Active Site Research Articles

Cleistopholis patens root bark extract exerts cardioprotective effect against doxorubicin-induced myocardial toxicity in rats

Abstract Background Myocardial Infarction still persists as the most prevalent cardiovascular disease and is a top cause of morbidity and mortality in doxorubicin treated cancer patients. This study evaluated the prophylactic effect of the ethanol root bark extract of Cleistopholis patens (ERBECP) against doxorubicin-induced myocardial infarction in wistar rats. Extraction, preliminary phytochemical analysis, acute toxicity study and body weight (b.w.) of ERBECP were achieved using standard methods. Phyto-constituents in ERBECP were indentified using Gas Chromatography-Mass Spectrometry (GC–MS) technique. Thirty (30) male albino Wistar rats of average b.w. ranging between 100 and 130 g were divided into six groups of five rats each. Groups I, II and III served as normal, doxorubicin (DOX) and standard (Vasoprin 150 mg/kg b.w) controls respectively, while groups IV, V and VI were orally pre-treated with the extract (200, 400 and 600 mg/kgb.w) for two weeks prior to intraperitoneal induction of cardiotoxicity with DOX (20 mg/kg bw) on day 14. Results Disturbances in serum cardiac function bio-markers such as; Cardiac Troponin-I (CTnI), Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT). Lipid profile markers such as; Total cholesterol (TC), Triacylglycerol (TAG), Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL). Oxidative stress markers such as; Malondialdehyde (MDA), Superoxide Dismutase (SOD), Catalase (CAT), Glutathione (GSH) confirmed the induction of myocardial infarction. Histological assessment of heart tissues was performed to validate biochemical results. The GC–MS analysis of ERBECP identified a total of 69 compounds. Safety profile of the aqueous extract was safe for the animals up to the highest dose of 5000 mg/kg b.w. Pre-treatment of DOX group with ERBECP could significantly increase the b.w. compared to the DOX-treated group during the experimental period of 2 weeks. There were significant (p < 0.05) alterations in the levels of CTnI, CK, LDH, AST, ALT and lipid profile indices in the DOX control rats. Also, significant (p < 0.05) increase was observed in MDA and decrease in SOD, CAT and GSH in the DOX control rats. However, administration of the extract significantly (p < 0.05) normalized these alterations and reversed the architectural changes in the heart. The 69 compounds were screened against the target protein (CBR1); we identified seven hits based on the docking score and interactions with the active site residues. All the C. patens constituents had MW (g/mol) less than 500, HBA < 10 and HBD not more than 5. Apart, 9-Octadecenoic acid (Z)-, 2,3-dihydroxy propyl ester and Estra-1,3,5(10)-trien-17. beta. -ol, all the constituents had LD50 lower than 2000 mg/kg. Conclusions The findings reveals ERBECP demonstrated promising potential and can be exploited in the development novel cardiac therapeutic agents.

Macromolecular crowding and bicarbonate enhance the hydrogen peroxide-induced inactivation of glyceraldehyde-3-phosphate dehydrogenase

The active site Cys residue in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to oxidation by hydrogen peroxide (H2O2), with this resulting in enzyme inactivation. This re-routes the carbon flux from glycolysis to the pentose phosphate pathway favoring the formation of NADPH and synthetic intermediates required for antioxidant defense and repair systems. Consequently, GAPDH inactivation serves as a redox switch for metabolic adaptation under conditions of oxidative stress. However, there is a major knowledge gap as to how GAPDH is efficiently oxidized and inactivated, when the increase in intracellular H2O2 is modest, and there is a high concentration of alternative (non-signaling) thiols and efficient peroxide removing systems. We have therefore explored whether GAPDH inactivation is enhanced by two factors of in vivo relevance: macromolecular crowding, an inherent property of biological environments, and the presence of bicarbonate, an abundant biological buffer. Bicarbonate is already known to modulate H2O2 metabolism via formation of peroxymonocarbonate. GAPDH activity was assessed in experiments with low doses of H2O2 under both dilute and crowded conditions (induced by inert high molecular mass polymers and small molecules), in both the absence and presence of 25 mM sodium bicarbonate. H2O2-induced inactivation of GAPDH was observed to be significantly enhanced under macromolecular crowding conditions, with bicarbonate having an additional effect. These data strongly suggest that these two factors are of major importance in redox switch mechanisms involving GAPDH (and possibly other thiol-dependent systems) within the cellular environment.

