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At least 541 records · Page 30
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

The JmjC-domain protein NO66/RIOX-1 affects the balance between proliferation and maturation in acute myeloid leukemia

Highlights: • NO66/RIOX1 is not expressed in the AML cell line KG1a regarded as a “stem cell-like” model system. • NO66/RIOX1 is not expressed in primary HSC-enriched CD34{sup +}/CD38{sup -} cells of healthy donors. • Ectopic expression of NO66/RIOX1 in KG1a cells inhibits proliferation and rDNA transcription. • NO66/RIOX1 impacts on the transition from stem cells to progenitors by regulation of gene expression. As epigenetic regulators are frequently dysregulated in acute myeloid leukemia (AML) we determined expression levels of the JmjC-protein NO66 in AML cell lines and sub fractions of healthy human hematopoietic cells. NO66 is absent in the AML cell lines KG1/KG1a which consist of cells with the immature CD34{sup +}/CD38{sup -} phenotype and is regarded as a “stem cell-like” model system. Similarly, NO66 is not detectable in CD34{sup +}/CD38{sup -} cells purified from healthy donors but is clearly expressed in the more committed CD34{sup +}/CD38{sup +} cell population. Loss of NO66 expression in KG1/KG1a cells is due to hyper-methylation of its promoter and is released by DNA-methyltransferase inhibitors. In KG1a cells stably expressing exogenous wild type (KG1a66wt) or enzymatically inactive mutant (KG1a66mut) NO66, respectively, the wild type protein inhibited proliferation and rDNA transcription. Gene expression profiling revealed that the expression of NO66 induces a transcriptional program enriched for genes with roles in proliferation and maturation (e.g.EPDR1, FCER1A, CD247, MYCN, SNORD13). Genes important for the maintenance of stem cell properties are downregulated (e.g. SIRPA, Lin28B, JAML). Our results indicate that NO66 induces lineage commitment towards myeloid progenitor cell fate and suggest that NO66 contributes to loss of stem cell properties.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial β-1 spirodienone linkage cleavage has not been elucidated. In this study, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a β-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Chemical-damage MINE: A database of curated and predicted spontaneous metabolic reactions

Spontaneous reactions between metabolites are often neglected in favor of emphasizing enzyme-catalyzed chemistry because spontaneous reaction rates are assumed to be insignificant under physiological conditions. However, synthetic biology and engineering efforts can raise natural metabolites' levels or introduce unnatural ones, so that previously innocuous or nonexistent spontaneous reactions become an issue. Problems arise when spontaneous reaction rates exceed the capacity of a platform organism to dispose of toxic or chemically active reaction products. While various reliable sources list competing or toxic enzymatic pathways' side-reactions, no corresponding compilation of spontaneous side-reactions exists, nor is it possible to predict their occurrence. We addressed this deficiency by creating the Chemical Damage (CD)-MINE resource. First, we used literature data to construct a comprehensive database of metabolite reactions that occur spontaneously in physiological conditions. We then leveraged this data to construct 148 reaction rules describing the known spontaneous chemistry in a substrate-generic way. We applied these rules to all compounds in the ModelSEED database, predicting 180,891 spontaneous reactions. The resulting (CD)-MINE is available at https://minedatabase.mcs.anl. gov/cdmine/#/home and through developer tools. We also demonstrate how damage-prone intermediates and end products are widely distributed among metabolic pathways, and how predicting spontaneous chemical damage helps rationalize toxicity and carbon loss using examples from published pathways to commercial products. We explain how analyzing damage-prone areas in metabolism helps design effective engineering strategies. Finally, we use the CD-MINE toolset to predict the formation of the novel damage product N-carbamoyl proline, and present mass spectrometric evidence for its presence in Escherichia coli.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13 C-metabolic flux analysis ( 13 C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13 C labeling data were used with 13 C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C–C bond formation. Here, in this study, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions – the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard β-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evolution and engineering of pathways for aromatic O -demethylation in Pseudomonas putida KT2440

In this study, biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ~1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.

