Association Constants For Binding Research Articles

Binding Mechanisms and Therapeutic Activity of Heterocyclic Substituted Arylazothioformamide Ligands and Their Cu(I) Coordination Complexes.

Finding new sources of biologically active compounds for anticancer or antimicrobial therapies remains an active area of research. Azothioformamides (ATFs) with a 1,3 N=N-C=S heterodiene backbone are a new class of biologically active compounds that chelate metals (e.g., Cu) forming stable ATF metal coordination complexes. In this study, ATF ligands were prepared with pyrrolidine, piperidine, N-methylpiperazine, and morpholine substituents on the formamide as to add more heterocyclic drug-like character for biological studies. Formamide derivatives were then complexed with various Cu(I) salts to form coordination complexes. Cu(I) salts were selected as to create potential bioactive compounds with less toxicity. Binding association constants of each Cu(I) salt to ATF ligands were extrapolated from UV-vis titration studies and were corroborated with DFT calculations using a hybrid functional B3LYP method. It was observed that the smaller pyrrolidine functionalized ATFs bound to the Cu(I) salts had stronger binding than any of the larger six-membered-ring heterocycles with association values in the 104 - 105 M-1 range. The ATF-Cu(I) salt coordination complexes were then evaluated for antimicrobial activity against two bacteria (Staphylococcus aureus, Escherichia coli), one yeast (Candida albicans), four human cancer lines (A-549, K-562, HT-1080, MDA-MB-231), and two normal human lines (MRC-5, HFF). The ATF ligands themselves were inactive against all microbes and most human lines except K-562 cells, which were sensitive to three of the four ligands (IC50's = 7.0-25.5 μM). Most ATF-Cu(I) complexes showed low to medium micromolar activity against Candida albicans (IC50's 2.6-24.8 μM) and Staphylococcus aureus (IC50's = 3.4-37.7 μM), with increasing activity corresponding to complexes with higher binding association constants. The antiproliferative properties of ATF-Cu(I) metal salt complexes against mammalian cells were mixed, with low to medium micromolar activity across all cell lines. Notably, several ATF-Cu(I) salt coordination complexes showed submicromolar activity against the HT-1080 fibrosarcoma line (0.52-0.69 μM). The results demonstrate promising activity of ATF-Cu(I) complexes, particularly with pyrrolidine as the formamide component. These studies suggest that the stronger binding association values correlate to higher levels of biological activity.

Inhibitory mechanism of arabinoxylan and β-glucan from hull-less barley on α-amylase activity: A comparison study

The inhibitory mechanisms of arabinoxylan (HBAX) and β-glucan (HBBG) from hull-less barley against pancreatic α-amylase were investigated using enzymatic inhibitory kinetics, multi-spectroscopy and thermodynamic analysis. Results indicated that both HBAX and HBBG effectively inhibited α-amylase activity, with HBAX exhibiting superior potency (IC50 of 3.26 mg/mL) compared to HBBG (IC50 of 13.23 mg/mL). Kinetic analysis revealed that HBAX displayed competitive and noncompetitive mixed inhibition, while HBBG predominantly exhibited noncompetitive inhibition. Interaction studies demonstrated that both HBAX and HBBG could spontaneously interact with α-amylase through hydrogen bonding and van der Waals forces, leading to structural adjustments of α-amylase, albeit without notable secondary structure alterations. However, HBAX showed stronger fluorescence quenching ability and microenvironmental modifications of α-amylase compared to HBBG. Bio-layer interferometry confirmed the stronger binding ability of HBAX to α-amylase, characterized by higher association binding constant (Ka) and lower dissociation constant (Kd) and affinity constant (KD). The lower equilibrium dissociation constant (K) and greater enthalpy change (ΔH) of HBAX to α-amylase further proved its stronger interaction with α-amylase. These differences were ascribed that HBAX inhibited α-amylase activity potentially through starch competition or physical interference, while HBBG might primarily act as physical barriers. This study enhances understanding of HBAX and HBBG in lowering starch digestion from an enzyme-inhibitory perspective.

Impact of bound ssRNA length on allostery in the Dengue Virus NS3 helicase.