A Novel Cold‐Adapted Catechol 1,2‐Dioxygenase From Antarctic Sea‐Ice Bacterium Halomonas sp. ANT108: Characterization and Immobilization

ABSTRACTThe enzyme catechol 1,2‐dioxygenase (CAT) plays a critical role in the biosynthesis pathway of cis, cis‐muconic acid (CCMA), which serves as an indispensable raw material for various industrial applications. In this research, we cloned a novel cold‐adapted CAT (HaCAT) from the Antarctic sea ice bacterium Halomonas sp. ANT108. Homology modeling analysis revealed that HaCAT possessed the characteristic Fe3+ binding site and catalytic active site of typical CATs, and it exhibited unique structural adaptations to cold environments. The optimal temperature and pH for recombinant HaCAT (rHaCAT) were found to be 25°C and 6.5, respectively. At 0°C, the enzyme retained a maximum activity of 43.6%, and in the presence of 1.0 M NaCl, its activity reached 173.9%, demonstrating significant salt tolerance. Additionally, the Vmax and Km of rHaCAT were 6.68 μmol/min/mg and 128.90 μM at 25°C, respectively. Furthermore, rHaCAT was successfully immobilized in the metal‐organic framework ZIF‐8 and retained almost 50% of its activity after five reuse cycles, demonstrating excellent reusability. Overall, these results provided a new resource and theoretical foundation for the industrial biocatalytic production and modification of CAT.

CoFe Hydroxide Nanopheres for Enhanced Alkaline Splitting and Seawater Oxidation:Anion Doping Effects of Fluorine and Carbonate

Green hydrogen production can be achieved through electrolysis of fresh water or the use of renewable energy to electrolyze seawater. However, due to the low activity and poor stability of oxygen evolution reaction catalysts, direct electrolysis of alkaline seawater faces significant challenges. Herein, The catalyst F‐CoFe(OH)‐CO3/NF with three‐dimensional nanosphere structure was prepared, The introduction of CO32‐ into the intermediate layer of CoFe Hydroxide improves the corrosion resistance of alkaline electrolyte and the doping of F‐ is to design three‐dimensional layered nanostructures, increase the active site, and accelerate the diffusion of the electrolyte. By in situ Raman analysis, partial oxidation of CoFe hydroxide to CoFe (oxy)hydroxide as the active center can accelerating the adsorption of oxygen‐related intermediates. In 1M KOH, it requires overpotentials of 210 mV and 251mV to drive current densities of 10 and 100 mA cm‐2, respectively. And it remained stable at the current density of 100 mA cm‐2 for 120 h in 1M KOH. F‐CoFe(OH)‐CO3/NF can also catalyzes the decomposition of electrolytic seawater. Compared with hydroxide, anion‐doped carbonate hydroxide is more efficient and stable in electrolyte solution, which is of great importance for the development of a new stable electrocatalyst for water decomposition.

