Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα.

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinact and reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kß-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.

Replicates Number for Drug Stability Testing during Bioanalytical Method Validation-An Experimental and Retrospective Approach.

BACKGROUND: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. METHODS: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes-tramadol and its major metabolite (O-desmethyl-tramadol)-in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). RESULTS: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85-115% acceptance criteria. CONCLUSIONS: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

The Aggregation Pattern of Aß1-40 is Altered by the Presence of N-Truncated Aß4-40 and/or CuII in a Similar Way through Ionic Interactions.

Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-ß (Aß) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of CuII on the aggregation of Aß1-40 and Aß4-40 , representing the two most prevalent families of Aß peptides, that is, the full length and N-truncated peptides. Both families are similarly abundant in healthy and AD brains. For either of the studied peptides, substoichiometric CuII concentrations accelerated aggregation, whereas superstoichiometric CuII inhibited fibril formation, likely by stabilizing the oligomers. The addition of either Aß4-40 or substoichiometric CuII affected the aggregation profile of Aß1-40 , by yielding shorter and thicker fibrils; amorphous aggregates were formed in the presence of a molar excess of CuII . The similarity of these two effects can be attributed to the increase in the positive charge on the Aß N terminus, caused both by CuII complexation and N truncation at position 4. Our findings provide a better understanding of the biological Aß aggregation process as these two Aß species and CuII coexist and interact under physiological conditions.

The Reactions of H2O2 and GSNO with the Zinc Finger Motif of XPA. Not A Regulatory Mechanism, But No Synergy with Cadmium Toxicity.

Tetrathiolate zinc fingers are potential targets of oxidative assault under cellular stress conditions. We used the synthetic 37-residue peptide representing the tetrathiolate zinc finger domain of the DNA repair protein XPA, acetyl-DYVICEECGKEFMSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) as a working model to study the reaction of its Zn(II) complex (ZnXPAzf) with hydrogen peroxide and S-nitrosoglutathione (GSNO), as oxidative and nitrosative stress agents, respectively. We also used the Cd(II) substituted XPAzf (CdXPAzf) to assess the situation of cadmium assault, which is accompanied by oxidative stress. Using electrospray mass spectrometry (ESI-MS), HPLC, and UV-vis and circular dichroism spectroscopies we demonstrated that even very low levels of H2O2 and GSNO invariably cause irreversible thiol oxidation and concomitant Zn(II) release from ZnXPAzf. In contrast, CdXPAzf was more resistant to oxidation, demonstrating the absence of synergy between cadmium and oxidative stresses. Our results indicate that GSNO cannot act as a reversible modifier of XPA, and rather has a deleterious effect on DNA repair.

Stochastic or Not? Method To Predict and Quantify the Stochastic Effects on the Association Reaction Equilibria in Nanoscopic Systems.

The stochastic nature of chemical reaction and impact of the stochasticity on their evolution is soundly documented. Both theoretical predictions and emerging experimental evidence indicate the influence of stochastic effects on the equilibrium state of association reaction. In this work simple mathematical formulas are introduced to estimate these effects. First, the dependence of the ratio of observed reactants (apparent association constant, equivalent of macroscopic association constant in stochastic analysis) on the volume and the number of molecules of reagents is discussed and the limiting factors of this effect are shown. Next, the apparent association constant is approximated for nanoscale systems by closed-form formulas derived for this purpose. Finally, an estimation for the macroscopic constant value from the apparent one is provided and validated on the published experimental data. This work was inspired by chemical reactions occurring in biological compartments, but the results can be used for all systems belonging to the stochastic regime of chemical reactions.

Ternary Zn(II) Complexes of Fluorescent Zinc Probes Zinpyr-1 and Zinbo-5 with the Low Molecular Weight Component of Exchangeable Cellular Zinc Pool.

