ATP-binding Site Research Articles

Siamenflavones A-C, three undescribed biflavonoids from Selaginella siamensis Hieron. and biflavonoids from spike mosses as EGFR inhibitor

Three undescribed biflavonoids (BFVs), siamenflavones A–C along with twelve BFVs were isolated from Selaginella siamensis Hieron. and Selaginella bryopteris (L.) Baker (Selaginellaceae). The chemical structures of undescribed compounds were established through comprehensive spectroscopic techniques, chemical correlations, and X-ray crystallography. The ten isolated BFVs, siamenflavones A–C, delicaflavone, chrysocauflavone, robustaflavone, robustaflavone-4-methylether, amentoflavone, tetrahydro-amentoflavone, and sciadopitysin were evaluated for the antiproliferative effects against four human cancer cell lines A549, H1975, HepG2 and T47D. Delicaflavone and robustaflavone 4′-methylether exerted strong effects on the four human cancer cell lines. Siamenflavone B, delicaflavone and robustaflavone 4′-methylether showed potent inhibitory activities against wild-type EGFR. The inhibition of the compounds was further supported by molecular docking and predictive intermolecular interactions. Molecular dynamics simulation studies of siamenflavone B and robustaflavone-4′-methylether complexed to EGFR-TK further supported inhibition of the compounds to the ATP binding site. Finally, analysis of pharmacokinetic and electronic properties using density-functional theory and known drug index calculations suggest that the compounds are pharmaceutically compatible for drug administration.

Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model.

O-GlcNAcylation is a reversible post-translational modification that regulates numerous cellular processes, including embryonic development as well as immune responses. However, its role in inflammation remains ambiguous. This study was designed to examine the role of O-GlcNAcylation in rheumatoid arthritis (RA) and its regulation using human RA patient-derived synovial fibroblasts (RASFs). The efficacy of penta-O-galloyl-beta-D-glucose (PGG), a potent anti-inflammatory molecule, in regulating inflammatory processes in human RASFs was also evaluated. Human synovial tissues and RASFs exhibited higher expression of O-GlcNAcylation compared to their non-diseased counterparts. Pretreatment of RASFs with Thiamet G, an inhibitor of O-GlcNAcase, markedly increased the O-GlcNAc-modified proteins and concomitantly inhibited the IL-1β-induced IL-6 and IL-8 production in human RASFs in vitro. Pretreatment of human RASFs with PGG (0.5-10 µM) abrogated IL-1β-induced IL-6 and IL-8 production in a dose-dependent manner. Immunoprecipitation analysis showed that PGG inhibited O-GlcNAcylation of TAB1 to reduce its association with TGF β-activated kinase 1 (TAK1) and its autophosphorylation, an essential signaling step in IL-1β-induced signaling pathways. Molecular docking in silico studies shows that PGG occupies the C174 position, an ATP-binding site in the kinase domain to inhibit TAK1 kinase activity. Oral administration of PGG (25 mg/kg/day) for 10 days from disease onset significantly ameliorated rat adjuvant-induced (AIA) in rats. PGG treatment reduced the phosphorylation of TAK1 in the treated joints compared to AIA joints, which correlated with the reduced disease severity and suppressed levels of serum IL-1β, GM-CSF, TNF-α, and RANKL. These findings suggest O-GlcNAcylation as a potential therapeutic target and provide the rationale for testing PGG or structurally similar molecule for their therapeutic efficacy.

Unveiling the Structure-Activity Relationships at the Orthosteric Binding Site of P2X Ion Channels: The Route to Selectivity.

The orthosteric ATP-binding site of the P2X receptors is poorly understood. Only a few compounds were well characterized for their P2X receptor functional activity and subtype selectivity. This study represents the first fully functional characterization of various ATP derivatives combined with in silico studies to advance the understanding of SARs at the orthosteric binding sites of P2X receptors leading to the identification of 2-chloro-3-trifluoromethylbenzoyl ATP ester as a novel pan-P2X receptor agonist and several subtype-selective P2X receptor agonists. Furthermore, esterification of both hydroxyl functions of ATP using 1-naphthoic acid has led to compound 26 acting as an antagonist at P2X1-4 and P2X2/3 receptors and an agonist at P2X7 receptors. This particular ATP derivative will allow interrogating the P2X7 receptor function while antagonizing all other P2X receptor subtypes and therefore serve as a valuable pharmacological tool in the future.