Electronic Modulation and Symmetry‐Breaking Engineering of Single‐Atom Catalysts Driving Long‐Cycling Li−S Battery

AbstractDeveloping efficient and durable single‐atom catalysts is vitally important for the sulfur redox reaction (SROR) in Li−S battery, while it remains enormous challenging. Herein, undercoordinated Ni−N3 moieties anchored on N,S‐codoped porous carbon (Ni−NSC) is obtained to enhance the SROR. The experiments and theoretical calculations indicate that the symmetry‐breaking charge transfer in Ni single‐atom catalyst originates from tuning effect of sulfur atoms mediated Ni−N3 moieties, which can both facilitate the chemical adsorption by formation of N−Ni⋅⋅⋅Sn2−, and achieve a rapid redox conversion of polysulfides because of the enhanced electron transfer. As results, the Ni−NSC based Li−S battery delivers a very high initial reversible capacity (1025 mAh g−1 at 1 C), as well as outstanding cycling‐stability for 2400 cycles at 2 C and 3 C, respectively. Noteworthy, the areal capacity can reach 7.8 mAh cm−2 at 0.05 C and a retention capacity of 4.7 mAh cm−2 after 100 cycles at 0.2 C for Ni−NSC based Li−S battery with sulfur loading of 5.88 mg cm−2. This work provides profound insight for rational optimizing microscopic electronic density of active site to promoting SROR in metal‐sulfur batteries.

Reduction of 4‐Nitrophenol Catalyzed by Gold Nanoclusters with Aggregation‐Induced‐Emission: Emission Intensity Correlated Activity and Mechanistic Exploration

AbstractThiolate‐protected Au nanoclusters (NCs) have developed as a promising class of model catalysts to achieve fundamental understanding of metal nanocatalysis. Whereas, the packing mode of peripheral ligand on metal core is changeful in the reaction medium and show elusive impact on catalytic activity. In this work, using glutathione (GSH) protected Au NCs (Au@GSH NCs) with aggregation‐induced‐emission (AIE) characteristics as model catalyst for the hydrogenation of 4‐nitrophenol (4‐NP), photoluminescence (PL) intensity correlated catalytic activity of Au@GSH NCs was successfully mediated by the addition of Ag+ in the preparation or poor solvent in the reaction medium, showing a relationship of “as one falls, another rises.” Au NCs with intense PL implied a dense packing of peripheral ligand, which hampered the accessibility of active site and thus exhibited slowest catalytic reaction kinetics of the reduction of 4‐NP and vice versa. Based on this methodology, a case study of the effect of salt additives on the catalytic activity is carried out, different mechanisms are distinguished by the change in the PL intensity, and with the combination of diagnostic deuterium isotope experiments, it has been demonstrated that the proton from water solvent is involved in the reaction and that the proton transfer process is the rate‐determining step, the contribution of ionic additives to the hydrogen bonding network determines their effect on the reaction kinetics. The correlation between PL intensity and catalytic activity of Au NCs could provide an efficient way to design highly active Au NC catalysts and give a new insight to understand the unique optoelectronic properties of Au NCs and reaction mechanism.

The Electron‐Rich Interface Regulated MBene by S‐Bridge Guided to Enhance Nitrogen Fixation under Environmental Conditions

AbstractThe underutilization of active sites limits the performance enhancement of 2D transition metal boride (MBene) in electrocatalytic nitrogen reduction reaction (NRR). Herein, a highly efficient NRR electrocatalyst with S atoms bridging Fe and Mo atoms on the surface of MBene is successfully constructed by using an active site electron optimization strategy, which increases the charge density around the Mo active site and enhances the activation ability of the catalyst to N2 molecules. It is noteworthy that FeS2‐MBene demonstrates a low intrinsic potential for NRR (−0.2 V vs RHE). It is more favorable for the adsorption of nitrogen atoms in comparison to hydrogen atoms, thereby it can effectively inhibit the hydrogen evolution reaction (HER). Under a potential of −0.2 V versus RHE, the ammonia yield rate is 37.13 ± 1.31 µg h−1 mg−1, and the FE is 55.97 ± 2.63%. Density functional theory (DFT) calculations demonstrate that Mo on the surface of MBene serves as a site for the adsorption of N2. The formation of the heterostructure enhances electron transfer, resulting in the Mo active site becoming an electron‐rich state in favor of subsequent hydrogenation steps. This work offers significant insights into the design and utilization of 2D MBene‐based catalysts in NRR.

