bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Inhibitor Targeting ALDOA at Cysteine 289 and the Regulation of its C‐terminal Tail
Authors: Stancu Gabriel , Devkota Ashwini , Aleshin Alexander , De Jong Petrus , Cho Eun Jeong , Liddington Robert , Powis Garth , Dalby Kevin 
Published: November 2023

Human fructose‐bisphosphate aldolases A (ALDOA) is a key glycolytic enzyme that catalyzes the conversion of fructose‐1,6‐bisphosphate to glyceraldehyde‐3‐phosphate and dihydroxyacetone phosphate. ALDOA has also been shown to play major moonlighting roles associated with cellular structure, mobility and proliferation. This aldolase isoenzyme is significantly overexpressed in a number of human tumors and is a potential oncogene, where it contributes to the Warburg effect in glycolysis. We recently identified a feed‐forward loop whereby ALDOA positively regulates the activity of hypoxia‐inducible transcription factor‐1 (HIF‐1) through AMPK, which in turn further activates ALDOA. This signaling pathway is a potential target in the irregular hypoxic environment of solid tumors. Through our University of Texas Targeted Therapeutic Drug Discovery & Development Program (TTP) we have identified a number of compounds that are able to target and inhibit aldolase enzymatic activity. Using mutational studies and high‐resolution X‐ray crystallography, we characterized a covalent inhibitor of ALDOA (TX1) which specifically targets the enzyme at Cysteine 289. Time dependent inhibition through TX1 at Cys289 leads to an approximate two‐fold decrease in the Km and kcat of ALDOA. When we treated MDA‐MB‐231 metastatic breast cancer cells with TX1 for 30 min, we observed an IC50 of 90 μM in cell lysates assayed for aldolase activity. Inhibition of ALDOA by TX1 appears to involve the complex regulation of the C‐terminal tail of the enzyme. We show that inhibition by TX1 is lessened when Cys289 is mutated to alanine and completely abrogated by removal of the C‐terminal tail. X‐ray crystallography reveals that targeting of Cys289 by TX1 significantly alters the conformation of the ALDOA C‐terminal tail with respect to the active site. This C‐term domain is important in controlling substrate specificity and enzymatic activity, and could play a major role in the moonlighting function of ALDOA. Moreover, the local environment of Cys289 makes it a prospective site for post‐translational modifications (PTMs) and an attractive target for mechanism based inhibition of ALDOA. We are working on understanding the redox and reactive oxygen species regulation of both Cys289 and the C‐terminal tail, which could shed light on the moonlighting potential of ALDOA within tumor signaling. In conclusion, we have shown that modulation of the C‐terminal domain of ALDOA through Cys289 represents a novel method of regulating its activity. Our work represents an important step in further defining ALDOA as a central signaling target in human cancer.Support or Funding InformationThis research was supported by the grant from CPRIT (RP110532‐P1).
Modulation of pTyr signaling by small molecule inhibitors of SH2‐lipid interaction
Authors: Hu Yusi 
Published: November 2023

The Src homology 2 (SH2) domain plays a pivotal role in phosphotyrosine (pTyr) signaling pathway. Our recent study showed that a majority of SH2 domains tightly bind lipids in the plasma membrane (PM) using a cationic site separate from the pTyr pocket, which is essential for their cellular protein‐protein interactions. To test the feasibility of modulating pTyr signaling activity by controlling SH2 domain‐lipid binding, we screened and optimized small molecule inhibitors for SH2 domain‐lipid binding. Small molecule inhibitors of the SH2 domain of spleen tyrosine kinase (Syk), which plays a key role in B cell signaling and has high affinity for PM, caused potent and specific inhibition of Syk signaling activities in DT40 B cells while exhibiting minimal activity against ZAP70 in Jurkat T cells.Support or Funding InformationNIH GM68849
Expression, Purification, And Characterization of W. Bancrofti DHFR and its Evaluation as a Possible Drug Target for Treating Lymphatic Filariasis
Authors: Tobias Andrew M , Gubler Ulrich A , Goodey Nina 
Published: November 2023