The intracellular exchangeable Zn(II) is usually measured with synthetic fluorescent zinc sensors. 4',5'-Bis[bis(2-pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein (Zinpyr-1) is a sensor containing the fluorescein platform and a duplicated chelating unit. Its advantages include brightness and a relatively high affinity for Zn(II), Kd = 0.7 nM. 2-(4,5-Dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole (Zinbo-5) is a member of a growing family of ratiometric synthetic Zn(II) probes, offering a possibility to determine Zn(II) concentration independently of the sensor concentration. Cells, however, contain high, millimolar or nearly millimolar concentrations of low molecular weight ligands (LMWLs) capable of binding Zn(II) ions. Previously, we demonstrated that such LMWLs can perturb the performance of some fluorescent zinc sensors by competition and formation of ternary Zn(sensor) (LMWL) complexes. Here we tested Zinpyr-1 and Zinbo-5 in this respect. Despite structural differences, both sensors formed such ternary complexes. We determined their stability constants CKtern and performed numerical simulations of Zn(II) distributions at physiological concentrations of selected LMWLs. Glutamic acid was found to provide the strongest ternary complexes with either of the studied sensors. Zn(Zinpyr-1)(Glu) was an absolutely dominant Zn(II)/Zinpyr-1 species (more than 96% of the exchangeable Zn(II)), and Zn(Zinbo-5)(Glu) was the most abundant one (more than 40%) in these simulations. Our results indicate that under cellular conditions these sensors are able to report Zn(II) complexed to LMWLs rather than free Zn2+ ions. On the other hand, the specific affinity of Zn(Zinpyr-1) and Zn(Zinbo-5) for Glu creates interesting opportunities for determining glutamic acid in biological samples.

Ternary Zn(II) Complexes of FluoZin-3 and the Low Molecular Weight Component of the Exchangeable Cellular Zinc Pool.

Knowledge of the nature of exchangeable (labile) intracellular Zn(II) is increasingly important for biomedical research. The detection and quantitative determination of Zn(II) ions is usually performed by using Zn(II)-specific fluorescent sensors, among which 2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) has been used most widely. Selectivity of this sensor for Zn(II) over other divalent cations was demonstrated, but possible interference in its performance by other compounds has not been investigated. Many potential low molecular weight ligands for Zn(II) ions (LMWLs) are abundant in the cell. In this study we demonstrate that FluoZin-3 is susceptible to competition for Zn(II) from LMWLs and also forms strong ternary complexes with some of them. We determined the set of conditional stability constants C Ktern for ternary Zn(FluoZin-3)(LMWL) complexes using fluorescence titrations and applied it to model the response of exchangeable zinc to FluoZin-3. We found that competition and formation of ternary complexes with LMWLs together strongly affect (net reduce) the Zn(FluoZin-3) fluorescence. This effect may cause a significant underestimation of exchangeable Zn(II). We also demonstrated a strong pH dependence of this effect. Reduced glutathione (GSH) emerged as the most important Zn(II) partner among the LMWLs, characterized with Ktern = 2.8 ± 0.2 × 106 M-1. Our experiments and calculations suggest that Zn(LMWL) complexes contribute to the exchangeable cellular zinc pool.

Numerical Simulations Reveal Randomness of Cu(II) Induced Aß Peptide Dimerization under Conditions Present in Glutamatergic Synapses.

The interactions between the Aß1-40 molecules species and the copper ions (Cu(II)) were intensively investigated due to their potential role in the development of the Alzheimer Disease (AD). The rate and the mechanism of the Cu(II)-Aß complexes formation determines the aggregation pathway of the Aß species, starting from smaller but more cytotoxic oligomers and ending up in large Aß plaques, being the main hallmark of the AD. In our study we exploit the existing knowledge on the Cu(II)-Aß interactions and create the theoretical model of the initial phase of the copper- driven Aß aggregation mechanism. The model is based on the direct solution of the Chemical Master Equations, which capture the inherent stochastics of the considered system. In our work we argue that due to a strong Cu(II) affinity to Aß and temporal accessibility of the Cu(II) ions during normal synaptic activity the aggregation driven by Cu(II) dominates the pure Aß aggregation. We also demonstrate the dependence of the formation of different Cu(II)-Aß complexes on the concentrations of reagents and the synaptic activity. Our findings correspond to recent experimental results and give a sound hypothesis on the AD development mechanisms.

Selenocysteine containing analogues of Atx1-based peptides protect cells from copper ion toxicity.