Densely populated single‐atom catalysts for boosting hydrogen generation from formic acid

AbstractThe single‐atom M‐N‐C (M typically being Co or Fe) is a prominent material with exceptional reactivity in areas of catalysis for sustainable energy. However, the formation of metal nanoparticles in M‐N‐C materials is coupled with high‐temperature calcination conditions, limiting the density of M‐Nx active sites and thus restricting the catalytic performance of such catalysts. Herein, we describe an effective decoupling strategy to construct high‐density M‐Nx active sites by generating polyfurfuryl alcohol in the MOF precursor, effectively preventing the formation of metal nanoparticles even with up to 6.377% cobalt loading. This catalyst showed a high H2 production rate of 778 mL gCat−1 h−1 when used in the dehydrogenation reaction of formic acid. In addition to the high density of the active site, a curved carbon surface in the structure is also thought to be the reason for the high performance of the catalyst.

Cu-Supported ZnO under Conditions of CO2 Reduction to Methanol: Why 0.2 ML Coverage?

By hydrogenating carbon dioxide to value-added products such as methanol, heterogeneous catalysts can lower greenhouse gas emissions and generate alternative liquid fuels. The most common commercial catalyst for the reduction of CO2 to methanol is Cu/ZnO/Al2O3, where ZnO improves conversion and selectivity toward methanol. The structure of this catalyst is thought to be Zn oxy(hydroxyl) overlayers on the nanometer scale on Cu. In the presence of CO2 and H2 under reaction conditions, the Cu substrate itself can be restructured and/or partially oxidized at its interface with ZnO, or the Zn might be reduced, possibly completely to a CuZn alloy, making the exact structure and stoichiometry of the active site a topic of active debate. In this study, we examine Zn3 clusters on Cu(100) and Cu(111), as a subnano model of the catalyst. We use a grand canonical genetic algorithm to sample the system structure and stoichiometry under catalytic conditions: T of 550 K, initial partial pressures of H2 of 4.5 atm and CO2 of 0.5 atm, and 1% conversion. We uncover a strong dependence of the catalyst stoichiometry on the surface coverage. At the optimal 0.2 ML surface coverage, chains of Zn(OH) form on both Cu surfaces. On Cu(100), the catalyst has many thermally accessible metastable minima, whereas on Cu(111), it does not. No oxidation or reconstruction of the Cu is found. However, at a lower coverage of Zn, Zn3 clusters take on a metallic form on Cu(100), and slightly oxidized Zn3O on Cu(111), while the surface uptakes H to form a variety of low hydrides of Cu. We thus hypothesize that the 0.2 ML Zn coverage is optimal, as found experimentally, because of the stronger yet incomplete oxidation afforded by Zn at this coverage.

Understanding the Carbyne Formation from C2H2 Complexes.

Nature chooses a high-valent tungsten center at the active site of the enzyme acetylene hydratase to facilitate acetylene hydration to acetaldehyde. However, the reactions of tungsten-coordinated acetylene are still not well understood, which prevents the development of sustainable bioinspired alkyne hydration catalysts. Here we report the reactivity of two bioinspired tungsten complexes with the acetylene ligand acting as a four-: [W(CO)(C2H2)(PymS)2] (1) and a two-electron donor: [WO(C2H2)(PymS)2] (3), with PMe3 as a nucleophile to simulate the enzyme's reactivity (PymS = 4-(trifluoromethyl)-6-methylpyrimidine-2-thiolate). In dichloromethane, compound 1 was found to react to the cationic carbyne [W≡CCH2PMe3(CO)(PMe3)2(PymS)]Cl (2-Cl) while 3 reacts to the vinyl compound [WO(CH═CHPMe3)(PMe3)3(PymS)]Cl (4-Cl). The formation of the latter follows the common rules applied to η2-alkyne complexes, whereas the carbyne formation was not expected due to the challenging 1,2-H shift. To understand these differences in behavior between seemingly similar acetylene complexes, stepwise addition of the nucleophile in various solvents was investigated by synthetic, spectroscopic, and computational approaches. In this manuscript, we describe that only a four-electron donor acetylene complex can react to the carbyne over the η1-vinyl intermediate and that 1,2-H shift can be assisted by an H-transfer reagent (in this case, the decoordinated PymS ligand). Furthermore, to favor the attack of PMe3 at W coordinated acetylene, the metal center needs to be electron-poor and crowded enough to prevent nucleophile coordination. Finally, the intricate role of the anionic PymS ligand in the vicinity of the first coordination sphere models the potential involvement of amino acid residues during acetylene transformations in AH.