Lymphatic filariasis is a disease caused by filarial nematodes that affects millions of individuals every year. A specific filarial nematode, W. bancrofti, is one organism that causes Lymphatic filariasis. This disease affects the lymphatic system and can cause severe swelling in the lower appendages. According to the World Health Organization, people living in Bangladesh, India, Indonesia, Myanmar, Nigeria, Nepal, the Philippines and others are at risk of infection by this neglected tropical disease. To help cure those infected, research is needed to find a drug able to inhibit the proliferation of W. bancrofti. The protein we are evaluating in order to possibly accomplish this task is WbDHFR. DHFR is an enzyme that reduces dihydrofolate into tetrahydrofolate (THF). THF and its derivatives are required for purine synthesis, making the enzyme crucial for cell proliferation. In this project, WbDHFR was expressed in LOBSTR strain E. coli and purified using a Hexa‐His tag and Ni‐NTA resin. The purified enzyme is active with a Vmax of 159 ± 9 nM/s and Km of 36 ± 4. The enzyme will be tested against several antifolate compounds as inhibitors.Support or Funding InformationScience Honors Innovation Program at Montclair State University, and the American Society for Biochemistry and Molecular Biology
Understanding the link between antimicrobial properties of safranal and microbial ATP synthase
Authors: Liu Mason , Ahmad Zulfiqar 
Published: November 2023

BackgroundA variety of phytochemicals with antimicrobial and antitumor properties have been identified to bind and inhibit ATP synthase. Saffron constituent safranal and its analogs are known to have antitumor, cytotoxic, and antimicrobial properties. Our lab has studied safranal and its structural analogs, thymol, cuminol, carvacrol, damascenone, 2,6,6‐trimethyl‐2‐cyclohexene‐1,4‐dione, and 4‐isoproply benzyl bromide induced‐inhibition of Escherichia coli ATP synthase and their effect on cell growth to investigate any link between antimicrobial properties of these compounds and ATP synthase inhibition.MethodsWild type and mutant E. coli strains were grown to late log phase. Cells were harvested by centrifugation and lysed by French Press to isolate the membrane bound F1FO ATP synthase. Null E. coli strain pUC118 with deleted ATPase gene was used as a negative control. Safranal and its analogs induced inhibitory studies were performed on membrane bound F1FO ATP synthase. Growth of wild‐type, mutant, and null strain in limiting glucose and succinate was also examined in presence and absence of safranal and its analogs.ResultsSafranal and its analogs inhibited wild‐type enzyme to variable degrees. While safranal caused 100% inhibition of wild‐type F1FO ATP synthase, only ~50% inhibition occurred for the mutant ATP synthase. Inhibitory profiles of mutant E. coli strains indicated the involvement of specific amino acids in the binding of safranal and its analogs to ATP synthase. Furthermore, safranal, thymol, cuminol, carvacrol, and damascenone fully abrogated the growth of wild‐type E. coli cells and had little or no effect on the growth of null and mutant E. coli strains.ConclusionsSafranal and its analogs appear to bind to the polyphenol binding pocket on F1 sector of ATP synthase. The antimicrobial properties of safranal and its analogs can be linked to their binding and inhibition of ATP synthase. Thus, our results support the idea that ATP synthase can be used as a molecular drug target to combat microbial infections.Support or Funding InformationThis work was supported by the National Institutes of Health Grant GM085771 to ZA and KCOM Biomedical Sciences Graduate Program Grant Award # 850‐619 to ML and ZA.
Inhibition of Myeloperoxidase by Staphylococcal SPIN Proteins
Authors: Ploscariu Nicoleta T. , de Jong Nienke W.M. , Ramyar Kasra X. , Garcia Brandon L. , Herrera Alvaro I. , Prakash Om , Haas Pieter‐Jan , van Strijp Jos A.G. , Geisbrecht Brian V. 
Published: November 2023

