Single Binding Site Research Articles

Transport mechanism and structural pharmacology of human urate transporter URAT1

Urate is an endogenous product of purine metabolism in the liver. High urate levels in the blood lead to gout, a very common and painful inflammatory arthritis. Excreted urate is reabsorbed in the kidney mainly by URAT1 antiporter, a key target for anti-gout drugs. To uncover the mechanisms of urate transport and drug inhibition, we determined cryo-EM structures of human URAT1 with urate, counter anion pyrazinoate, or anti-gout drugs of different chemotypes — lesinurad, verinurad, and dotinurad. We captured the outward-to-inward transition of URAT1 during urate uptake, revealing that urate binds in a phenylalanine-rich pocket and engages with key gating residues to drive the transport cycle. In contrast to the single binding site for urate, pyrazinoate interacts with three distinct, functionally relevant sites within URAT1, a mechanism that has not yet been observed in other anion antiporters. In addition, we found that while all three drugs compete with substrates and halt the transport cycle, verinurad and dotinurad further hijack gating residues to achieve high potency. These insights advance our understanding of organic anion transport and provide a foundation for designing improved gout therapeutics.

Multi-target anti-diabetic styrylpyrones from Phellinus igniarius: Inhibition of α-glucosidase, protein glycation, and oxidative stress

Bioactivity screening revealed that the EtOAc extract from the culture broth of Phellinus igniarius SY489 exhibited remarkable α-glucosidase inhibitory activity, with an IC50 value of 1.92 μg/mL. Activity- and ultraviolet (UV) profile-guided isolation led to the discovery of four anti-diabetic styrylpyrones (1–4), including two novel compounds, phelignidins A (1) and B (2). Compounds 1 and 2 represent a rare structural type of styrylpyrone dimer, in which one of the pyrone moieties exists in an open-ring state. The absolute configurations of the new compounds 1 and 2, as well as the previously unresolved compound 3, were established. Compounds 1–4 were effective in α-glucosidase inhibition, anti-glycation, and antioxidant assays, surpassing or being comparable to the positive control drugs, with minimal cytotoxicity. Furthermore, studies on α-glucosidase inhibition mechanisms suggested that these compounds interact with α-glucosidase at a single binding site, causing secondary structure unfolding and exerting inhibitory activity via a mixed-type mechanism. These results provide an important basis for developing novel, low-toxicity, multi-target anti-diabetic drugs from edible and medicinal fungi.

Ketanserin exhibits dose- and concentration-proportional serotonin 2A receptor occupancy in healthy individuals: Relevance for psychedelic research

The serotonin 2A (5-HT2A) receptor is an important target for drug development and the main receptor through which classical psychedelics elucidate their hallucinogenic effects. The 5-HT2A receptor antagonist ketanserin has frequently been used as a tool to block the receptor.Here, we establish the dose-occupancy relation of ketanserin and the cerebral 5-HT2A receptor in healthy participants by conducting a positron emission tomography (PET) study. 120-min PET scans using the 5-HT2A receptor agonist radiotracer [11C]Cimbi-36 were conducted at baseline and after oral doses of either 10, 20, or 40 mg of ketanserin; each participant underwent one or two scans after ketanserin administration. Occupancy was defined as the percent change in neocortex binding potential (BPND), estimated using the simplified reference tissue model (SRTM) with the cerebellum as reference region.Peroral ketanserin intake resulted in a plasma concentration-related increase in cerebral 5-HT2A receptor occupancy with the highest plasma ketanserin concentrations measured after ∼2 h. The relation between mean plasma ketanserin concentrations and 5-HT2A receptor occupancy conformed to a single-site binding model with an estimated EC50 (95 % CI) of 2.52 (0.75; 8.1) ng/mL, which corresponds to a peroral dose of ketanserin of approximately 10 mg.These data elucidate for the first time in humans the cerebral pharmacodynamics of ketanserin, both benefitting its use as a pharmacological tool for probing brain function and adding to its potential for therapeutic use in rescuing a bad psychedelic experience.