Modulating Electronic Structure of MoS2 nanosheets by Sulfide‐Enrichment‐Induced Vacancies for Enhanced Photocatalytic Hydrogen Production

Enhancing photocatalytic activity by changing the electronic structure of the catalyst is an effective strategy. 2H‐MoS2 has semiconductor properties, and its unsaturated side S atom is the main active site for its photocatalytic activity. However, due to the weak S‐H bond energy, its hydrogen adsorption capacity is weak. In this work, the electronic structure of MoS2 is adjusted by S‐rich treatment, resulting in the formation of Mo vacancy (MoS2‐VMo). The level of Mo vacancies was assessed using UV‐visible diffuse reflectance spectroscopy (UV‐vis DRS). As these vacancies can capture certain photogenerated electrons, thereby reducing the recombination of electron‐hole pairs. Through DFT calculation, it is found that the anti‐bonding orbital electron filling of MoS2‐VMo is weakened, the bond energy of S‐Mo bond is weakened, and the bond energy between S‐H bond is enhanced, which is more conducive to proton adsorption. The photocatalytic activity for hydrogen evolution of MoS2‐3, which underwent the optimal S‐rich treatment, achieved a remarkable rate of 2404.6 μmol g‐1 h‐1 in dye eosin Y (EY) sensitization system, significantly surpassing that of MoS2 lacking the S‐enriched treatment. This study introduces a novel concept for enhancing the photocatalytic hydrogen evolution performance of metal sulfides via defect engineering.

Investigating the Effect of Carbon Nanotubes Decorated SmVO4-MoS2 Nanocomposite for Energy Storage Enhancement via VARTM-Fabricated Solid-State Structural Supercapacitors Using Woven Carbon Fiber.

Traditional supercapacitors are cumbersome and need separate enclosures, which add weight and reduce space efficiency. In contrast, structural supercapacitors combine energy storage with load-bearing materials, optimizing space and weight for automotive and aerospace applications. This study investigates the synthesis of SmVO4-MoS2 and SmVO4-MoS2-CNT nanocomposites, focusing on optimizing CNT concentration in SmVO4-MoS2-CNT for high-performance supercapacitors. The optimal concentration of SmVO4-MoS2-CNT is identified and used to fabricate structural supercapacitor devices via the vacuum-assisted resin transfer molding (VARTM) technique. The results indicate that the specific capacitance of Sm-Mo-C5, using a three-electrode system, reached 1.01Fcm-2 at a current density of 2.187mAcm-2. The performance improvement is attributed to the synergistic interaction among SmVO4, MoS2, and CNTs, collectively enhancing conductivity and active site availability. The practical application of this study is demonstrated by synthesizing Sm-Mo-C5 on woven carbon fiber (WCF) and subsequently fabricating a structural supercapacitor device (SSD) using the VARTM. The SSD, produced via VARTM, exhibited a specific capacitance of 0.287Fcm- 2 at a current density of 2Acm-2. The device showcased exceptional cyclic stability, maintaining 72.5% of its initial capacitance after 50,000 charge-discharge cycles. Additionally, it achieved a maximum energy density of 79.86Whkg-1 at a power density of 1017.69Wkg-1.

