study will help to develop more selective HDAC2 inhibitors for targeted cancer therapies.Support or Funding InformationWilliam Randolph Hearst Foundation and Rhodes College Department of Chemistry
Development and Mechanism of Small‐Molecule SIRT6 Activators
Authors: Klein Mark , Liu Can , Camacho Beatriz , Tang Weiping , Denu John M. 
Published: November 2023

Sirtuin 6 (SIRT6) is a histone deacetylase that regulates numerous biological functions including transcriptional repression, DNA repair, and telomere maintenance. SIRT6 imparts its function primarily through deacetylation of histone 3 at lysine 9 and 56 (H3K9 and H3K56) however also functions as a non‐histone deacetylase with targets including GCN5 and CtIP. In recent years our understanding of the complexities of SIRT6 enzymatic and biological functions has expanded and its protective roles against carcinogenesis, non‐alcoholic fatty liver disease, and obesity are now commonly accepted. This mounting evidence implicates SIRT6 as an untapped, yet valuable, therapeutic target in combating numerous human diseases. In the work described here our lab has identified a number of SIRT6 activating compounds through targeted in vitro screening. Subsequent characterization of these molecules suggests that activation of SIRT6 deacetylation occurs through increased affinity for acetylated substrate. In collaboration with Professor Weiping Tang, we performed structure‐activity relationship studies to further develop an identified activator and demonstrated its capacity to activate SIRT6 deacetylation on extracted mammalian histones. Current and ongoing work of this project aims to provide a detailed mechanistic understanding of SIRT6 activation by small‐molecules, evaluate our compounds in cell‐based disease models, and identify endogenous SIRT6 activating compounds.Support or Funding InformationNational Institutes of Health (NIH)Chemistry Biology Interface Training Program (CBITP)University of Wisconsin Institute for Clinical and Translational Research (ICTR)
Rational Design of Epitope Binding Monobodies: New Tools for Autoimmune Therapy
Authors: Zaravar AliReza , May Xavier , Stoddard Shana 
Published: November 2023

Autoimmune Disease is the misguided targeting of self‐proteins or tissues by immune cells and is the number two cause of chronic long‐term disease. Many therapeutic options for autoimmune disease involve the use of immunosuppressive medicines that reduce the immune system's abnormal response. This approach is not specific to the individual autoimmune disorder and as a result these drugs weaken the entire immune system leaving the patient more prone to infection. Therefore, more targeted approaches to treatment of autoimmune disorders is needed. In our work we are developing an antigen specific approach to autoimmune therapy by hiding the epitope (specific auto‐antibody binding site) on the antigen to which an antibody is binding. Using the model system, idiopathic membranous nephropathy (IMN), we have developed epitope binding nanobodies which cover up the epitope region. Twenty‐five epitope binding nanobodies were designed and docked to the epitope region. Binding scores to the epitope regions of the IMN antigen phospholipase A2 receptor were evaluated. Our results show that several prominent interactions lead to improved binding scores compared to the starting nanobody template. Gel analysis of the protein expression of the ySMB, SH13‐M001, and SH13‐M003 epitope binding nanobody candidates, demonstrates successful protein expression and purification. Future studies underway are ELISA assays to confirm binding of epitope binding nanobodies and their ability to inhibit autoantibody binding in vitro.Support or Funding InformationWilliam Randolph Hearst Foundation and Rhodes College department of Chemistry
Prolonged Half‐life of Recombinant Immunotoxins in Mice by Adding an Albumin Binding Domain
Authors: Onda Mansanori , Pastan Ira 
Published: November 2023