Separation and purification of bovine nasal cartilage-derived chondroitin sulfate and evaluation of its binding to bovine serum albumin

This study employs an optimized and environmentally friendly method to extract and purify chondroitin sulfate (CS) from bovine nasal cartilage using enzymatic hydrolysis, ethanol precipitation, and DEAE Sepharose Fast Flow column chromatography. The extracted CS, representing 44.67 % ± 0.0016 of the cartilage, has a molecular weight of 7.62 kDa. Characterization through UV, FT-IR, NMR spectroscopy, and 2-aminoacridone derivatization HPLC revealed a high content of sulfated disaccharides, particularly ΔDi4S (73.59 %) and ΔDi6S (20.61 %). Interaction studies with bovine serum albumin (BSA) using fluorescence spectroscopy and molecular docking confirmed a high-affinity, static quenching interaction with a single binding site, primarily mediated by van der Waals forces and hydrogen bonding. The interaction did not significantly alter the polarity or hydrophobicity of BSA aromatic amino acids. These findings provide a strong foundation for exploring the application of CS in tissue engineering and drug delivery systems, leveraging its unique interaction with BSA for targeted delivery and enhanced efficacy.

Multispectroscopic and Theoretical Investigation on the Binding Interaction of a Neurodegenerative Drug, Lobeline with Human Serum Albumin: Perturbation in Protein Conformation and Hydrophobic-Hydrophilic Surface.

Lobeline (LOB), a naturally occurring alkaloid, has a broad spectrum of pharmacological activities and therapeutic potential, including applications in central nervous system disorders, drug misuse, multidrug resistance, smoking cessation, depression, and epilepsy. LOB represents a promising compound for developing treatments in various medical fields. However, despite extensive pharmacological profiling, the biophysical interaction between the LOB and proteins remains largely unexplored. In the current article, a range of complementary photophysical and cheminformatics methodologies were applied to study the interaction mechanism between LOB and the carrier protein HSA. Steady-state fluorescence and fluorescence lifetime experiments confirmed the static-quenching mechanisms in the HSA-LOB system. "K" (binding constant) of the HSA-LOB system was determined to be 105 M-1, with a single preferable binding site in HSA. The forces governing the HSA-LOB stable complex were analyzed by thermodynamic parameters and electrostatic contribution. The research also investigated how various metal ions affect complex binding. Site-specific binding studies depict Site I as probable binding in HSA by LOB. We conducted synchronous fluorescence, 3D fluorescence, and circular dichroism studies to explore the structural alteration occurring in the microenvironment of amino acids. To understand the robustness of the HSA-LOB complex, we used theoretical approaches, including molecular docking and MD simulations, and analyzed the principal component analysis and free energy landscape. These comprehensive studies of the structural features of biomolecules in ligand binding are of paramount importance for designing targeted drugs and delivery systems.

The fully reduced terminal oxidase bd-I isolated from Escherichia coli binds cyanide

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b558, b595 and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d2+ in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d2+ α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (Kd) of the cyanide complex of heme d2+ is ∼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d2+ but not to b5582+ or b5952+. This agrees with the published structural data that the enzyme's active site is not a di-heme site. The observed rate of binding (kobs) increases as the concentration of cyanide is increased, giving a second-order rate constant (kon) of ∼0.1 M−1 s−1.

Assessment of cerebral drug occupancy in humans using a single PET-scan: A [11C]UCB-J PET study