Structural basis for coupled ATP-driven electron transfer in the double-cubane cluster protein

Electron transfers coupled to the hydrolysis of ATP allow various metalloenzymes to catalyze reductions at very negative reduction potentials. The double-cubane cluster protein (DCCP) catalyzes the reduction of small molecules, such as acetylene and hydrazine, with electrons provided by its cognate ATP-hydrolyzing reductase (DCCP-R). How ATP-driven electron transfer occurs is not known. To resolve the structural basis for ATP-driven electron transfer, we solved the structures of the DCCP:DCCP-R complex in three different states. The structures show that the DCCP-R homodimer is covalently bridged by a [4Fe4S] cluster that is aligned with the twofold axis of the DCCP homodimer, positioning the [4Fe4S] cluster to enable electron transfer to both double-cubane clusters in the DCCP dimer. DCCP and DCCP-R form stable complexes independent of oxidation state or nucleotides present, and electron transfer requires the hydrolysis of ATP. Electron transfer appears to be additionally driven by modulating the angle between the helices binding the [4Fe4S] cluster. We observed hydrogen bond networks running from the ATP binding site via the [4Fe4S] cluster in DCCP-R to the double-cubane cluster in DCCP, allowing the propagation of conformational changes. Remarkable similarities between the DCCP:DCCP-R complex and the nonhomologous nitrogenases suggest a convergent evolution of catalytic strategies to achieve ATP-driven electron transfers between iron-sulfur clusters.

A drug and ATP binding site in type 1 ryanodine receptor

A drug and ATP binding site in type 1 ryanodine receptor

Prediction of protein mononucleotide binding sites using AlphaFold2 and machine learning

In this study, we developed a system that predicts the binding sites of proteins for five mononucleotides (AMP, ADP, ATP, GDP, and GTP). The system comprises two machine learning (ML)-based predictors using a convolutional neural network and a gradient boosting machine, two template-based predictors based on sequence and structure alignment, and a predictor that performs ensemble learning of these four predictors. In this study, data augmentation of ligand binding sites with similar ligand structures was performed. For example, in the prediction of ADP-binding sites using ML methods, the binding sites of AMP and ATP, which have similar structures, are considered. In addition, we constructed the structure models using AlphaFold2, a highly accurate protein prediction method. The secondary structure and dihedral angle information obtained using the model structures were used as ML predictor features. Additionally, in the template-based predictor, the structures of the binding sites were used as templates to be explored based on structure alignment to identify the binding site of the target. Consequently, the template-based predictor based on structure alignment showed the best performance among the four individual predictors, and the ensemble predictor achieved the best performance, with an area under the curve of 0.958 for all mononucleotides.

Mechanistic Insights into the Mechanism of Inhibitor Selectivity toward the Dark Kinase STK17B against Its High Homology STK17A.

As a member of the death-associated protein kinase (DAPK) family, STK17B plays an important role in the regulation of cellular apoptosis and has been considered as a promising drug target for hepatocellular carcinoma. However, the highly conserved ATP-binding site of protein kinases represents a challenge to design selective inhibitors for a specific DAPK isoform. In this study, molecular docking, multiple large-scale molecular dynamics (MD) simulations, and binding free energy calculations were performed to decipher the molecular mechanism of the binding selectivity of PKIS43 toward STK17B against its high homology STK17A. MD simulations revealed that STK17A underwent a significant conformational arrangement of the activation loop compared to STK17B. The binding free energy predictions suggested that the driving force to control the binding selectivity of PKIS43 was derived from the difference in the protein–ligand electrostatic interactions. Furthermore, the per-residue free energy decomposition unveiled that the energy contribution from Arg41 at the phosphate-binding loop of STK17B was the determinant factor responsible for the binding specificity of PKIS43. This study may provide useful information for the rational design of novel and potent selective inhibitors toward STK17B.

Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells.

Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

Comparison of ATP-binding pockets and discovery of homologous recombination inhibitors

The ATP binding sites of many enzymes are structurally related, which complicates their development as therapeutic targets. In this work, we explore a diverse set of ATPases and compare their ATP binding pockets using different strategies, including direct and indirect structural methods, in search of pockets attractive for drug discovery. We pursue different direct and indirect structural strategies, as well as ligandability assessments to help guide target selection. The analyses indicate human RAD51, an enzyme crucial in homologous recombination, as a promising, tractable target. Inhibition of RAD51 has shown promise in the treatment of certain cancers but more potent inhibitors are needed. Thus, we design compounds computationally against the ATP binding pocket of RAD51 with consideration of multiple criteria, including predicted specificity, drug-likeness, and toxicity. The molecules designed are evaluated experimentally using molecular and cell-based assays. Our results provide two novel hit compounds against RAD51 and illustrate a computational pipeline to design new inhibitors against ATPases.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria.

The F1FO-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the “forward” direction, but it can also consume the same ATP pools by rotating in “reverse.” To prevent futile F1FO-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF1), and chloroplasts (ε and γ disulfide) emerged to block the F1FO-ATPase activity selectively. In this study, we analyze how these F1FO-ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans. Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F1FO-ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF1 likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria.