09 BIOMASS FUELS↗
DOE OSTI2020

Mechanism of Catalysis by L -Asparaginase

Two bacterial type II L -asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II L -asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. In this work, we provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) L -asparaginases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials has potential applications in sensing, catalysis and medicine. Whereas numerous designs have been implemented for the bottom-up construction of novel protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. Here, the site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small-molecule- and protein-functionalized surfaces with no loss in crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces towards the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Reversal of Catalytic Material Substrate Selectivity through Partitioning of Polymers in Hierarchically Ordered Virus-like Particle Frameworks

Control over the selectivity of catalytic materials is a topic of growing interest. Virus-like particle (VLP) based materials such as protein macromolecular frameworks (PMFs) are promising for catalytic applications due to their ease of assembly, modular ability to encapsulate a variety of enzymes, and ease of separation from a reaction mixture. Here we demonstrate the reversal of the initially negative material charge through the titration of a positively charged polymer into the material, causing the reversal of guest molecule uptake and enzymatic activity of PMFs. The charge-inverse material partitions a charged enzyme substrate to concentrate the substrate near an enzyme incorporated within the material, generating up to 5.9-fold increases in enzyme activity toward the partitioned substrate over the excluded substrate. Here we also show that the polymer distributes heterogeneously through the material up to a point of saturation and the effects of guest macromolecules on the lattice parameters of PMFs.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Naphthalene-DNA Adduct Formation in a Lung Airway Explant Model: The Role of Bioactivation and Naphthalene Metabolites

Humans are widely exposed to naphthalene. Once inhaled or ingested, naphthalene is metabolized by cytochrome P450 and other enzymes to form toxic metabolites known to harm lung epithelial cells. Naphthalene metabolites circulate in the blood. Chronic naphthalene inhalation promotes lesions in the epithelium of the mouse lung and rat nose. Oral naphthalene exposure leads to DNA adduct formation in mouse lung, but the contributions of different enzymatic pathways and the metabolites they generate are not fully understood. This study explores the influence of naphthalene metabolites on DNA adduct formation in the lungs of two species (mice and primates). To isolate the lung response, conducting airway explants containing Club cells, a target for pulmonary naphthalene toxicity, were microdissected from live lung tissue and incubated with 14 C-naphthalene or its metabolites: 14 C-1,2-naphthoquinone or 14 C-naphthalene-1,2-dihydrodiol. Explants were incubated for 1 h, then processed immediately (T1), or were transferred to clean media for the remainder of the 24 h (T24), to monitor 14 C in DNA over time. Accelerator mass spectrometry analysis revealed the formation of DNA adducts by all three radiolabeled compounds by T24. Our results support the notion that P450 enzymes of the Cyp2abfgs subfamily contribute to naphthalene-induced DNA adduct formation (approximately 4-fold reduction in male mice lacking the Cyp2abfgs genes, P < 0.01). The finding that naphthalene-1,2-dihydrodiol, a stable metabolite, formed DNA adducts (102–117 adducts/10 8 nucleotides) at 24 h following addition to the culture media validates the concern that circulating naphthalene metabolites can contribute to DNA adduct formation in the lung. DNA adducts persisted to 24 h after exposure in both mouse and primate airways and at comparable levels between species (77.8 vs 129 adducts/10 8 nucleotides, respectively). Together, these results support the importance of a potential genotoxic mechanism of naphthalene and its metabolites in vivo in both mice and nonhuman primates, and possibly also in humans.