The presence of ATP is known to stimulate helicase activity of the Dengue Virus Non-structural protein 3 helicase (NS3h), and the presence of RNA stimulates NS3h ATPase activity, however this coupling is still mechanistically unclear. Here we use atomistic models and molecular dynamics simulations to evaluate the single-stranded RNA(ssRNA)-length dependence of the NS3h-ssRNA binding affinity and its modulation by bound ATP. Considering complexes with 7, 11, 16and 26 nucleotides (nts), we observe that both the binding affinity and its modulation by bound ATP are augmented with increased ssRNA lengths. In models with at least 11 nts bound, the binding of ATP results in a shift from a tightly bound to a weakly bound state. We find that the weakly bound state persists during both the ADP-Pi- and ADP-bound stages of the catalytic cycle. We obtain the equilibrium association constants for NS3h binding to an ssRNA 10-mer in vitro, both in the absence and presence of ADP, which further support the alternation between tightlyand weaklybound states during the catalytic cycle. The length of bound ssRNA is critical for understanding the NS3h-RNA interaction as well as how it is modulated during the catalytic cycle.

Calorimetric analysis of AdcR and its interactions with zinc(II) and DNA

Zinc(II) ions play critical roles in all known life as structurally important stabilizing ions in proteins, catalytically active metals in enzymes, and signaling agents impacting physiological changes. To maintain homeostasis, the intracellular concentration of zinc(II) is strictly controlled by a family of metal-regulatory proteins in both prokaryotic and eukaryotic organisms. In S. pneumoniae, there are two proteins that share responsibility for Zn2+ homeostasis, one of them is the Adhesin Competence Repressor (AdcR) and it binds to a specific double-stranded DNA binding domain (dsDNA). AdcR has been structurally characterized containing two zinc(II) metal centers per monomeric unit. Here we report data collected from differential scanning calorimetry (DSC) experiments aimed to measure the structural stability of AdcR, the fully complimented Zn2AdcR complex, and the protein/DNA complex Zn2AdcR/dsDNA. Thermograms collected from DSC experiments yielded endothermic unfolding events for AdcR, Zn2AdcR, and Zn2AdcR/dsDNA complex at 55.6, 70.2, and 56.6 °C, respectively. A non-two state unfolding model best fits the data, giving ΔH terms associated with these thermal unfolding events of 5.1, 7.1, and 4.9 kcal/mol. These data allow for the development of a thermodynamic cycle connecting both zinc(II) and DNA binding to AdcR. Furthermore, pairing this newly reported data with known association constants for zinc(II) and DNA binding allowed for the generation of thermodynamic profiles for both zinc(II) binding to AdcR and Zn2AdcR binding to DNA, which show both are decisively entropy-driven processes.

Theory of Ethidium Binding to DNA under Tension and Torque: Possible Role of Cruciforms at Inverted Repeat Sequences.

Celedon et al. reported an unexpectedly low slope of applied torque vs turns (or apparent torsional rigidity) for a long DNA subject to 0.8 pN tension and modest negative torques (up to approximately -5 pN nm) in 3.4 × 10-9 M ethidium (J. Phys. Chem. B 2010, 114, 16929-16935). Extrusion of inverted repeat sequences to create cruciforms with anomalously large association constants for binding 4 ethidiums to the cruciform arms is investigated as a possible explanation for this observation and also for its compatibility with other observations of Celedon et al. The equilibrium between the linear main chain and cruciform states of an inverted repeat sequence under the prevailing tension, torque, and ethidium concentration is treated by first computing the free energy per bp of the linear main chain. This is done for a complex model, wherein every bp in the linear main chain participates in both the recently reviewed cooperative two-state a ⇔ b equilibrium (Quarterly Reviews of Biophysics 2021, 54, e5, 1-25) and in ethidium binding with a modest relative preference for either the a- or b-state. Plausible assumptions are made concerning the relative populations of the cruciform and linear main chain states of an inverted repeat, and also the relative populations of cruciform states with and without 4 bound ethidiums in the presence of tension, torque, and 3.4 × 10-9 M ethidium. Besides a large drop in slope (or apparent torsional rigidity) from 10-9 to 10-8 M ethidium, this theory also predicts maxima between 6.4 × 10-8 and 2.0 × 10-7 M ethidium, a region where no measurements were made. Overall agreement between theoretical and experimental values of the slope (or apparent torsional rigidity), and also the number of negative turns due to bound ethidium at zero torque, is fairly good for all of the ethidium concentrations studied by Celedon et al., provided that there is a modest preference for binding to the b-state. When there is a modest preference for binding to the a-state, the theory significantly underestimates experimental values at the higher ethidium concentrations, likely ruling out that possibility.