PurposeHere, we evaluate a PET displacement model with a Single-step and Numerical solution in healthy individuals using the synaptic vesicle glycoprotein (SV2A) PET-tracer [11C]UCB-J and the anti-seizure medication levetiracetam (LEV). We aimed to (1) validate the displacement model by comparing the brain LEV-SV2A occupancy from a single PET scan with the occupancy derived from two PET scans and the Lassen plot and (2) determine the plasma LEV concentration-SV2A occupancy curve in healthy individuals.MethodsEleven healthy individuals (five females, mean age 35.5 [range: 25–47] years) underwent two 120-min [11C]UCB-J PET scans where an LEV dose (5–30 mg/kg) was administered intravenously halfway through the first PET scan to partially displace radioligand binding to SV2A. Five individuals were scanned twice on the same day; the remaining six were scanned once on two separate days, receiving two identical LEV doses. Arterial blood samples were acquired to determine the arterial input function and plasma LEV concentrations. Using the displacement model, the SV2A-LEV target engagement was calculated and compared with the Lassen plot method. The resulting data were fitted with a single-site binding model.ResultsSV2A occupancies and VND estimates derived from the displacement model were not significantly different from the Lassen plot (p = 0.55 and 0.13, respectively). The coefficient of variation was 14.6% vs. 17.3% for the Numerical and the Single-step solution in Bland-Altman comparisons with the Lassen plot. The average half maximal inhibitory concentration (IC50), as estimated from the area under the curve of the plasma LEV concentration, was 12.5 µg/mL (95% CI: 5–25) for the Single-Step solution, 11.8 µg/mL (95% CI: 4–25) for the Numerical solution, and 6.3 µg/mL (95% CI: 0.08-21) for the Lassen plot. Constraining Emax to 100% did not significantly improve model fits.ConclusionPlasma LEV concentration vs. SV2A occupancy can be determined in humans using a single PET scan displacement model. The average concentration of the three computed IC50 values ranges between 6.3 and 12.5 µg/mL. The next step is to use the displacement model to evaluate LEV occupancy and corresponding plasma concentrations in relation to treatment efficacy.Clinical trial registrationNCT05450822. Retrospectively registered 5 July 2022 https://clinicaltrials.gov/ct2/results? term=NCT05450822&Search=Search.

Abstract 3081: Competitive Inhibition Of Tlt-1/fibrinogen Molecular Interaction Disrupts Platelet Function In Ex Vivo Platelet Assays

Background: Platelets facilitate thrombosis, hemostasis, and immunity through their interaction with fibrinogen. Fibrinogen is the ligand for the platelet receptors αIIbβ3 and T REM- L ike T ranscript-1 (TLT-1), TLT-1 is an Ig-domain containing receptor released from platelet α-granules upon platelet activation. Unlike αIIbβ3, little is known about the TLT-1/fibrinogen interaction. Aims: Delineate the TLT-1/fibrinogen molecular interaction and determine if, peptides representing this interaction can regulate platelet function and inflammatory hemostasis. Methods: Octet Qke Bio-layer Interferometry and atomic force microscopy (AFM) were used to evaluate soluble TLT-1 binding to fibrinogen. Immunoprecipitation and a peptide array were used to determine which fibrinogen peptides bound TLT-1. Flow cytometry using FITC-labelled fibrinogen binding to activated human platelets and human platelet aggregometry in the presence of these peptides were used in competitive inhibition assays. Additionally, competitive ELISAs using peptides representing regions the TLT-1 extracellular domain incubated with soluble TLT-1 in a fibrinogen coated wells were used to identify the region(s) on TLT-1 interaction with fibrinogen. Results: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1/Fibrinogen interaction. AFM suggests that sTLT-1 mediates fibrinogen polymerization. Competitive inhibition assays suggest complementary determining loop-1 and LP-17 (a 17 amino acid region of TLT-1) mediate TLT-1’s interaction to fibrinogen. The 3D modeling of our identified peptides show that they form a binding complex on the B module of the D-domain rather than a single binding site. These peptides competitively reduce platelet aggregation in aggregation assays and decrease fibrinogen binding to platelets in flow cytometry assays compared to controls. Cell binding and clotting assays are underway and the current status of projected will be reported here. Conclusions: TLT-1 uses LP17 and CDR-1 to interact with the newly identified TLT-1 binding complex on fibrinogen. This TLT-1 binding surface may represent a targetable intervention to regulate thrombosis and inflammation.

Elucidating the Mechanism of Copper-Induced Photoluminescence Quenching in 2-Phenylbenzimidazole-5-Sulfonic Acid.

To explore the possible impact of 2-Phenylbenzimidazole-5-sulfonic acid (PBSA) on the function of a sunscreen, in this work we investigate the binding of copper metal ions (Cu2+) to PBSA. Due to the existence of an intrinsic interaction phenomenon between Cu2+ ions and PBSA molecules, the photoluminescence (PL) quenching arises owing to the charge transfer from PBSA to Cu2+ ions. The mechanism of fluorescence quenching is probed experimentally following excitation at 306 nm by evaluating various quenching parameters with the help of the Stern-Volmer plot. Through the assessment of the values of the Stern-Volmer constant ( ) and bimolecular quenching rate constant ( ), it is deduced that the dynamic mode of PL quenching is operative between PBSA and Cu2+ ions. We evaluate the number of binding sites (n = 1) that advocate the presence of a single binding site in PBSA for Cu2+ ions. The numerical value of standard Gibbs free energy change, ~ -27.485 kJ.mol-1 implies the spontaneous binding between Cu2+ ions and PBSA molecules. The results obtained give an insight into the mechanism of metal-induced PL quenching of water soluble PBSA sunscreen.