Biological and medical sciences↗
DOE OSTI2024

Impacts of Hydrogen Bond Donor Structures in Phenolic Aldehyde Deep Eutectic Solvents on Pretreatment Efficiency

As a green solvent for biomass processing, deep eutectic solvents (DESs) have shown effectiveness in biomass processing. Here, in this study, phenolic aldehydes with different numbers of methoxy groups, including 4-hydroxybenzaldehyde (HBA, no methoxy), vanillin (VA, monomethoxy), and syringaldehyde (SA, dimethoxy) were employed to synthesize DESs with choline chloride (ChCl). The presence of methoxy groups in the hydrogen bond donor structure affected DES properties, as well as biomass pretreatment performance. The high thermal stability of phenolic aldehyde DESs was shown with over 225 °C onset temperature. The hydrogen bond donor with one aldehyde and one hydroxyl group at the para position without a methoxy group (ChCl-HBA) showed the highest xylan removal and delignification, reaching 59.3 and 88.0%, respectively, leading to the highest enzymatic hydrolysis yield. Sonication after pretreatment further enhanced the hydrolysis yields, achieving 83.3% glucan conversion and 50.1% xylan conversion. In the lignin-rich fraction, the recovered lignin showed a low weight–average molecular weight under 2100 g/mol with a relatively uniform molecular weight dispersity below 1.5. This study provides insights into how the chemical structure of hydrogen bond donors in DESs affects biomass processing and paves the way for designing effective lignin-derived DES in future biorefinery processes.

09 BIOMASS FUELS↗
DOE OSTI2022

Effect of Redox-Inactive Metal Ion–Nickel(III) Interactions on the Redox Properties and Proton-Coupled Electron Transfer Reactivity

We report Mononuclear nickel(II) and nickel(III) complexes of a bisamidate-bisalkoxide ligand, (NMe 4 ) 2 [Ni- II (HMPAB)] (1) and (NMe 4 )[Ni III (HMPAB)] (2), respectively, have been synthesized and characterized by various spectroscopic techniques including X-ray crystallography. The reaction of redox-inactive metal ions (M n+ = Ca 2+ , Mg 2+ , Zn 2+ , Y 3+ , and Sc 3+ ) with 2 resulted in 2-M n+ adducts, which was assessed by an array of spectroscopic techniques including X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR), and reactivity studies. The X-ray structure of Ca 2+ coordinated to Ni(III) complexes, 2Ca 2+ T, was determined and exhibited an average Ni-Ca distance of 3.1253 angstrom, close to the metal ions' covalent radius. XAS analysis of 2-Ca 2+ and 2-Y 3+ in solution further revealed an additional coordination to Ca and Y in the 2-M n+ adducts with shortened Ni-M distances of 2.15 and 2.11 Å, respectively, implying direct bonding interactions between Ni and Lewis acids (LAs). Such a short interatomic distance between Ni(III) and M is unprecedented and was not observed before. EPR analysis of 2 and 2-M n+ species, moreover, displayed rhombic signals with g av > 2.12 for all complexes, supporting the +III oxidation state of Ni. The Ni III /Ni II redox potential of 2 and 2-M n+ species was determined, and a plot of E 1/ 2 of 2-M n+ versus pK a of [M(H 2 O) n ] m+ exhibited a linear relationship, implying that the Ni III /Ni II potential of 2 can be tuned with different redox-inactive metal ions. Reactivity studies of 2 and 2-M n+ with different 4-X-2,6-ditert-butylphenol (4-XDTBP) and other phenol derivatives were performed, and based on kinetic studies, we propose the involvement of a proton-coupled electron transfer (PCET) pathway. Analysis of the reaction products after the reaction of 2 with 4-OMe-DTBP showed the formation of a Ni(II) complex (1a) where one of the alkoxide arms of the ligand is protonated. A pK a value of 24.2 was estimated for 1a. The reaction of 2-M n+ species was examined with 4-OMe-DTBP, and it was observed that the k 2 values of 2-M n+ species increase by increasing the Lewis acidity of redox-inactive metal ions. However, the obtained k 2 values for 2-M n+ species are much lower compared to the k 2 value for 2. Such a variation of PCET reactivity between 2 and 2-M n+ species may be attributed to the interactions between Ni(III) and LAs. Our findings show the significance of the secondary coordination sphere effect on the PCET reactivity of Ni(III) complexes and furnish important insights into the reaction mechanism involving high-valent nickel species, which are frequently invoked as key intermediates in Ni-mediated enzymatic reactions, solar-fuel catalysis, and biomimetic/synthetic transformation reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-β-Dglucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-β-D-glucuronide (4), 3-O-methyl-quercetin-3'-O-β-D-glucuronide (5), and 4'-O-methylquercetin-3'-O-β-D-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. Furthermore, a mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Discovery of Novel Rhizoctonia solani DHFR Inhibitors as Fungicides Using Virtual Screening