Dehydroisohispanolone as a Promising NLRP3 Inhibitor Agent: Bioevaluation and Molecular Docking.

Dehydroisohispanolone (DIH), is a labdane diterpene that has exhibited anti-inflammatory activity via inhibition of NF-κB activation, although its potential effects on inflammasome activation remain unexplored. This study aims to elucidate whether DIH modulates NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome in macrophages. Our findings show that DIH inhibited NLRP3 activation triggered by Nigericin (Nig), adenosine triphosphate (ATP) and monosodium urate (MSU) crystals, indicating broad inhibitory effects. DIH significantly attenuated caspase-1 activation and secretion of the interleukin-1β (IL-1β) in J774A.1 cells. Interestingly, the protein expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), pro-caspase-1 and pro-IL-1β were not affected by DIH treatment. Furthermore, we found that DIH pretreatment also inhibited the lipopolysaccharide (LPS)-induced NLRP3 inflammasome priming stage. In addition, DIH alleviated pyroptosis mediated by NLRP3 inflammasome activation. Similar results on IL-1β release were observed in Nig-activated bone marrow-derived macrophages (BMDMs). Covalent molecular docking analysis revealed that DIH fits well into the ATP-binding site of NLRP3 protein, forming a covalent bond with Cys415. In conclusion, our experiments show that DIH is an effective NLRP3 inflammasome inhibitor and provide new evidence for its application in the therapy of inflammation-related diseases.

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more ‘open’ allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P–31P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

SY31-1 Molecular targeted therapy with FLT3 inhibitor in AML

Two main types of FLT3 mutations are commonly identified in AML, internal tandem duplications (ITD) and kinase domain (KD) mutations. FLT3 mutations result in constitutive kinase activation and are associated with poor outcome. FLT3 inhibitors (FLT3i) include type 1 inhibitors (e.g., gilteritinib, lestaurtinib, midostaurin, crenolanib) that bind to the ATP binding site in both its active and inactive configuration, inhibiting both ITD and KD mutations. Type 2 inhibitors (e.g., quizartinib, sorafenib, ponatinib) bind to a hydrophobic domain adjacent to the ATP binding are only accessible in its inactive configuration.

Triiodothyronine Acts as a Smart Influencer on Hsp90 via a Triiodothyronine Binding Site.

Microarray-based experiments revealed that thyroid hormone triiodothyronine (T3) enhanced the binding of Cy5-labeled ATP on heat shock protein 90 (Hsp90). By molecular docking experiments with T3 on Hsp90, we identified a T3 binding site (TBS) near the ATP binding site on Hsp90. A synthetic peptide encoding HHHHHHRIKEIVKKHSQFIGYPITLFVEKE derived from the TBS on Hsp90 showed, in MST experiments, the binding of T3 at an EC50 of 50 μM. The binding motif can influence the activity of Hsp90 by hindering ATP accessibility or the release of ADP.

Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect.

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure- and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H+ current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic β-cells, immune cells, and cancer cells.

Structure Characterization of Escherichia coli Pseudouridine Kinase PsuK.

Pseudouridine (Ψ) is one of the most abundant RNA modifications in cellular RNAs that post-transcriptionally impact many aspects of RNA. However, the metabolic fate of modified RNA nucleotides has long been a question. A pseudouridine kinase (PsuK) and a pseudouridine monophosphate glycosylase (PsuG) in Escherichia coli were first characterized as involved in pseudouridine degradation by catalyzing the phosphorylation of pseudouridine to pseudouridine 5′-phosphate (ΨMP) and further hydrolyzing 5′-ΨMP to produce uracil and ribose 5′-phosphate. Recently, their homolog proteins in eukaryotes were also identified, which were named PUKI and PUMY in Arabidopsis. Here, we solved the crystal structures of apo-EcPsuK and its binary complex with Ψ or N1-methyl-pseudouridine (m1Ψ). The structure of EcPsuK showed a homodimer conformation assembled by its β-thumb region. EcPsuK has an appropriate binding site with a series of hydrophilic and hydrophobic interactions for Ψ. Moreover, our complex structure of EcPsuK-m1Ψ suggested the binding pocket has an appropriate capacity for m1Ψ. We also identified the monovalent ion-binding site and potential ATP-binding site. Our studies improved the understanding of the mechanism of Ψ turnover.

Molecular Pharmacology of P2X Receptors: Exploring Druggable Domains Revealed by Structural Biology.