Improved charge extraction in inverted perovskite solar cells with dual-site-binding ligands.

Inverted (pin) perovskite solar cells (PSCs) afford improved operating stability in comparison to their nip counterparts but have lagged in power conversion efficiency (PCE). The energetic losses responsible for this PCE deficit in pin PSCs occur primarily at the interfaces between the perovskite and the charge-transport layers. Additive and surface treatments that use passivating ligands usually bind to a single active binding site: This dense packing of electrically resistive passivants perpendicular to the surface may limit the fill factor in pin PSCs. We identified ligands that bind two neighboring lead(II) ion (Pb2+) defect sites in a planar ligand orientation on the perovskite. We fabricated pin PSCs and report a certified quasi-steady state PCE of 26.15 and 24.74% for 0.05- and 1.04-square centimeter illuminated areas, respectively. The devices retain 95% of their initial PCE after 1200 hours of continuous 1 sun maximum power point operation at 65°C.

Exploring the interaction of a potent anti-cancer drug Selumetinib with bovine serum albumin: Spectral and computational attributes.

The binding of drugs to plasma proteins determines its fate within the physiological system, hence profound understanding of its interaction within the bloodstream is important to understand its pharmacodynamics and pharmacokinetics and thereby its therapeutic potential. In this regard, our work delineates the mechanism of interaction of Selumetinib (SEL), a potent anti-cancer drug showing excellent effect against multiple solid tumors, with plasma protein bovine serum albumin (BSA), using methods such as absorption, steady-state fluorescence, time-resolved, fluorescence resonance energy transfer, Fourier transform infrared spectra (FTIR), circular dichroism (CD), synchronous and 3D-fluorescence, salt fluorescence, molecular docking and molecular dynamic simulations. The BSA fluorescence intensity was quenched with increasing concentration of SEL which indicates interactions of SEL with BSA. Stern-Volmer quenching analysis and lifetime studies indicate the involvement of dynamic quenching. However, some contributions from the static quenching mechanism could not be ruled out unambiguously. The association constant was found to be 5.34 × 105 M-1 and it has a single binding site. The Förster distance (r) indicated probable energy transmission between the BSA and SEL. The positive entropy changes and enthalpy change indicate that the main interacting forces are hydrophobic forces, also evidenced by the results of molecular modeling studies. Conformation change in protein framework was revealed from FTIR, synchronous and 3D fluorescence and CD studies. Competitive binding experiments as well as docking studies suggest that SEL attaches itself to site I (subdomain IIA) of BSA where warfarin binds. Molecular dynamic simulations indicate the stability of the SEL-BSA complex. The association energy between BSA and SEL is affected in the presence of different metals differently.

Supramolecular Modulation of Fluid Flow in a Self-Powered Enzyme Micropump.

Regulating macroscopic fluid flow by catalytic harnessing of chemical energy could potentially provide a solution for powerless microfluidic devices. Earlier reports have shown that surface-anchored enzymes can actuate the surrounding fluid in the presence of the respective substrate in a concentration-dependent manner. It is also crucial to have control over the flow speed of a self-powered enzyme micropump in various applications where controlled dosing and mixing are required. However, modulating the flow speed independent of the fuel concentration remains a significant challenge. In a quest to regulate the fluid flow in such a system, a supramolecular approach has been adopted, where reversible regulation of enzyme activity was achieved by a two-faced synthetic receptor bearing sulfonamide and adamantane groups. The bovine carbonic anhydrase (BCA) enzyme containing a single binding site favorable to the sulfonamide group was used as a model enzyme, and the enzyme activity was inhibited in the presence of the two-faced inhibitor. The same effect was reflected when the immobilized enzyme was used as an engine to actuate the fluid flow. The flow velocity was reduced up to 53% in the presence of 100 μM inhibitor. Later, upon addition of a supramolecular "host" CB[7], the inhibitor was sequestered from the enzyme due to the higher binding affinity of CB[7] with the adamantane functionality of the inhibitor. As a result, the flow velocity was restored to ∼72%, thus providing successful supramolecular control over a self-powered enzyme micropump.