BackgroundReconbinant immunotoxins (RITs) are potent anti‐cancer agents, that are now in clinical trials in tumors that express CD25, CD22 or Mesothelin. The entry of antibodies and antibody‐based therapeutics into tumors is affected by their half‐life in the circulation and the size of the protein. A longer half‐life should increase tumor penetration and efficacy.GoalBecause RITs have a relatively short half‐life in the circulation, we have explored the possibility of adding albumin‐binding domains (ABDs) to change their pharmacokinetics and increase their half‐life in the circulation.ApproachWe have inserted two different ABDs into a recombinant immunotoxin targeting CD25. These modifications can be done genetically, and require no additional step in the modified immunotoxins for the clinical use. We have measured their cytotoxic activities in vitro, half‐life in the circulation and anti‐tumor activity in mice.ResultsTwo different expression plasmids were constructed with 2 types of ABDs. These RITs were refolded from the inclusion bodies expressed in E. coli according to the standard procedure used for other immunotoxins in clinical use. M1(dsFv)‐PE38, targeting CD25, was used as the parental molecule. The ABDs were inserted near the C‐treminus of domain III, but before the REDLK sequence essential for toxin activity. The pahamacokinetics experiments in mice showed the prolonged retention of the modified immunotoxins as expected by 3‐fold of the half‐life. Also specific cytotoxic activities of the ABD‐RITs in vitro were 2‐fold less comparing to parental unmodified RIT. Moreover, these ABD‐RITs showed similar level of nonspecific acute in mice in spite of the prolonged retention time. Antitumor experiments indicated that no significant improvement was observed by the ABD addition.ConclusionIncreasing the half‐life of an immunotoxin by the insertion of an ABD does not increase its anti‐tumor activity. Insertion of an ABD into regions of the molecule that are not necessary for cytotoxicity may overcome this loss of activity.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Conductive Gel Polymers as an Extracellular Matrix Mimic and Cell Vehicle for Cardiac Tissue Engineering
Authors: Maldonado Marcos A , Bonham Andrew J 
Published: November 2023

Cardiomyopathies, diseases of the heart, are one of the major causes of death in the United States, and thus research into preventing and treating these complications is imperative. To do this, further improvements in culturing human tissues/tissue‐derived cells and development of new techniques in tissue engineering are needed, particularly in finding a suitable extracellular environment in which cardiac cells such as cardiomyocytes can be developed and maintained. As such, a great deal of effort has gone into producing polymer mimics of the natural cell environment in properties such as binding sites, stiffness, reactivity, and hydration. Here, we present work on the facile fabrication of high purity gold nanorods with carboxylate functionalization to aid in the development and characterization of conductive polymer scaffolds for cardiac tissue support. Such conductive polymers have been shown to have key roles in properly accommodating the needs of cardiac cells to build functional cardiac tissue constructs and further improve cell retention, spreading, homogenous distribution of cardiac specific markers, cell‐cell coupling and synchronized beating behavior at tissue level. We illustrate high purity gold nanorod synthesis and purification that yields high aspect ratio (> 10:1), stable gold nanorods. These were crosslinked into functional polyethylene glycol‐based scaffolds for cardiomyocyte differentiation from human induced pluripotent stem cells. This conductive extracellular matrix mimic shows great promise in improvement in control of cellular differentiation for cardiac tissue engineering.Support or Funding InformationSupport for this work was provided by the Metropolitan State University of Denver Provost's and LAS Dean's offices.
Translational Top Down Proteomics of KRAS‐driven Cancers and Models Thereof
Authors: Ntai Ioanna , Fornelli Luca , Hutton Josiah , LeDuc Richard , Fellers Ryan , Haverland Nicole , Compton Philip , Whiteley Gordon , Boja Emily , Rodriguez Henry , Kelleher Neil 
Published: November 2023