Seleno-substituted model peptides of copper metallochaperone proteins were analyzed for the metal affinity and in vitro anti-oxidative reactivity. An acyclic MTCXXC (X is any amino acid) reference peptide previously analyzed as a potent inhibitor of ROS production underwent substitution of the cysteine residues with selenocysteine to give two singly substituted derivatives C3U and C6U and the doubly substituted analogue C3U/C6U. Presumably due to the softer nature of Se vs. S, all selenocysteine containing peptides demonstrated high affinity to Cu(i), higher than that of the reference peptide, and in the same order of magnitude as that measured for the native protein, Atox1. A stronger impact of residue 3 confirmed previous findings on its more dominant role in metal coordination. In vitro studies on the HT-29 human colon cancer cell line, MEF mice embryonic fibroblasts, and MEF with the knocked-out Atox1 gene (Atox1-/-) consistently identified C3U/C6U as the most potent inhibitor of ROS cellular production based on the 2',7'-dichlorodihydrofluorescin diacetate (H2DCF-DA) assay, also in comparison with known drugs employed in the clinic for Wilson's disease. The selenocysteine containing peptides are thus promising drug candidates for chelation therapy of Wilson's disease and related conditions relevant to excessive copper levels.

On the ability of CuAß1-x peptides to form ternary complexes: Neurotransmitter glutamate is a competitor while not a ternary partner.

In the light of conflicting reports on the ability of copper(II) complexes of amyloid beta (Aß) peptides to form ternary complexes with small molecules co-present in the biological milieu, we performed a study of coordination equilibria in the system containing Cu(II) ions, the Aß1-16 peptide, glutamic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, HEPES) buffer. Using potentiometry, isothermal titration calorimetry (ITC), UV-visible spectroscopy and EPR, we concluded that glutamic acid was not able to form such a ternary complex, but can efficiently compete for the Cu(II) ion with the Aß peptide at Glu concentrations relevant for the synaptic cleft. We also found that the literature constants for Cu(II) complexes with Glu were overestimated, but this effect was partially compensated by the formation of a ternary Cu(Glu)(HEPES) complex. Our results indicate that small molecules co-present with Cu(II) ions and Aß peptides in the synaptic cleft are not very likely to enhance Cu(II)/Aß interactions, but instead should be considered as a Cu(II) buffering system that may help prevent these interactions and participate in Cu(II) clearance from the synaptic cleft.

Unbound position II in MXCXXC metallochaperone model peptides impacts metal binding mode and reactivity: Distinct similarities to whole proteins.

The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural analysis and affinity and oxidation kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidation of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidation rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport.

Unusual Zn(II) Affinities of Zinc Fingers of Poly(ADP-ribose)Polymerase 1 (PARP-1) Nuclear Protein.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a key eukaryotic enzyme,catalyzing the NAD+ dependent poly(ADP-ribosyl)ation of protein substrates, crucial for major DNA repair pathways, and involved in other fundamental cellular processes, such as transcription, cell cycle control, and apoptosis. Its ability to bind DNA depends on two CCHC zinc finger domains, in short, PARPzf1 and PARPzf2. Using spectroscopic methods and competitive titrations with Zn(II), Co(II), and Ni(II) ions, we determined conditional dissociation constants for Zn(II) complexes of PARPzf1 and PARPzf2 at pH 7.4 (HEPESbuffer) as 26 ± 4 nM and 4 ± 1 pM, respectively. The former value indicates an extremely low affinity of PARPzf1 toward metal ions, meaning that under cellular conditions PARP1zf might be largely present in a "metal-free" state. This finding provides a clue to the high susceptibility of PARP-1 to oxidative stress but also raises questions regarding the activation of PARPzf1 under cellular conditions. We also determined conditional dissociation constants for Ni(II) complexes of PARPzf1 and PARPzf2 under the same conditions as 0.78 ± 0.04 µM and 0.26 ± 0.05 nM, respectively.

Human annexins A1, A2, and A8 as potential molecular targets for Ni(II) ions.

Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1) and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and circular dichroism (CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and electrospray ionization mass spectrometry (ESI-MS). We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2 bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies.

cis-Urocanic acid as a potential nickel(II) binding molecule in the human skin.

cis-Urocanic acid, a derivative of histidine, is one of the essential components of human skin. We found that it can bind nickel(II) ions in a pH-dependent manner, with the dissociation constant in the low millimolar range, as revealed by potentiometry, and confirmed by isothermal titration calorimetry and UV-vis spectroscopy. The binding occurs within the physiological skin pH range. Considering the fact that cis-urocanic acid is present in the human skin in concentrations as high as millimolar, this molecule may be a physiologically important player in nickel trafficking in the human organism.