Synthetic Receptors with Micromolar Affinity for Chloride in Water.

A water-soluble coordination cage was obtained by reaction of Pd(NO3 )2 with a 1,3-di(pyridin-3-yl)benzene ligand featuring a short PEG chain. The cavity of the metal-organic cage contains one nitrate anion, which is readily replaced by chloride. The apparent association constant for chloride binding in buffered aqueous solution is Ka =1.8(±0.1)×105 M-1 . This value is significantly higher than what has been reported for other macrocyclic chloride receptors. The heavier halides Br- and I- compete with binding or self-assembly, but the receptor displays very good selectivity over common anions such as phosphate, acetate, carbonate, and sulfate. A further increase of the chloride binding affinity by a factor of 3 was achieved using a fluorinated dipyridyl ligand.

Synthetic Receptors with Micromolar Affinity for Chloride in Water

AbstractA water‐soluble coordination cage was obtained by reaction of Pd(NO3)2with a 1,3‐di(pyridin‐3‐yl)benzene ligand featuring a short PEG chain. The cavity of the metal‐organic cage contains one nitrate anion, which is readily replaced by chloride. The apparent association constant for chloride binding in buffered aqueous solution isKa=1.8(±0.1)×105 M−1. This value is significantly higher than what has been reported for other macrocyclic chloride receptors. The heavier halides Br−and I−compete with binding or self‐assembly, but the receptor displays very good selectivity over common anions such as phosphate, acetate, carbonate, and sulfate. A further increase of the chloride binding affinity by a factor of 3 was achieved using a fluorinated dipyridyl ligand.

Ninhydrin and isatin appended 2-Hydrazinobenzothiazole based simple Schiff bases for colorimetric selective detection of Cr3+ and Pb2+ ions

A simple and easily synthesizable chromogenic small molecules probe L4(2-(2-(benzo[d]thiazol-2-yl)hydrazono)-1H-indene-1,3(2H)-dione) and L5 (2-(2-(benzo[d]thiazol-2-yl)hydrazono)indolin-3-one) have been synthesized and characterized by various spectroscopy techniques such as FT-IR, NMR, and HRMS. The colorimetric naked eye detection of probe L4 showed a color change towards Cr3+ from orange color to pale yellow, whereas in case of L5 Pb2+ yellow to orange color was witnessed. The jobs plot analyses indicate the 1:1 binding stoichiometric for Cr3+ and 1:2 binding stoichiometric for Pb2+ ions with probes L4 and L5 respectively. The binding association constant (ka) values for L4 and L5 were found to be 6.651 × 10-5 M for Cr3+ and 6.493 × 10-5 M for Pb2+ ions. The limit of detection was found to be for Cr3+ (1.69 × 10−7 M) and Pb2+ (1.208 × 10−7 M) are far below than the limit set by the World Health Organization (WHO) for drinking water. Finally, the designed probes were applied for visible colorimetric test kits and real samples for rapid detection of Cr3+ and Pb2+ for all practical purposes, indicating their potential use in environmental samples.