Extracellular ATP is a critical signaling molecule that is found in a wide range of concentrations across cellular environments. The family of nonselective cation channels that sense extracellular ATP, termed P2X receptors (P2XRs), is composed of seven subtypes (P2X1-P2X7) that assemble as functional homotrimeric and heterotrimeric ion channels. Each P2XR is activated by a distinct concentration of extracellular ATP, spanning from high nanomolar to low millimolar. P2XRs are implicated in a variety of physiological and pathophysiological processes in the cardiovascular, immune, and central nervous systems, corresponding to the spatiotemporal expression, regulation, and activation of each subtype. The therapeutic potential of P2XRs is an emerging area of research in which structural biology has seemingly exceeded medicinal chemistry, as there are several published P2XR structures but currently no FDA-approved drugs targeting these ion channels. Cryogenic electron microscopy is ideally suited to facilitate structure-based drug design for P2XRs by revealing and characterizing novel ligand-binding sites. This review covers structural elements in P2XRs including the extracellular orthosteric ATP-binding site, extracellular allosteric modulator sites, channel pore, and cytoplasmic substructures, with an emphasis on potential therapeutic ligand development.

Acridone Derivatives from Atalantia monophyla Inhibited Cancer Cell Proliferation through ERK Pathway.

The present study aimed to investigate the effect of acridone alkaloids on cancer cell lines and elucidate the underlying molecular mechanisms. The ten acridone alkaloids from Atalantia monophyla were screened for cytotoxicity against LNCaP cell lines by a WST-8 assay. Then, the most potential acridone, buxifoliadine E, was evaluated on four types of cancer cells, namely prostate cancer (LNCaP), neuroblastoma (SH SY5Y), hepatoblastoma (HepG2), and colorectal cancer (HT29). The results showed that buxifoliadine E was able to significantly inhibit the proliferation of all four types of cancer cells, having the most potent cytotoxicity against the HepG2 cell line. Western blotting analysis was performed to assess the expression of signaling proteins in the cancer cells. In HepG2 cells, buxifoliadine E induced changes in the levels of Bid as well as cleaved caspase-3 and Bax through MAPKs, including Erk and p38. Moreover, the binding interaction between buxifoliadine E and Erk was investigated by using the Autodock 4.2.6 and Discovery Studio programs. The result showed that buxifoliadine E bound at the ATP-binding site, located at the interface between the N- and C-terminal lobes of Erk2. The results of this study indicate that buxifoliadine E suppressed cancer cell proliferation by inhibiting the Erk pathway.

In silico screening of c-Met tyrosine kinase inhibitors targeting nucleotide and drug-substrate binding sites of ABCB1 as potential MDR reversal agents

Purpose Cancer is a significant public health problem and ranks as a leading cause of death globally. Multidrug resistance (MDR) affects the therapeutic potential of conventional chemotherapeutic agents in cancer chemotherapy. Receptor tyrosine kinases (RTKs) are enzymes whose aberrant activation contributes to the tumorigenesis of various types of cancers. The ability of several RTKs, such as c-Met, to reverse ABC transporters mediated MDR was shown before. We aimed to explore the ability of c-Met inhibitors to circumvent MDR in cancer by inhibiting the ABCB1 transporter using in silico studies. Methods Docking virtual screening of several potent and structurally diverse c-Met inhibitors were applied to find repurposed candidates to target the ATP binding sites and drug-substrate binding pockets of the ABCB1 transporter. The selected candidate was subjected to molecular dynamics simulations. Results Based on docking findings, among 19 clinical c-Met inhibitors, several drugs, particularly golvatinib, exerted the affinity to both ATP binding sites in the nucleotide-binding domains (NBDs) as well as the drug-substrate binding site in the transmembrane domains (TMDs). Moreover, several non-clinical c-Met inhibitors obtained from the ChEMBL database had strong interactions with TMDs and NBDs, among which CHEMBL1950194 and CHEMBL2385194 compounds showed the highest binding affinity, respectively. Additionally, as a potential repositioning drug, MD simulation studies of golvatinib, corroborated the docking results. Conclusion We applied docking and molecular dynamics simulations to screen the potential c-Met inhibitors as the MDR reversing agents targeting ATP and drug-substrate binding sites, and the results suggested several repurposed candidate drugs.

Discovery of CC-99677, a selective targeted covalent MAPKAPK2 (MK2) inhibitor for autoimmune disorders

As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (SNAr) mechanism. This irreversible mechanism translates biochemical potency to cells shown by potent inhibition of heat shock protein 27 (HSP27) phosphorylation in LPS-activated monocytic THP-1 cells. The cytokine inhibitory profile of CC-99677 differentiates it from known p38 inhibitors, potentially suppressing a p38 pathway inflammatory response while avoiding tachyphylaxis. Dosed orally, CC-99677 is efficacious in a rat model of ankylosing spondylitis. Single doses, 3 to 400 mg, in healthy human volunteers show linear pharmacokinetics and apparent sustained tumor necrosis factor-α inhibition, with a favorable safety profile. These results support further development of CC-99677 for autoimmune diseases like ankylosing spondylitis.