Molecular interaction of human serum albumin with paracetamol: Spectroscopic and molecular modeling studies

The interaction between paracetamol and human serum albumin (HSA) under physiological conditions has been investigated by fluorescence, circular dichroism (CD) and docking. Fluorescence data revealed that the fluorescence quenching of HSA by paracetamol was the result of the formed complex of HSA–paracetamol, and the binding constant (K_a) and binding number obtained is 1.3 × 10⁴ at 298 K and 2, respectively for the primary binding site. Circular dichorism spectra showed the induced conformational changes in HSA by the binding of paracetamol. Moreover, protein–ligand docking study indicated that paracetamols (two paracetamols bind to HSA) bind to residues located in the subdomain IIIA.     

Triazolopyridyl ketones as a novel class of antileishmanial agents. DNA binding and BSA interaction

A new series of triazolopyridyl pyridyl ketones has been synthetized by regioselective lithiation of the corresponding [1,2,3]triazolo[1,5-a]pyridine at 7 position followed by reaction with different electrophiles. The in vitro antileishmanial activity of these compounds was evaluated against Leishmania infantum, Leishmania braziliensis, Leishmania guyanensis and Leishmania amazonensis. Compounds 6 and 7 were found to be the most active leishmanicidal agents. Both of them showed activities at micromolar concentration against cultured promastigotes of Leishmania spp. (IC₅₀ = 99.8–26.8 μM), without cytotoxicity on J774 macrophage cells. These two compounds were also tested in vivo in a murine model of acute infection by L. infantum. The triazolopyridine derivative 6 was effective against both spleen and liver parasites forms, while 7 was inactive against liver parasites. Mechanistic aspects of the antileishmanial activity were investigated by means of DNA binding studies (UV-titration and viscosimetry). Results have revealed that these active ligands are able to interact strongly with DNA [K_b = 1.14 × 10⁵ M⁻¹ (6) and 3.26 × 10⁵ M⁻¹ (7)]. Moreover, a DNA groove binding has been proposed for both 6 and 7. To provide more insight on the mode of action of compounds 6 and 7 under biological conditions, their interaction with bovine serum albumin (BSA) was monitored by fluorescence titrations and UV–visible spectroscopy. The quenching constants and binding parameters were determined. Triazolopyridine ketones 6 and 7 have exhibited significant affinity towards BSA [K_b = 2.5 × 10⁴ M⁻¹ (6) and 1.9 × 10⁴ M⁻¹ (7)]. Finally, to identify the binding location of compounds 6 and 7 on the BSA, competitive binding experiments were carried out, using warfarin, a characteristic marker for site I, and ibuprofen as one for site II. Results derived from these studies have indicated that both compounds interact at BSA site I and, to a lesser extent, at site II.     

Kinetics parameter estimation for the binding process of salicylic acid to human serum albumin (HSA) with capacitive sensing technique

A novel method for real-time investigating the binding interaction between human serum albumin (HSA) and salicylic acid with capacitive sensing technique was successfully proposed. HSA was immobilized on the surface of a gold electrode modified with an insulating poly (o-phenylenediamine) (o-PD) film and colloid Au nanoparticles layers. The bioactivity of HSA was remained and major binding sites were available because of the excellent biocompatibility of gold nanoparticles. The capacitance and interfacial electron resistance of the sensor were altered, owing to the binding of HSA to salicylic acid. The time courses of the capacitance change were acquired with capacitive sensing technique during the binding process. Based on the capacitance response curves with time, the response model for the binding was derived in theory and the corresponding regression parameters were determined by fitting the real-time experimental data to the model. The binding and the dissociation rate constants (k₁ and k_− 1) were estimated to be 54.8 (mol l^− 1)^− 1 s^− 1 and 2.9 × 10^− 3 s^− 1, respectively. And the binding equilibrium constant (K_a) was calculated to be 1.89 × 10⁴ (mol l^− 1)^− 1.     