Enzyme stabilisation due to incorporation of a fluorinated non-natural amino acid at the protein surface.

We have previously engineered E. coli transketolase (TK) enzyme variants that accept new substrates such as aliphatic or aromatic aldehydes, and also with improved thermal stability. Irreversible aggregation is the primary mechanism of deactivation for TK in the buffers used for biocatalysis, and so we were interested in determining the extent to which this remains true in more complex media, crude cell lysates or even in vivo. Such understanding would better guide future protein engineering efforts. NMR offers a potential approach to probe protein structure changes, aggregation, and diffusion, and19F-labelled amino acids are a useful NMR probe for complex systems with little or no background signal from the rest of the protein or their environment. Here we labelled E. coli TK with two different19F probes, trifluoromethyl-L-phenylalanine (tfm-Phe), and 4-fluoro phenylalanine (4F-Phe), through site specific non-natural amino acid incorporation. We targeted them to residue K316, a highly solvent exposed site located at the furthest point from the enzyme active sites. Characterisation of the19F-labelled TK variants revealed surprising effects of these mutations on stability, and to some extent on activity. While variant TK-tfm-Phe led to a 7.5°C increase in the thermal transition midpoint (Tm) for denaturation, the TK-4F-Phe variant largely abolished the aggregation of the enzyme when incubated at 50°C19. F-NMR revealed different behaviours in response to temperature increases for the two TK variants, displaying opposite temperature gradient chemical shifts, and diverging motion regimes, suggesting that the mutations affected differently both the local environment at this site, and its temperature-induced dynamics. A similar incubation of TK at 40-55°C is also known to induce higher cofactor-binding affinities, leading to an apparent heat activation under low cofactor concentration conditions. We have hypothesised previously that a heat-inducible conformational change in TK leads to this effect1. H-NMR revealed a temperature-dependent re-structuring of methyl groups, also at 30-50°C, which may be linked to the heat activation. While our kinetic studies were not expected to observe the heat activation event due to the high cofactor concentrations used, this was not the case for TK-4F-Phe, which did appear to heat activate slightly at 45°C. This implied that the mutations at K316 could influence cofactor-binding, despite their location at 47 Å from either active site. Such long-distance effects of mutations are not unprecedented, and indeed we have previously shown how distant mutations can influence active-site loop stability and function in TK, mediated via dynamically coupled networks of residues. Molecular dynamics simulations of the two19F containing variants similarly revealed networks of residues that could couple the changes in dynamics at residue K316, through to changes in active site dynamics. These results independently highlight the sensitivity of active-site function to distant mutations coupled through correlated dynamic networks of residues. They also highlight the potential influence of surface-incorporated probes on protein stability and function, and the need to characterise them well prior to further studies.

Evolution Enhances Kemp Eliminase Activity by Optimizing Oxyanion Stabilization and Conformational Flexibility.

The base-promoted Kemp elimination reaction has been used as a model system for enzyme design. Among the multiple computationally designed and evolved Kemp eliminases generated along the years, the HG3-to-HG3.17 evolutionary trajectory is particularly interesting due to the high catalytic efficiency of HG3.17 and the debated role of glutamine 50 (Gln50) as potential oxyanion stabilizer. This study aims to elucidate the structural and dynamic changes along the evolutionary pathway from HG3 to HG3.17 that contribute to improved catalytic efficiency. In particular, we evaluate key variants along the HG3 evolutionary trajectory via molecular dynamics simulations coupled to non-covalent interactions and water analysis. Our computational study indicates that HG3.17 can adopt a catalytically competent conformation promoted by a water-mediated network of non-covalent interactions, in which aspartate 124 is properly positioned for proton abstraction and both glutamine 50 and mutation cysteine 84 contribute to oxyanion stabilization. We find that HG3.17 exhibits a rather high flexibility of Gln50, which is regulated by the conformation adopted by the active site residue Trp44. This interplay between Gln50 and Trp44 positioning induced by distal active site mutations affects the water-mediated network of non-covalent interactions, Gln50 preorganization, and water content of the active site pocket.