Probing the interaction mechanism of SDBS with AtPrxQ from Arabidopsis thaliana: Insight into the molecular toxicity to plants

As the most universally used anionic surfactant, ubiquitous existence and accumulation of sodium dodecyl benzene sulfonate (SDBS) in the environment has inevitably imposed the associated harmful impacts to plants due to producing excessive reactive oxygen species. However, the underlying hazardous mechanism of the SDBS-induced oxidative stress to plants at molecular level has never been reported. Here, the molecular interaction of AtPrxQ with SDBS was explored for the first time. The intrinsic fluorescence of AtPrxQ was quenched based on static quenching, and a single binding site of AtPrxQ towards SDBS and the potential interaction forces driven by hydrophobic interactions were predicted from thermodynamic parameters and molecular docking results. Besides, the interaction pattern of AtPrxQ and SDBS was also confirmed by the bio-layer interferometry with moderate binding affinity. Moreover, the structural changes of AtPrxQ along with the destructions of the protein framework and the hydrophobic enhancement around aromatic amino acids were observed upon binding with SDBS. At last, the toxic effects produced by SDBS on peroxidase activities and Arabidopsis seedlings growth were also characterized. Thus this work may provide insights on the molecular interactions of AtPrxQ with SDBS and assessments on the biological hazards of SDBS to plants even for the agriculture.

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA.

Timely initiation of chromosomal DNA replication in Escherichia coli is achieved by cell cycle-coordinated regulation of the replication origin, oriC, and the replication initiator, ATP-DnaA. Cellular levels of ATP-DnaA increase and peak at the time for initiation at oriC, after which hydrolysis of DnaA-bound ATP causes those to fall, yielding initiation-inactive ADP-DnaA. This hydrolysis is facilitated by the chromosomal locus datA located downstream of the tRNA-Gly (glyV-X-Y) operon, which possesses a cluster of DnaA-binding sequences and a single binding site (IBS) for the DNA bending protein IHF (integration host factor). While IHF binding activates the datA function and is regulated to occur specifically at post-initiation time, the underlying regulatory mechanisms remain obscure. Here, we demonstrate that datA-IHF binding at pre-initiation time is down-regulated depending on the read-through transcription of datA IBS initiated at the glyV-X-Y promoter. During the cell cycle, the level of read-through transcription, but not promoter activity, fluctuated in a manner inversely related to datA-IHF binding. Transcription from the glyV-X-Y promoter was predominantly interrupted at datA IBS by IHF binding. The terminator/attenuator sequence of the glyV-X-Y operon, as well as DnaA binding within datA overall, contributed to attenuation of transcription upstream of datA IBS, preserving the timely fluctuation of read-through transcription. These findings provide a mechanistic insight of tRNA transcription-dependent datA-IHF regulation, in which an unidentified factor is additionally required for the timely datA-IHF dissociation, and support the significance of datA for controlling the cell cycle progression as a connecting hub of tRNA production and replication initiation.

The Nicotinic Acetylcholine Receptor and Its Pentameric Homologs: Toward an Allosteric Mechanism of Signal Transduction at the Atomic Level.

The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.

Elucidating the influence of some metal ions on the binding of antimalarial drugs to human serum albumin: spectroscopic approach