Mathematical and experimental validation of an approach for simultaneously determining the binding parameters of two drugs to a receptor

Quantifying drug-protein interactions has a pivotal role in both early phase drug development and clinical processes. Diverse affinity chromatographic methods like nonlinear chromatography can realize such quantification, however, their throughputs are challenged due to the loading of a single ligand during each run. This work derivatized a new equation for simultaneously determining the bindings of two ligands to a protein relying on assumption that the retention factors of the ligands are dependent on their injection amounts. Experimental validation of the derivatization was performed on an immobilized endothelin A receptor (ETAR) column taking ambrisentan, bosentan, and macitentan as injecting solutes. All three ligands presented a decrease in retention times along with increasing moles of injection when they were singly injected into the column. Likewise, negative relationships between the retentions and the injection amounts were observed when co-injection of ambrisentan/bosentan or bosentan/macitentan was performed, thus confirming the assumption of the derivatization. The association constants of ambrisentan, bosentan, and macitentan binding to ETAR were (1.42 ± 0.78)×104, (1.81 ± 0.22)×104, and (1.71 ± 0.41)×104 L/mol when each of them was singly loaded on the column. Such data displayed insignificant changes in four weeks thereby providing a proof of good stability of the column during the period. Co-injections of the two ligand pairs resulted in the association constants of (2.97 ± 0.13)×104 for ambrisentan, (2.51 ± 0.87)×104 for bosentan, and (2.88 ± 0.34)×104 L/mol for macitentan. These results were in good agreement with the calculation when each of the ligands was injected alone into the column and demonstrated little differences from the data by nonlinear chromatography. Owning to the simultaneous analysis of two ligands, the throughput of the proposed method was twofold higher than the typical assays including frontal analysis, zonal elution, and nonlinear chromatography. It is possible to become an alternative for rapid analysis of drug-protein interaction.

Impact of pectin and whey minerals solubilized by lime juice on calcium bioaccessibility in yogurt based snacks

Pectin is used in dairy products to improve texture, but it may also affect calcium absorption. The influence of pectins with different degrees of esterification on calcium bioaccessibility in yogurt snacks enriched with or without whey mineral concentrates and lime juice was quantified using a three-step in vitro digestion including electrochemical determination of free and complex bound calcium. Calcium from whey minerals became bioaccessible under intestinal conditions when combined with lime juice, as a consequence of a large fraction of complex bound calcium by citrate. Pectin improved the texture of the snacks but lowered the calcium bioaccessibility by 6%–16% depending on the degree of esterification through endothermic binding of calcium to carboxylate groups of pectins as evidenced by higher yield stress of the snack bases. Decreasing degree of esterification and increasing blockiness promoted calcium binding, as indicated by the estimated binding sites and association constants. Increasing temperature from 25 °C to 37 °C and pH from 4 to 7 (intestinal conditions) also increased the interactions between calcium and pectins. For low methoxyl pectin, the complex binding of calcium to pectin carboxylates was found more evident than binding to citrate from the lime juice reducing calcium bioaccessibility. For high methoxyl pectin with fewer binding sites, citrate binding kept calcium available for uptake through intestinal digestion. A balanced formulation with whey minerals, lime juice and calcium-insensitive high methoxyl pectin yielded crispy yogurt snacks with high calcium bioaccessibility, which provides a new perspective to design dairy products with high nutritional value. • Pectin improves hardness and crispness of freeze-dried yogurt snacks. • Whey minerals with lime juice increase Ca bioaccessibility by citrate binding. • LM and to a lesser degree of HM pectin decreases Ca bioaccessibility. • LM pectin reduces calcium binding to citrate decreasing Ca bioaccessibility. • HM pectin and whey minerals lead to crispy snacks and high Ca bioaccessibility.

Rapid, Selective Fluorescent Determination of Copper (II) in Aqueous Solution and Living Cells Using a Dansyl-Based Click Probe

A novel 1,2,3-triazole based dansyl fluorophore has been designed and synthesized for detecting Cu2+ as a fluorescent probe (P1). The probe P1 exhibited high sensitivity and selectivity for Cu2+ compared to other selected species in aqueous solution within a wide pH span (5 to 14), which showed that P1 may be applied in the entire alkaline pH window. The limit of detection (LOD) was measured to be as low as 5 × 10−7 mol/L for Cu2+. A 1:2 binding ratio and association constant of 4.6 × 104 M−1 between P1 and Cu2+ were determined. Additionally, rapid response to Cu2+ was demonstrated in less than 3 seconds and the linear concentration range was from 0.5 to 2.5 µmol/L. Moreover, probe P1 showed good biocompatibility and cell membrane permeability and was successfully applied for imaging Cu2+ in water samples and living cells, which demonstrates the application of P1 in complicated environments.