Neutrophils are the most abundant type of white blood cells in humans and the most prominent cellular component of the innate immune system. They can be rapidly activated by various biochemical stimuli during the earliest phase of bacterial invasion and recruited into the sites of infection. Neutrophils contain subcellular granules that are replete with vital anti‐bacterial enzymes, including a series of chymotrypsin‐like Neutrophil Serine Proteases (NSPs) as well as a heme‐containing Myeloperoxidase (MPO). Work from the last decade has demonstrated that the pathogenic bacterium Staphylococcus aureus produces an array of virulence proteins that disrupt normal function of the human innate immune system. The combined activities of these proteins are believed to prolong bacterial survival within the host, allowing for infections to take hold. Given the critical role of neutrophils in responding to the initial stages of staphylococcal infection, we predicted that S. aureus might also express proteins that inhibit function of neutrophil granule components. Indeed, we recently identified two novel classes of S. aureus secreted proteins, EAP domain proteins and SPIN, which act as inhibitors of NSPs and MPO, respectively.SPIN (Staphylococcal Peroxidase INhibitor) is a previously uncharacterized protein found only in Staphylococci. SPIN potently inhibits MPO activity in a number of functional assays by forming a nanomolar affinity complex with MPO. To gain insight into the structure/function relationships of SPIN, we determined its structure bound to MPO by X‐ray crystallography. The structure of this complex implicates the N‐terminal ~10 amino acids as essential for SPIN function. To identify and further characterize key residues for SPIN function in more detail, we examined three types of SPIN proteins using structural methods, direct binding assays, and functional assays for MPO activity: (i) deletion mutants, (ii) SPIN proteins originating from divergent staphylococcal species, and (iii) a synthetically‐designed SPIN protein. The differences in binding and inhibition of MPO by these proteins not only provide a route for identifying the structure/function relationships in SPIN, they also constitute a template for the design of high‐affinity, synthetic MPO inhibitors based upon the properties of this unique immune evasion protein.Support or Funding InformationAI11120302
Biophysical Characterization and Catalytic Reactivity of Rubrerythrin and Symerythrin Model Proteins
Authors: Pellegrino Jenna , Bell Katherine A. , Polinski Rachel Z. , Cimerol Sabrina N. , Jacobs Ari , Solomon Edward I. , Reig Amanda 
Published: November 2023

Rubrerythrins (Rbr) and symerythrins (Sym) are diiron carboxylate enzymes that function as both ferroxidases and peroxidases in two‐electron reduction reactions. This peroxidase activity, uniquely preferential in Rbr and Sym as compared to other members of the ferritin‐like superfamily (FLSF), is thought to result from the one or two additional carboxylate active site amino acids, compared to the FLSF archetype. The relationship between these structural features and the altered function is currently not well understood. To investigate these phenomena, a de novo‐designed protein model system was computationally designed to demonstrate the canonical 4‐helix bundle and 2His/4‐carboxylate active site structure of FLSF enzymes and to mimic their reactivities. Modifications to this model resulted in G4DFsc, which exhibits higher solubility and stability as well as increased active site accessibility. Using the G4DFsc system, Rbr‐ and Sym‐like proteins were achieved by introducing either aspartate (D) or glutamate (E) residues at active site positions 14, 43, and/or 47. The structural and catalytic properties of these systems were investigated using metal‐binding, protein‐folding, and reactivity assays at pH 7 and pH 7.5. Data show that the double mutants exhibit the weakest metal‐binding capacity at pH 7. These proteins also show slower rates of 4‐aminophenol oxidation than the original G4DFsc protein. The G47D variant shows the greatest catalytic capacity, with 4‐aminophenol oxidation rates increased 11‐fold over control reactions and 2‐fold compared to G4DFsc. These results provide insight into how particular carboxylate residues in the G4DFsc active site affect its ability to react with dioxygen and hydrogen peroxide. Preliminary cobalt titration curves show the tightest binding between the metal and G4DFsc. Interestingly, the dissociation constant of G47D is 4 times greater.Support or Funding InformationHoward Hughes Medical Institute (HHMI), National Institutes of Health (NIH) R15GM110657
Development of Phospholipase A2 Receptor Antigen Binding Monobodies for the Inhibition of the Idiopathic Membranous Glomerulonephritis Auto‐antibody Binding
Authors: Stoddard Shana V , Zaravar AliReza , May Xavier , Goldman Liam 
Published: November 2023