Scalable Thiol Reactivity Profiling Identifies Azetidinyl Oxadiazoles as Cysteine-Targeting Electrophiles.

Cysteine reactive groups are a mainstay in the design of covalent drugs and probe molecules, yet only a handful of electrophiles are routinely used to target this amino acid. Here, we report the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. High throughput screening using STRP identified the azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. We show the utility of this reactive group with the discovery of an azetidinyl oxadiazole containing a small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site. This study adds a novel cysteine targeting group to the electrophilic lexicon and provides robust methodology to rapidly surveil libraries for reactivity with cysteine.

Discovery and binding mode of small molecule inhibitors of the apo form of human TDO2.

Tryptophan-2,3-dioxygenase (TDO2) and indoleamine-2,3-dioxygenase (IDO1) are structurally distinct heme enzymes that catalyze the conversion of L-tryptophan to N-formyl-kynurenine, and play important roles in metabolism, inflammation, and tumor immune surveillance. The enzymes can adopt an inactive, heme-free (apo) state or an active, heme-containing (holo) state, with the balance between them varying dynamically according to biological conditions. Inhibitors of holo-TDO2 are known but, despite several advantages of the heme-free state as a drug target, no inhibitors of apo-TDO2 have been reported. We describe the discovery of the first apo-TDO2 binding inhibitors, to our knowledge, and their inhibition of cellular TDO2 activity at low nanomolar concentrations. The crystal structure of a potent, small molecule inhibitor bound to apo-TDO2 reveals its detailed binding interactions within the large, hydrophobic heme binding pocket of the active site.

Discovery of highly potent and ALK2/ALK1 selective kinase inhibitors using DNA-encoded chemistry technology

Activin receptor type 1 (ACVR1; ALK2) and activin receptor like type 1 (ACVRL1; ALK1) are transforming growth factor beta family receptors that integrate extracellular signals of bone morphogenic proteins (BMPs) and activins into Mothers Against Decapentaplegic homolog 1/5 (SMAD1/SMAD5) signaling complexes. Several activating mutations in ALK2 are implicated in fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine gliomas, and ependymomas. The ALK2 R206H mutation is also present in a subset of endometrial tumors, melanomas, non-small lung cancers, and colorectal cancers, and ALK2 expression is elevated in pancreatic cancer. Using DNA-encoded chemistry technology, we screened 3.94 billion unique compounds from our diverse DNA-encoded chemical libraries (DECLs) against the kinase domain of ALK2. Off-DNA synthesis of DECL hits and biochemical validation revealed nanomolar potent ALK2 inhibitors. Further structure-activity relationship studies yielded center for drug discovery (CDD)-2789, a potent [NanoBRET (NB) cell IC50: 0.54 μM] and metabolically stable analog with good pharmacological profile. Crystal structures of ALK2 bound with CDD-2281, CDD-2282, or CDD-2789 show that these inhibitors bind the active site through Van der Waals interactions and solvent-mediated hydrogen bonds. CDD-2789 exhibits high selectivity toward ALK2/ALK1 in KINOMEscan analysis and NB K192 assay. In cell-based studies, ALK2 inhibitors effectively attenuated activin A and BMP-induced Phosphorylated SMAD1/5 activation in fibroblasts from individuals with FOP in a dose-dependent manner. Thus, CDD-2789 is a valuable tool compound for further investigation of the biological functions of ALK2 and ALK1 and the therapeutic potential of specific inhibition of ALK2.

Identification of Potential Inhibitors of TGFβR1 for the Treatment of Cancer through Structure-Based Virtual Screening and Molecular Dynamics Simulations.