Isothermal Titration Calorimetry Directly Measures the Selective Swelling of Block Copolymer Vesicles in the Presence of Organic Acid.

Block copolymer (BCP) vesicles loaded with drug molecules may have a nonidentical swelling behavior due to the strong interactions between BCP vesicles and loaded molecules. A thermodynamic study of the swelling for such a system is of great importance in clarifying their pH-gated drug delivery behavior. In this study, the selective swelling of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP) vesicles in the presence of different acids was compared using dynamic light scattering, zeta-potential, and isothermal titration calorimetry (ITC) measurements. Transmission electron microscopy observation verified that these PS-b-P2VP vesicles were mainly multilamellar. Importantly, using the ITC measurement, we first compared the thermodynamic parameters, including ΔH, ΔG, and ΔS, association binding sites (N), and binding association constants (Ka) in the selective swelling of the PS-b-P2VP vesicles in low pH (pH ∼3.5), with or without a hydrogen bonding interaction. We observed that the existence of a hydrogen bonding interaction between tartaric acid/malic acid and PS-b-P2VP generates a limitation to the selective swelling of PS-b-P2VP vesicles, in which conditions will depend on the molecular structures of the organic acids and PS-b-P2VP. This work first provides a quantitative insight on the swelling of BCP vesicles in the presence of hydrogen bonding and highlights the power of ITC measurements for investigating the structural transformation of polymer nanostructures.

Structure and biological activity of particles produced from highly activated carbon adsorbent

Over the recent years, carbon particles have gained relevance in the field of biomedical application to diminish the level of endo-/exogenous intoxication and oxidative stress products, which occur at different pathological states. However, it is very important that such carbon particles, specially developed for parenteral administration or per oral usage, possess a high adsorption potential and can remove hazard toxic substances of the hydrophilic, hydrophobic and amphiphilic nature usually accumulated in the blood due to the disease, and be absolutely safe for normal living cells and tissues of organism. In this work, the stable monodisperse suspension containing very small-sized (Dhydro = 1125.3 ± 243.8 nm) and highly pure carbon particles with an excellent accepting ability were obtained. UV-spectra, fluorescence quenching constant and binding association constant were provided by the information about conformational alterations in an albumin molecule in presence of carbon particles, about the dynamic type of quenching process and low binding affinity between carbon and protein. The later was confirmed by DSC method. In vitro cell culture experiments showed that carbon particles did not possess any cytotoxic effect towards all testing the normal cell lines of different histogenesis, did not show genotoxic effects and were absolutely safe for experimental animals during and after their parenteral administration. These observations may provide more information about how to develop a safe preparation of carbon particles for different biomedical applications, in particular, as a mean for intracorporeal therapy of various heavy diseases accompanied by the increased endogenous intoxication and the level of oxidative stress.

On the Effect of pH, Temperature, and Surfactant Structure on Bovine Serum Albumin-Cationic/Anionic/Nonionic Surfactants Interactions in Cacodylate Buffer-Fluorescence Quenching Studies Supported by UV Spectrophotometry and CD Spectroscopy.

Due to the fact that surfactant molecules are known to alter the structure (and consequently the function) of a protein, protein–surfactant interactions are very important in the biological, pharmaceutical, and cosmetic industries. Although there are numerous studies on the interactions of albumins with surfactants, the investigations are often performed at fixed environmental conditions and limited to separate surface-active agents and consequently do not present an appropriate comparison between their different types and structures. In the present paper, the interactions between selected cationic, anionic, and nonionic surfactants, namely hexadecylpyridinium chloride (CPC), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyethylene glycol sorbitan monolaurate, monopalmitate, and monooleate (TWEEN 20, TWEEN 40, and TWEEN 80, respectively) with bovine serum albumin (BSA) were studied qualitatively and quantitatively in an aqueous solution (10 mM cacodylate buffer; pH 5.0 and 7.0) by steady-state fluorescence spectroscopy supported by UV spectrophotometry and CD spectroscopy. Since in the case of all studied systems, the fluorescence intensity of BSA decreased regularly and significantly under the action of the surfactants added, the fluorescence quenching mechanism was analyzed thoroughly with the use of the Stern–Volmer equation (and its modification) and attributed to the formation of BSA–surfactant complexes. The binding efficiency and mode of interactions were evaluated among others by the determination, comparison, and discussion of the values of binding (association) constants of the newly formed complexes and the corresponding thermodynamic parameters (ΔG, ΔH, ΔS). Furthermore, the influence of the structure of the chosen surfactants (charge of hydrophilic head and length of hydrophobic chain) as well as different environmental conditions (pH, temperature) on the binding mode and the strength of the interaction has been investigated and elucidated.