The use of non‐specific treatment options in autoimmune therapy today unfortunately causes patients to be prone to other infections. This non‐specific therapy suppresses the entire patient's immunes system also making it harder to fight of basic infections. Thus therapies need to be developed which can accurately target only the aberrant portion of the immune system that contributes to the autoimmune disorder (AD) specific to the individual. The work here focuses on design of antigen specific binding monobodies as a targeted means to treat AD, and specifically idiopathic membranous glomerulonephritis. Sixty monobodies were designed and optimized using in silico mutagenesis. Protein‐protein docking simulations were performed using Rosetta to evaluate for maximum binding efficiency against the ricin and the CTLD2 epitopes present on the PLA2R antigen. Our results show improved binding scores and selectivity of monobodies against the phospholipase A2 receptor (PLA2R) antigens. Initial binding energy, measured in rosetta energy units (REU), were −2.863 and −2.633 for the SH13 template and ySMB‐1 templates respectively. The M005 mutation designed in the SH13 improved binding scores to −5.32 REU. The M006 ySMB‐1 mutant showed the most in silico selectivity between the two epitope regions on the PLA2R antigen having a score of −2.966 REU against the CTLD2 epitope and a better score of −3.736 against the ricin epitope. The SH13 M005 mutant showed the greatest binding efficiency having a score of −5.319 REU. However, this mutant also binds the CTLD2 epitope (binding energy score produced was −5.32 REU), suggesting that it will not have selectivity. Several molecular interactions were identified that facilitated increased binding of the monobodies to the epitope regions compared to the original template monobodies. Molecular interactions leading to selectivity are also being evaluated. Seven monobody candidates were selected for expression which was performed in E. coli followed by subsequent purification using affinity chromatography for experimental verification of computational results is using ELISA. This work has implications for new antigen specific approach to AD therapy.Support or Funding InformationWilliam Randolph Hearst Foundation and Rhodes College Department of Chemistry
Modification of Bovine Serum Albumin (BSA) with a Maleimido‐functionalized, 8‐arm Polyethylene Glycol Backbone (Mal‐PEG8) as a Drug Delivery System
Authors: Hill Jonathan , Huskic Dzenita , Parker Jacob , Carpino Ross , Olsen Kenneth W. 
Published: November 2023

Due to its ability to transport hydrophobic materials through the bloodstream, human serum albumin (HSA) has become an intriguing drug delivery system with several formulations currently available. Key residues in the two main drug binding sites, DS1 and DS2, are conserved in BSA, providing a cheaper, more readily available alternative to HSA. Additionally, BSA may be selectively modified at its lone free sulfhydryl (Cys34), requiring no additional modifications. A multi‐BSA PEG8 complex (PEG8‐BSAx) would reduce the effect on osmotic pressure, avoid immunogenic response, and decrease the risk of drug toxicity while maintaining ligand binding capabilities. Several BSA molecules were successfully conjugated onto Mal‐PEG8 in a one‐pot reaction, requiring only BSA, Mal‐PEG8 and MOPS/NaCl buffer at pH 7.2. High molecular weight species (HMW) of PEG8‐BSAx were separated from the reaction mixture via Size Exclusion Chromatography (SEC). Helicity was maintained following PEGylation, as determined by Circular Dichroism (CD). Using a 4–15% gradient SDS‐PAGE gel, three bands of HMW material were visualized. Two bands corresponding to molecular weights of PEG8 complexed with two and three BSA molecules were seen in the separation zone. A heavy band was maintained in the stacking zone, indicating the presence of at least one other species of PEG8‐BSAx. Thermal stability of PEGylated‐BSA complexes was evaluated using UV‐VIS and CD. Fluorimetric assays were performed with Sulfamethoxazole (SMZ), Phenytoin (PHT) and 8‐Anilino‐1‐naphthalenesulfonic acid (ANS). SMZ has shown preference for DS1 while PHT prefers DS2. ANS is a fluorescent probe commonly used in binding studies. Dissociation constants (Kd) and the number of binding sites were consistent between unmodified BSA and PEG8‐BSAx. These results suggest that PEG8‐BSAx is a viable delivery system for water‐insoluble pharmaceuticals.Support or Funding InformationLoyola University Chicago
Using Multivalency to Improve the Function of Synthetic Epigenetic Proteins
Authors: Tekel Stefan Josef 
Published: November 2023