Schiff’s base derivatives bearing nitroimidazole and quinoline nuclei: New class of anticancer agents and potential EGFR tyrosine kinase inhibitors

New Schiff’s base derivatives 5a–j have been synthesized by reaction between 2-phenoxyquinoline-3-carbaldehydes 3a–j and 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide 4 in presence of nickel(II) nitrate as a catalyst in ethanol under reflux in good yield (78–92%). All compounds were tested for anticancer and inhibition of EGFR. Of the compounds studied, majority of the compounds showed effective antiproliferation and inhibition of EGFR and HER-2 activities. Compound 5h showed most effective inhibition (IC₅₀ = 0.12 ± 0.05 μM) by binding in to the active pocket of EGFR receptor with minimum binding energy (ΔG_b = −58.3691 kcal/mol). The binding was stabilized by two hydrogen bonds, two π–cation and one π–sigma interactions. Compound 5d showed most effective inhibition (IC₅₀ = 0.37 ± 0.04 μM).     

DNA binding acridine–thiazolidinone agents affecting intracellular glutathione

Three new acridine–thiazolidinone derivatives (2a–2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovarian cancer cell lines A2780) were studied. The compounds 2a–2c possessed high affinity to calf thymus DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 × 10⁶–5.89 × 10⁶ M^?1. All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activity in cytotoxic tests was found for 2c with IC₅₀ = 1.3 ± 0.2 μM (HL-60), 3.1 ± 0.4 μM (L1210), and 7.7 ± 0.5 μM (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with their reactivity towards thiols and DNA binding activity.     

Investigation of Foscan® interactions with plasma proteins

The present study investigates the interaction of the second generation photosensitizer Foscan® with plasma albumin and lipoproteins. Spectroscopic studies indicated the presence of monomeric and aggregated Foscan® species upon addition to plasma protein solutions. Kinetics of Foscan® disaggregation in albumin-enriched solutions were very sensitive to the protein concentration and incubation temperature. Kinetic analysis demonstrated that two types of Foscan® aggregated species could be involved in disaggregation: dimers with a rate constant of k₁ = (2.30 ± 0.15) × 10⁻³ s⁻¹ and higher aggregates with rate constants varying from (0.55 ± 0.04) × 10⁻³ s⁻¹ for the lowest to the (0.17 ± 0.02) × 10⁻³ s⁻¹ for the highest albumin concentration. Disaggregation considerably increased with the temperature rise from 15 °C to 37 °C. Compared to albumin, Foscan® disaggregation kinetics in the presence of lipoproteins displayed poorer dependency on lipoprotein concentrations and smaller variations in disaggregation rate constants. Gel-filtration chromatography analysis of Foscan® in albumin solutions demonstrated the presence of aggregated fraction of free, non-bound to protein Foscan® and monomeric Foscan®, bound to protein.     

Bacterial Peptide deformylase inhibition of cyano substituted biaryl analogs: Synthesis,in vitro biological evaluation,molecular docking study and in silico ADME prediction

Herein, we report the synthesis and screening of cyano substituted biaryl analogs 5(a–m) as Peptide deformylase (PDF) enzyme inhibitors. The compounds 5a (IC₅₀ value = 13.16 μM), 5d (IC₅₀ value = 15.66 μM) and 5j (IC₅₀ value = 19.16 μM) had shown good PDF inhibition activity. The compounds 5a (MIC range = 11.00–15.83 μg/mL), 5b (MIC range = 23.75–28.50 μg/mL) and 5j (MIC range = 7.66–16.91 μg/mL) had also shown potent antibacterial activity when compared with ciprofloxacin (MIC range = 25–50 μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF, the synthesized compounds 5(a–m) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates.     

Analysis of lidocaine interactions with serum proteins using high-performance affinity chromatography

High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α₁-acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (K_a) of 1.1–1.7 × 10⁵ M^?1 at 37 °C and pH 7.4. Lidocaine had weak to moderate binding to HSA, with a K_a in the range of 10³ to 10⁴ M^?1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes.     