Synthesis of new anthracene-conjugated calix[4]arene as highly selective fluorescent chemosensor for determination of CN− ion

A new calix[4]arene derivative substituted with anthracene at its 1,3-distal position was synthesized via a simple Schiff base reaction, and its structure was fully characterized by using ATR-FTIR, 1H-NMR, 13C-NMR, ESI-MS and elemental analysis techniques. Furthermore, the ion binding feature of the anthracene substituted calix[4]arene (Ant-Calix) was evaluated against Br−, Cl−, F−, HSO4−, NO3− and CN− ions. Various aspects of anion sensing phenomena were deeply assessed, and the findings addressed that Ant-Calix exhibited excellent selectivity for CN− and could detect in a very low detection limit 92.4 nM. Beside, when CN− ion was bound to the probe Ant-Calix, a significant color change from light green to pink color was observed under UV light. The fluorescence data was also employed to calculate the binding stoichiometry and association constant of the complex obtained between Ant-Calix and cyanide ion. In addition, the 1H-NMR technique was also used to elucidate the mechanism of the complex formation between Ant-Calix and CN−.

Binding Methylarginines and Methyllysines as Free Amino Acids: A Comparative Study of Multiple Host Classes**

Methylated free amino acids are an important class of targets for host‐guest chemistry that have recognition properties distinct from those of methylated peptides and proteins. We present comparative binding studies for three different host classes that are each studied with multiple methylated arginines and lysines to determine fundamental structure‐function relationships. The hosts studied are all anionic and include three calixarenes, two acyclic cucurbiturils, and two other cleft‐like hosts, a clip and a tweezer. We determined the binding association constants for a panel of methylated amino acids using indicator displacement assays. The acyclic cucurbiturils display stronger binding to the methylated amino acids, and some unique patterns of selectivity. The two other cleft‐like hosts follow two different trends, shallow host (clip) following similar trends to the calixarenes, and the other more closed host (tweezer) binding certain less‐methylated amino acids stronger than their methylated counterparts. Molecular modelling sheds some light on the different preferences of the various hosts. The results identify hosts with new selectivities and with affinities in a range that could be useful for biomedical applications. The overall selectivity patterns are explained by a common framework that considers the geometry, depth of binding pockets, and functional group participation across all host classes.

A Pyrazolo Imine-based Colorimetric and Turn-on Fluorescent Sensor Probe for Determination of Hg2+ Ion and its Application in Test Paper Strips.

In this work, we synthesized diethylamino substituted pyrazolo imine (3) fluorescent probe for recognition of Hg2+ ion.The sensor probe 3 can detect Hg2+ by colorimetric method, and there is a 10-fold enhancement in fluorescence response. When the fluorescent probe bound with Hg2+ ion, turn-on fluorescence was observed via the coordination. Probe 3 has an excellent selectivity toward Hg2+ in the CH3 CN/H2 O (8:2, v/v) solution with low limit of detection and high binding association constant of 551 parts per billion (ppb) and 6.6067 × 106 m-1 for 3+Hg2+ , respectively. Furthermore, the formation of 3+Hg2+ complex with 1:1 binding mode was evidenced by Job's plot, 1 H NMR spectroscopy and Mass analysis. In addition, probe 3 is a feasible option to detect Hg2+ in various sources of water samples. Bio-imaging experiments have demonstrated that probe 3 can be used to monitor Hg2+ in Escherichia coli bacterial cell. The sensor 3 was also used for paper strip application to detect Hg2+ ion.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid–catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