Epigenetic protein engineering is a valuable new tool for studying cell development and cancer, with the potential for novel medical applications. One major advantage to using synthetic epigenetic proteins with transcriptional effector domains over DNA binding domains is the scale of downstream effects. Typically, CRISPR, TALEs, and zinc finger based fusions influence one target gene, while epigenetic‐based proteins are capable of binding across the genome. However, many natural epigenetic binding proteins have poor binding affinity and multiple ligands, making their use in synthetic systems challenging. In one such case with a fusion of the natural methyl‐histone binding protein CBX8 and the activation domain VP64, reactivation of an artificially silenced gene was modest compared to reactivation from a DNA‐binding domain fused to the same activator. In an attempt to increase effector robustness, we built a library of multivalent constructs inspired by multivalent histone‐binding proteins found in nature (e.g. BPTF). Here, we present an in vitro pipeline for screening synthetic multivalent histone binding proteins for target binding, which is followed by an in cell assay to verify gene reactivation potential. First, the library of fusion proteins, each fused to a C‐terminal mCherry RFP tag, were expressed in an in vitro transcription and translation (IVTT) system. RFP signal indicated protein production efficiency. An ELISA was run to screen the proteins for target ligand (H3K27me3) and off‐target binding and narrow the pool for in‐cell characterization. The candidates with the best relative target binding were then expressed in‐frame with a C‐terminal VP64 activation domain in cells that carried an H3K27me3‐enriched luciferase reporter system and levels of gene reactivation were compared. The novelty of our pipeline lies in the low cost IVTT system coupled with a quantifiable reporter allowing for monitoring of in vitro translation efficiency, as well as in vivo transfection efficiency and nuclear localization of the fusions. Cellular luciferase assays indicated a significant level of gene reactivation with a multivalent fusion that contained a flexible linker (4x glycine‐serine‐serine‐serine) compared to a single domain fusion or a double domain with a rigid, helical linker (4x glutamic acid‐alanine‐alanine‐alanine‐arginine). These results demonstrate the ability to optimize synthetic multivalent proteins to increase effector output, as well as highlights the importance of linkers between functional domains.Support or Funding InformationS. Tekel is supported by ASU SBHSE
Discovery and Development of Allosteric Pantothenate Kinase Activators
Authors: Sharma Lalit Kumar , Subramanian Chitra , Yun Mi‐Kyung , White Stephen , Jackowski Suzanne , Lee Richard E. , Rock Charles O. 
Published: November 2023

The central position of coenzyme A (CoA) in intermediary metabolism and its role in human neurodegenerative disease, led us to develop chemical probes targeting pantothenate kinase (PANK), the regulatory enzyme that controls CoA biosynthesis. There are four mammalian PANK isoforms that exhibit tissue‐specific expression and differ in their sensitivity to feedback inhibition by CoA and its thioesters. A high‐throughput screen and further medicinal chemistry efforts identified a novel class of orally bioavailable PanK modulators called pantazines. Optimization of the series based on lipophilic ligand efficiency (LipE) culminated in PZ‐2891, a drug‐like molecule that binds to PANK3 with nM affinity. Enzyme kinetics, biophysical studies, and X‐ray crystallography demonstrate that PZ‐2891 binds to the pantothenate substrate site of the PANK3•ATP•Mg2+ complex and extends across the dimer interface to form an induced fit, high‐affinity quaternary complex. PANK3 is a highly cooperative dimer and both protomer active sites simultaneously switch between the closed, active conformation that binds ATP, and the open, inactive conformation that binds acetyl‐CoA. PZ‐2891 inactivates the protomer to which it is bound and locks the opposite protomer in an active conformation that is refractory to feedback regulation by acetyl‐CoA. This allosteric property means that PZ‐2891 is a pantothenate‐dependent activator of CoA biosynthesis in cultured cells, and substantially increases intracellular CoA levels as a function of the media pantothenate supplement. Administration of PZ‐2891 to animals by oral gavage recapitulated the pantothenate‐dependent modulation of CoA levels in liver. PZ‐2891 dose‐response experiments also demonstrated the elevation of brain CoA illustrating the ability of PZ‐2891 to cross the blood brain barrier. PZ‐2891 and its inactive analogs are ready to be deployed as a toolkit to manipulate CoA levels in cultured cells and animals to analyze the role of CoA concentration in the control of intermediary metabolism. These nanomolar PANK allosteric effectors are being further developed as preclinical leads to validate PANK as a target to treat neurologic and metabolic disorders.Support or Funding InformationNIH, ALSAC/St. Jude