Influence of six metal ions viz. Mg2+, K+, Ca2+, Mn2+, Cu2+ and Ba2+ on the binding affinity of human serum albumin (HSA) towards three antimalarial drugs, namely, sulfadoxine (SDN), mefloquine (MEF) and lumefantrine (LUM) was investigated using fluorescence quenching titrations. The effects of metal ions (Mn2+, Cu2+ and Ba2+) were similar on the affinity of HSA towards SDN, MEF and LUM. Values of the association constant, Ka, for SDN/MEF/LUM ̶ HSA interactions in the presence of these metal ions, decreased significantly in the order: Mn2+ > Cu2+ > Ba2+. On the other hand, the presence of Mg2+, K+, and Ca2+ affected the binding affinity for these drugs differently. Whereas a slight increase in the Ka value of the SDN ̶ HSA complex was seen in the presence of Mg2+, Ca2+ produced a similar effect on the LUM ̶ HSA system. For MEF ̶ HSA and LUM ̶ HSA systems, effects of K+ and Mg2+ were similar, showing a decrease in the Ka value, being more effective with K+; however, this decrease was more significant than Mn2+, Cu2+ and Ba2+. Values of ‘n’ remained approximately equal to 1, hinting at a single binding site for each drug.

Multivalent Calixarene Complexation of a Designed Pentameric Lectin.

We describe complex formation between a designed pentameric β-propeller and the anionic macrocycle sulfonato-calix[8]arene (sclx8), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15N HSQC experiments reveal a single calixarene binding site in the concave pocket of the β-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.

Investigating the binding of caffeic acid to human hemoglobin using multispectroscopic and molecular docking approaches

Caffeic acid (CA) is a hydroxycinnamic acid derivative that possess a wide variety of biological properties and is non-toxic to humans. The binding mechanism of CA with human hemoglobin (HHb) was studied for the first time using multispectroscopic and molecular docking methods. The UV–visible absorption spectra showed that CA interacts with HHb through the formation of ground state complex. The fluorescence studies revealed that CA quenches HHb fluorescence by the static mechanism and confirmed the presence of a single CA binding site on the HHb molecule. The negative values of thermodynamics parameters (ΔH,ΔSand ΔG) showed that the binding of CA to HHb is a spontaneous and exothermic process. Hydrogen bonds and van der Waal interactions are the major intermolecular forces that stabilize the HHb-CA complex. Synchronous fluorescence experiments showed that binding of CA altered the microenvironment around tryptophan residues of HHb. Fluorescence resonance energy transfer studies indicated a high propensity of energy transfer from tryptophan residues of HHb to CA. Circular dichroism spectra and catalytic esterase activity confirmed that only minor changes occur in the secondary structure of HHb in the presence of CA. Molecular docking study identified the HHb amino acid residues involved in binding to CA. Analysis of the binding mode of CA with HHb represents an important step in elucidating the molecular mechanism of the protection of HHb from oxidative damage. This will help in understanding the potential role of CA and related antioxidants as therapeutic agents in various blood-related diseases.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M−1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M−1. In the plot of ΔH-T*ΔS, the of Enthalpy−Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

Multitracer Approach to Understanding the Complexity of Reactive Astrogliosis in Alzheimer's Brains.

A monoamine oxidase B (MAO-B) selective positron emission tomography (PET) tracer [11C]-deuterium-l-deprenyl holds promise for imaging reactive astrogliosis in neurodegenerative diseases, such as Alzheimer's disease (AD). Two novel PET tracers ([11C]-BU99008 and [18F]-SMBT-1) have recently been developed to assess the complexity of reactive astrogliosis in the AD continuum. We have investigated the binding properties of SMBT-1, l-deprenyl, and BU99008 in AD and cognitively normal control (CN) brains. Competition binding assays with [3H]-l-deprenyl and [3H]-BU99008 versus unlabeled SMBT-1 in postmortem AD and CN temporal and frontal cortex brains demonstrated that SMBT-1 interacted with [3H]-deprenyl at a single binding site (nM range) and with [3H]-BU99008 at multiple binding sites (from nM to μM). Autoradiography studies on large frozen postmortem AD and CN hemisphere brain sections demonstrated that 1 μM SMBT-1 almost completely displaced the [3H]-l-deprenyl binding (>90%), while SMBT-1 only partly displaced the [3H]-BU99008 binding (50-60% displacement) in cortical regions. In conclusion, SMBT-1, l-deprenyl, and BU99008 interact at the same MAO-B binding site, while BU99008 shows an additional independent binding site in AD and CN brains. The high translational power of our studies in human AD and CN brains suggests that the multitracer approach with SMBT-1, l-deprenyl, and BU99008 could be useful for imaging reactive astrogliosis.