Plastic bioconversion: Reaction mechanism of PETases

The enzyme IsPETase can efficiently degrade polyethylene terephthalate (PET) at room temperature and is an attractive method of plastic bioconversion. Based on mutagenesis experiments on IsPETase, we propose a physical model which predicts that its reaction mechanism can be described by the Michaelis-Menten model with a catalytic efficiency of Ka∙k2, where Ka is the association constant of substrate binding and k2 is the rate constant to form the acyl-enzyme intermediate. This model verifies the assumption of Michaelis-Menten kinetics in previous studies on PETases and has novel applications in deriving the enzyme activity using computational molecular dockings. By computationally docking bis-(2-hydroxyethyl) terephthalic acid (BHET) on the surface of IsPETase mutants, we studied the catalytic effects of various side chains and observed that their predicted activities are consistent with experimental data, with a correlation coefficient in the range of 0.79∼0.88. Based on this study, our model presents an analytical interpretation for the reaction mechanism of PETases and provides an efficient method for computing their catalytic efficiency for identifying enzymes with a better catalytic performance from numerous protein sequences in open databases.

Abstract 1145: AGX323 - A SAM-competitive, orally available inhibitor of protein arginine methyltransferase 5 (PRMT5) with potent cellular antiproliferative and in vivo antitumor activity against selected solid cancer types

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is an attractive therapeutic target because of its involvement in key oncogenic mechanisms including epigenetics, DNA response and RNA processing. Early development of PRMT5 inhibitors has focused on hematological malignancies although more recently several compounds have entered the clinic for both liquid and solid malignancies. Early Phase I clinical data indicate dose-limiting hematological toxicity, particularly anemia and thrombocytopenia. We focused our effort in discovering compounds with potent inhibitory activity against cells from solid cancer types known to overexpress PRMT5 and which correlated with poor prognosis (NSCLC, breast, bladder and pancreatic cancers). Methods: Enzyme inhibition was conducted using a PRMT5:MEP50 flash plate assay, with S-adenosylmethionine (SAM) as the methyl donor and histone H4 as substrate. Inhibition of cell proliferation was assessed in various cancer lines using a 7-day cellTitre-Glo (CTG) assay. Binding mode of the inhibitors to PRMT5 was investigated using surface plasmon resonance (SPR). In vivo antitumor activity was evaluated in various human cancer models grown in immunocompromised mice. Results: Structure activity relationship of a series of over 50 close analogs demonstrated that in vitro anticancer activities correlate poorly with enzyme inhibition potency. To understand this discrepancy we used SPR to study the PRMT5 binding mode of cell active and inactive compounds in the presence of various endogenous substrates (SAM, SAH or MTA). We obtained binding affinity (KD), association constant (Ka) and dissociation constant (Kd) of exemplars of active and inactive inhibitors and correlated their relationship to cell activity. The lead compound AGX323 has a KD of 1.8 x 10-10 M to SAM-bound PRMT5 and exhibited potent antiproliferative activity against a panel of over 20 human solid epithelial cancer cell lines from lung, breast, bladder and pancreas, with nM potency. For example, in the H1048 human lung cancer model it has an enzyme inhibition IC50 of 0.8 nM, a cell proliferation IC50 of 1.7 nM and when given to tumor bearing mice it demonstrated robust antitumor activity at well-tolerated dose levels. In nonclinical ADME studies, AGX323 demonstrated favorable drug-like properties: including acceptable oral bioavailability (in mouse, dog and monkey) and systemic exposure following oral administration, excellent metabolic stability, lack of significant drug-drug interaction liabilities (transporters, CYP inhibition, CYP induction). Conclusion: AGX323 demonstrated potentially best-in-class activity in PRMT5 dependent human solid cancer models and exhibited excellent drug-like properties. IND enabling studies are currently planned. Citation Format: Francis Y. F. Lee, Wen-Lian Wu, Zhiqiang Yang, John Tan. AGX323 - A SAM-competitive, orally available inhibitor of protein arginine methyltransferase 5 (PRMT5) with potent cellular antiproliferative and in vivo antitumor activity against selected solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1145.