Dihydrofolate reductase (DHFR) is an essential enzyme in the folate pathway and has been recognized as a well-known target for antibacterial and antifungal drugs. We discovered eight compounds from the ZINC database using virtual screening to inhibit Rhizoctonia solani (R. solani), a fungal pathogen in crops. These compounds were evaluated with in vitro assays for enzymatic and antifungal activity. Among these, compound Hit8 is the most active R. solani DHFR inhibitor, with the IC 50 of 10.2 μM. The selectivity of inhibition is 22.3 against human DHFR with the IC 50 of 227.7 μM. Moreover, Hit8 has higher antifungal activity against R. solani (EC 50 of 38.2 mg L –1 ) compared with validamycin A (EC 50 of 67.6 mg L –1 ), a well-documented fungicide. These results suggest that Hit8 may be a potential fungicide. Finally, our study exemplifies a computer-aided method to discover novel inhibitors that could target plant pathogenic fungi.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Taste Perception of Branched Maltooligosaccharides

Oral processing of starch generates linear and branched maltooligosaccharides (MOS). This study investigates the impact of branching and the degree of polymerization (DP) on MOS taste detection. Three branched MOS samples (A−C) were enzymatically prepared from gelatinized waxy corn starch and characterized using reducing-end assays, MALDI-TOF MS, β-amylase hydrolysis, and GC-MS linkage analysis. Samples A, B, and C had average DPs of 6, 10, and 18, respectively, with α-1,6 branch points comprising 9−13% of glycosidic bonds; linear MOS were negligible. In triangle tests, participants evaluated MOS samples and a maltose control at 75 mM, with and without lactisole, a sweet receptor inhibitor. All samples were detectable without lactisole (d′ = 1.81−2.9; p < 0.05), but not with lactisole (d′ = 0−0.61; p > 0.05). These findings demonstrate that α-1,6 branching enables MOS detection via the sweet receptor, not the putative “starchy” receptor, underscoring the importance of glycosidic structure in carbohydrate taste perception.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Discovery of TAK-981, a First-in-Class Inhibitor of SUMO-Activating Enzyme for the Treatment of Cancer

SUMOylation is a reversible post-translational modification that regulates protein function through covalent attachment of small ubiquitin-like modifier (SUMO) proteins. The process of SUMOylating proteins involves an enzymatic cascade, the first step of which entails the activation of a SUMO protein through an ATP-dependent process catalyzed by SUMO-activating enzyme (SAE). Here, we describe the identification of TAK-981, a mechanism-based inhibitor of SAE which forms a SUMO–TAK-981 adduct as the inhibitory species within the enzyme catalytic site. Optimization of selectivity against related enzymes as well as enhancement of mean residence time of the adduct were critical to the identification of compounds with potent cellular pathway inhibition and ultimately a prolonged pharmacodynamic effect and efficacy in preclinical tumor models, culminating in the identification of the clinical molecule TAK-981.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Identification of Potent and Selective Inhibitors of Acanthamoeba : Structural Insights into Sterol 14α-Demethylase as a Key Drug Target

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC 50 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

60 APPLIED LIFE SCIENCES↗