KRAS is a frequently mutated oncogene and activating missense mutations have been shown to have a causative role in tumor formation. More than a third of human colon cancers are driven by KRAS mutations. Here, we integrated genomic, transcriptomic and proteomic information to study isogenic cell lines and primary tumor samples with defined KRAS genotypes. We combined RNA‐seq data with top‐down and bottom‐up proteomic analyses to measure differential expression within the proteome, and KRAS proteoforms specifically. Integration of these three complementary omics datasets provided us with a list of molecular entities showing differential expression in response to the mutational status of KRAS. Overall, the results show a strong enrichment for kinases, consistent with the established effect of KRAS mutations in constitutive activation of the MAPK signaling pathway. In addition, proteins associated with cytoskeleton reorganization seemed to be upregulated in the presence of the KRAS G13D mutant allele. This could be involved in increasing cell motility and lead to a more invasive phenotype. Furthermore, we developed a targeted top‐down assay for the detection and quantitation of endogenous KRAS proteoforms to measure allele‐specific modification patterns. Such quantitative measurements (both targeted and untargeted) provide new insights into colorectal cancer phenotypes through the measurement of cancer‐specific protein variants.Support or Funding InformationThis work was supported by Federal Funds from the National Cancer Institute (Office of Cancer Clinical Proteomics Research), National Institutes of Health, under Contract No. HHSN261200800001E, and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health.
Analysis of proteins and protein interactions by size exclusion chromatography – high resolution mass spectrometry
Authors: Busch Florian M , Sahasrabuddhe Aniruddha , vanAernum Zac , Rivera Brian , Wysocki Vicki H 
Published: November 2023

Size exclusion chromatography (SEC) is the most commonly used technique for the determination of protein heterogeneity derived from the presence of multiple biomolecules and their interaction via non‐covalent interactions. On‐line detection of eluting species by native mass spectrometry (MS) provides a clear advantage over the traditionally used UV‐spectroscopic detection as it allows for the direct and unambiguous molecular weight measurement of the eluting species. Here, we demonstrate that the combination of sub 2μm particle SEC separation with high‐resolution mass detection is suitable for the analysis of a wide range of proteins. The possibility to detect as little as 100 ng protein makes it possible to analyze complex mixtures at low samples consumption. Non‐ideal interactions of proteins with the stationary phase as indicated by a non‐linear relationship between the logarithm of the molecular weight and the elution time within the column separation range make it challenging to find standard calibration proteins. We show that a mixture of commercially available proteins can be used to assess and monitor the column performance and reproducibility under MS‐compatible mobile phase conditions. The ability to resolve slight molecular weight changes by high resolution MS makes it possible to detect a variety of biomolecular interactions, which can be further studied by surface induced dissociation (SID) and collision induced dissociation (CID). The suitability of this technique to monitor a protein‐ligand interaction in a protein mixture is demonstrated in the case of the biotin‐streptavidin interaction.Support or Funding InformationWe thank the National Institutes of Health (NIH) for funding [grant R01 GM113658].
Cytotoxicity of Urban Dust and Diesel Exhaust Particulates in Murine Astrocyte Cells
Authors: Fortuna Tyler R , Price Peyton C , Lopez Kevin J , Schwader Andrew J , Mazzer Paula 
Published: November 2023

Alzheimer's and related neurodegenerative diseases affect as many as 24 million individuals world‐wide—a number which will increase with our increasing global population. In a recent survey of epidemiological evidence, a panel of experts concluded that the existing data suggest a lower incidence of age‐related dementia in developing regions. A potential link between urban environments and neurodegenerative diseases was found by a series of studies conducted in dogs and humans in Mexico City, Mexico, which found markers of amyloid‐β accumulation and oxidative stress in their brains. Dogs and humans from rural areas did not show these neuropathological findings. These findings suggest that the difference may be due to the heavy airborne particular matter in Mexico City.We studied the ability of urban dust (NIST SRM 1649a) and diesel exhaust particulate matter (NIST SRM 2975) to induce cytotoxicity and oxidative stress in murine astrocyte cells (ATCC C8‐D1A). Diesel exhaust particulate matter (DEP) and urban dust (UD) have very different organic components, with UD containing a higher concentration of oxidized aromatic compounds. Therefore we hypothesized that these two airborne particulate types would produce different toxicological effects in our astrocyte cells.LC50 determinations showed DEP to be more toxic with an LC50 of 35μg/mL versus the LC50 of UD at 52μg/mL. A caspase 3 activity assay showed that the enzyme was activated by DEP but not by UD. These data suggest that DEP is inducing apoptotic cell death while UD is inducing necrotic cell death. The difference in cell death suggests that UD could potentially be more harmful in vivo due to the release of intracellular components caused by the rupture of the cell membrane. The necrotic cell death could lead to the oxidative stress found in previous studies of brains form polluted environments.Support or Funding InformationThis work was sponsored by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443.