Human growth hormone-specific aptamer identification using improved oligonucleotide ligand evolution method

LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoiding cross reactions of aptamers with Bacillus subtilis extracellular proteins by negative SELEX, the effects of time and pH on aptamer binding to rhGH were investigated. The highest binding efficiency of aptamers on rhGH-immobilized microparticles was obtained at pH 7.0. The aptamers that interacted with rhGH were eluted by a multi-stage step-up temperature gradient in ΔT = 10 °C increments within the range T = 55–95 °C; and the strongest affinity binding was disrupted at T = 85 °C where C_Apt = 0.16 μM was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K_D = 218 nM rhGH. RhGH was separated from the fermentation broth with 99.8% purity, indicating that the method developed is properly applicable even for an anionic protein.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.     

Towards understanding the unbound state of drug compounds: Implications for the intramolecular reorganization energy upon binding

There has been an explosion of structural information for pharmaceutical compounds bound to biological targets, but the conformations and dynamics of compounds free in solution are poorly characterized, if at all. Yet, knowledge of the unbound state is essential to understand the fundamentals of molecular recognition, including the much debated conformational intramolecular reorganization energy of a compound upon binding (ΔE_Reorg). Also, dependable observation of the unbound compounds is important for ligand-based drug discovery, e.g. with pharmacophore modelling. Here, these questions are addressed with long (⩾0.5 μs) state-of-the-art molecular dynamics (MD) simulations of 26 compounds (including 7 approved drugs) unbound in explicit solvent. These compounds were selected to be chemically diverse, with a range of flexibility, and good quality bioactive X-ray structures. The MD-simulated free compounds are compared to their bioactive structure and conformers generated with ad hoc sampling in vacuo or with implicit generalized Born (GB) aqueous solvation models. The GB conformational models clearly depart from those obtained in explicit solvent, and suffer from conformational collapse almost as severe as in vacuo. Thus, the global energy minima in vacuo or with GB are not suitable representations of the unbound state, which can instead be extensively sampled by MD simulations. Many, but not all, MD-simulated compounds displayed some structural similarity to their bioactive structure, supporting the notion of conformational pre-organization for binding. The ligand–protein complexes were also simulated in explicit solvent, to estimate ΔE_Reorg as an enthalpic difference ΔH_Reorg between the intramolecular energies in the bound and unbound states. This fresh approach yielded ΔH_Reorg values ⩽ 6 kcal/mol for 18 out of 26 compounds. For three particularly polar compounds 15 ⩽ ΔH_Reorg ⩽ 20 kcal/mol, supporting the notion that ΔH_Reorg can be substantial. Those large ΔH_Reorg values correspond to a redistribution of electrostatic interactions upon binding. Overall, the study illustrates how MD simulations offer a promising avenue to characterize the unbound state of medicinal compounds.     

Schiff’s base derivatives bearing nitroimidazole moiety: New class of antibacterial,anticancer agents and potential EGFR tyrosine kinase inhibitors

New Schiff’s base derivatives 5a–5h have been synthesized by reaction between 1-(4-bromophenyl)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanone 3 and various benzohydrazide 4a–4h in presence of nickel (II) nitrate as a catalyst in ethanol at room temperature in good yield (54–88%). All compounds were tested for antibacterial as well as anticancer and inhibition of EGFR. Of the compounds studied, compounds 5d, 5f and 5g in the case of antiproliferation and inhibition of EGFR as well as compounds 5b, 5c, 5e and 5h in the case of antibacterial activity were found to be most effective compounds in the series. Compound 5f shows effective inhibition (IC₅₀ = 0.21 ± 0.02 μM) by binding in to the active pocket of EGFR receptor with minimum binding energy (ΔG_b = ?49.4869 kcal/mol).     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Synthesis and characterization of novel 1,2,4-triazine derivatives with antiproliferative activity

A series of novel small molecules with a 1,2,4-triazine scaffold was obtained according to a recently published and highly efficient synthetic route. Screening for antiproliferative and cytotoxic activity revealed distinct anticancer effects against the human leukemia cell line K-562 combined with a remarkable low cytotoxicity. All compounds were in agreement with the ‘rule-of-five’ claims by Lipinski and calculated log P_calc values were experimentally confirmed (log P_exp). For the most active compounds, in vitro serum albumin binding was investigated and structure–activity relationships were established.     