Globally, cancer is one of the leading causes of death. Resistance to conventional medications, such as chemotherapy and radiation, continues to be a significant challenge in the treatment of cancer despite the availability of numerous medicines. Therefore, the highest priority is to hunt for new therapeutic agents. Transforming growth factor-beta is a pivotal regulatory cytokine that exerts significant influence over cellular processes, particularly emphasizing its role in facilitating and modulating cell proliferation. TGF-β receptor 1, identified as the most promising active site of the TGF-β signaling, is a potent drug target site that has garnered wide attention for developing new anticancer agents. The present investigation investigates the potential natural products as TGFβR1 inhibitors. The SB431542 complexed TGFβR1 protein model was used to screen the natural product database to obtain a compound with high binding potential. NPC247629 has emerged as the best-scored compound among all the screened compounds, demonstrating the highest affinity towards the TGFβR1 regarding docking score -17.54 kcal/mol. The all-atoms MD simulation study indicated that all proposed hits are retained inside the receptor in dynamic states. Additionally, principal component and free energy landscape analysis were performed to explore the binding mechanism of top-hit natural products. The best-screened hits, NPC247629 and NPC60735, have excellent binding affinity and hold a massive potential for TGFβR1 inhibition, paving the way for promising future investigations in cancer treatment.

The apoptotic and anti-proliferative effects of Neosetophomone B in T-cell acute lymphoblastic leukaemia via PI3K/AKT/mTOR pathway inhibition.

The phosphatidylinositol 3-kinase/Protein Kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is pivotal in various cancers, including T-cell acute lymphoblastic leukaemia (T-ALL), a particularly aggressive type of leukaemia. This study investigates the effects of Neosetophomone B (NSP-B), a meroterpenoid fungal metabolite, on T-ALL cell lines, focusing on its anti-cancer mechanisms and therapeutic potential. NSP-B significantly inhibited the proliferation of T-ALL cells by inducing G0/G1 cell cycle arrest and promoting caspase-dependent apoptosis. Additionally, NSP-B led to the dephosphorylation and subsequent inactivation of the PI3K/AKT/mTOR signalling pathway, a critical pathway in cell survival and growth. Molecular docking studies revealed a strong binding affinity of NSP-B to the active site of AKT, primarily involving key residues crucial for its activity. Interestingly, NSP-B treatment also induced apoptosis and significantly reduced proliferation in phytohemagglutinin-activated primary human CD3+ T cells, accompanied by a G0/G1 cell cycle arrest. Importantly, NSP-B did not affect normal primary T cells, indicating a degree of selectivity in its action, targeting only T-ALL cells and activated T cells. In conclusion, our findings highlight the potential of NSP-B as a novel therapeutic agent for T-ALL, specifically targeting the aberrantly activated PI3K/AKT/mTOR pathway and being selective in action. These results provide a strong basis for further investigation into NSP-B's anti-cancer properties and potential application in T-ALL clinical therapies.

Crystal structure of β-d-galactofuranosidase from Streptomyces sp. JHA19 in complex with an inhibitor provides insights into substrate specificity.

d-Galactofuranose (Galf) is widely distributed in glycoconjugates of pathogenic microbes. β-d-Galactofuranosidase (Galf-ase) from Streptomyces sp. JHA19 (ORF1110) belongs to glycoside hydrolase (GH) family 2 and is the first identified Galf-specific degradation enzyme. Here, the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, d-iminogalactitol (Ki = 65 μm) was solved. ORF1110 binds to the C5-C6 hydroxy groups of d-iminogalactitol with an extensive and integral hydrogen bond network, a key interaction that discriminates the substrates. The active site structure of ORF1110 is largely different from those of β-glucuronidases and β-galactosidases in the same GH2 family. A C-terminal domain of ORF1110 is predicted to be a carbohydrate-binding module family 42 that may bind Galf. The structural insights into Galf-ase will contribute to the investigation of therapeutic tools against pathogens.