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This paper describes the design, development, synthesis, in silico, and in vitro evaluation of fourteen novel heterocycle hybrids as inhibitors of the α-glucosidase enzyme. The primary aim of this study was to explore the potential of novel pyrazole-phthalazine hybrids as selective inhibitors of α-glucosidase, an enzyme involved in carbohydrate metabolism, which plays a key role in the management of type 2 diabetes. The rationale for this study stems from the need for new, more effective inhibitors of α-glucosidase with improved efficacy and safety profiles compared to currently available therapies like Acarbose. The synthesized compounds were tested against the yeast α-glucosidase enzyme and showed significantly higher activity than the standard drug Acarbose. The IC50 values ranged from 13.66 ± 0.009 to 494 ± 0.006 μM, compared to the standard drug Acarbose (IC50 = 720.18 ± 0.008). The most effective α-glucosidase inhibitor, 2-acetyl-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8l) , was identified through a kinetic binding study that yielded an inhibition constant, Ki, of 34.75 µM. All of the pharmacophoric features used in the hybrid design were found to be involved in the interaction with the enzyme’s active site, as expected. Moreover, molecular dynamic simulation and the absorption, distribution, metabolism, and excretion (ADME) have been performed.

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides, and Aβ oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aβ peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods—mainly spectroscopy and imaging techniques—to characterize Aβ/Ni(II) interactions in vitro, for different Aβ variants: Aβ(1–40), Aβ(1–40)(H6A, H13A, H14A), Aβ(4–40), and Aβ(1–42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aβ monomers. Equimolar amounts of Ni(II) ions retard Aβ aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aβ binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aβ dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aβ monomers, while in a membrane-mimicking environment (SDS micelles) coil–coil helix interactions appear to be induced. For SDS-stabilized Aβ oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aβ aggregation processes that are involved in AD brain pathology.

Disulfide bond engineering is a promising strategy for enhancing the stability and functional lifespan of enzymes in therapeutic and industrial applications. In this study, we applied computational modeling to introduce interchain disulfide bonds in Aspergillus flavus uricase to increase its stability without compromising catalytic efficiency. Six uricase muteins were engineered with targeted disulfide bonds at positions selected based on energetic frustration, structural integrity, and tunnel profiling analyses. By employing frustration density mapping, Root Mean Square Fluctuation (RMSF) profiling, and tunnel analysis, we evaluated the structural stability, flexibility, and substrate accessibility of each variant. Our findings revealed that muteins with disulfide bonds between residues such as Ala6-Cys290 and Ser119-Cys220 exhibited significant reductions in highly frustrated regions, enhancing the enzyme’s structural resilience. RMSF analysis indicated decreased local flexibility near disulfide sites, contributing to increased stability. Tunnel profiling further demonstrated that muteins with strategically placed disulfide bonds maintained favorable substrate access and low-energy barriers, critical for catalytic turnover. These results underscore the potential of targeted disulfide bond engineering for optimizing enzyme stability, offering valuable insights for the development of stable, high-performance biocatalysts suitable for therapeutic and industrial use.

[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN) 2 CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN) 2 CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN) 2 CO group. Using native mass spectrometry of an anaerobically isolated HybG–HypD complex we show that HybG carries the Fe(CN) 2 CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO 2 -related, covalent modifications that depended on the presence of the N -terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN) 2 CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN) 2 CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation. G-quadruplex (G4) forming sequences are highly enriched in the human genome and function as important regulators of diverse range of biological processes. Here the authors show that while G4 structures on template strand block transcription, folding on the non-template strand enhances transcription by means of successive R-loop formation.

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease. The SARS-CoV-2 main protease is an important target for the development of COVID-19 therapeutics. Here, the authors combine X-ray crystallography and mass spectrometry and performed a large scale fragment screening campaign, which yielded 96 liganded structures of this essential viral protein that are of interest for further drug development efforts.

The widening gap between current supply of meat and its future demand has increased the need to produce plant-based meat analogs. Despite ongoing technical developments, one of the unresolved challenges of plant-based meat analogs is to safely and effectively imitate the appearance of raw and cooked animal-based meat, especially the color. This study aimed to develop a more effective and safe browning system for beet red (BR) in plant-based meat analog patties using laccase (LC) and sugar beet pectin (SBP). First, we investigated the synergistic effects of SBP and LC on BR decolorization of meat analog patties. We discovered that the red tones of LC-treated patties containing BR and SBP were remarkably browned after grilling, compared to patties that did not contain SBP. Notably, this color change by LC + SBP was similar to that of beef patties. Additionally, the hardness of LC-treated meat analog patties containing BR was higher than those that did not contain BR. Interestingly, the presence of SBP and LC enhanced the browning reaction and functional properties of meat analogs containing BR. This is the first report on a browning system for meat analogs containing BR using enzymatic methods to the best of our knowledge.

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal ‘fastener’ helix retains PAF and is followed by a ‘latch’ that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.Cryo-EM elucidation of a fully reconstituted Pol II–DSF–PAF1–SPT6 elongation complex defines the position of PAF1 subunit RTF1 and reveals contacts with the Pol II bridge helix that may allosterically stimulate transcription elongation.

Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc) n (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios . Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc) n translocation based on experiments using a recombinant Vc CBP, SBP specific for (GlcNAc) n from Vibrio cholerae . X-ray crystal structures of unliganded or (GlcNAc) 3 -liganded Vc CBP solved at 1.2–1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc) 3 -liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.

Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo. Some transcription factors have been proposed to functionally interact with RNA to facilitate proper regulation of gene expression. Here the authors demonstrate that human Sox2 interact directly and with high affinity to RNAs through its HMG DNA-binding domain.