Syntheses of 4,6-dihydroxypyrimidine diones,their urease inhibition,in vitro,in silico,and kinetic studies

A library of 4,6-dihydroxypyrimidine diones (1–35) were synthesized and evaluated for their urease inhibitory activity. Structure-activity relationships, and mechanism of inhibition were also studied. All compounds were found to be active with IC₅₀ values between 22.6 ± 1.14–117.4 ± 0.73 µM, in comparison to standard, thiourea (IC₅₀ = 21.2 ± 1.3 µM). Kinetics studies on the most active compounds 2–7, 16, 17, 28, and 33 were performed to investigate their modes of inhibition, and dissociation constants K_i. Compounds 2, 3, 7, 16, 28, and 33 were found to be mixed-type of inhibitors with K_i values in the range of 7.91 ± 0.024–13.03 ± 0.013 µM, whereas, compounds 4–6, and 17 were found to be non-competitive inhibitors with K_i values in the range of 9.28 ± 0.019–13.05 ± 0.023 µM. In silico study was also performed, and a good correlation was observed between experimental and docking studies. This study is continuation of our previously reported urease inhibitory activity of pyrimidine diones, representing potential leads for further research as possible treatment of diseases caused by ureolytic bacteria.     

Synthesis and structure–activity relationship of 3β-(4-alkylthio, -methylsulfinyl,and -methylsulfonylphenyl)tropane and 3β-(4-alkylthiophenyl)nortropane derivatives for monoamine transporters

Early studies led to the identification of 3β-(4-methoxyphenyl)tropane-2β-carboxylic acid methyl ester (5) with high affinity at the DAT (IC₅₀ = 6.5 nM) and 5-HTT (K_i = 4.3 nM), while having much less affinity at the NET (K_i = 1110 nM). In the present study, we replaced the 4′-methoxy group of the 3β-phenyl ring with a bioisosteric 4′-methylthio group to give 7a. We also synthesized a number of 3β-(4-alkylthiophenyl)tropanes 7b–e, 3β-(4-methylsulfinylphenyl) and 3β-(4-methylsulfonylphenyl)tropane analogues 7f–h as well as the 3β-(4-alkylthiophenyl)nortropane derivatives 8–11 to further characterize the structure–activity relationship of this type of compound for binding at monoamine transporters. With exception of the 4′-methylsulfonyl analogue 7h, all the tested compounds possessed high binding affinities at the 5-HTT. The K_i values ranged from 0.19 nM to 49 nM. The 3β-(4-methylthiophenyl)tropane 7a and its N-(3-fluoropropyl) analogue 9a and N-allyl analogue 10a are the most selective compounds for the 5-HTT over the NET (NET/5-HTT = 314–364) in the series. However, none of the compounds showed selectivity similar to 5 for both the DAT and 5-HTT relative to the NET. This study provided useful SAR information for rational design of potent and selective monoamine transporter inhibitors.     

5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human carbonic anhydrases I,II, IX and XII: Solution and X-ray crystallographic studies

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors (K_Is in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.     

Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II,IX and XII

A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (K_Is in the range of 87 nM–4.35 μM), hCA II was moderately inhibited by most of the new compounds (K_Is in the range of 12.5–130 nM), whereas the tumor associated isoforms were potently inhibited, with K_Is in the range of 1.2–34.1 nM against hCA IX and of 2.1–33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.     

Synthesis,X-ray crystal structure,DNA/protein binding and cytotoxicity studies of five α-aminophosphonate N-derivatives

Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal structures of 1–5 are monoclinic or triclinic system with the space group P 21/c, P − 1, P − 1, P2(1)/c and P − 1, respectively. All P atoms of 1–5 have tetrahedral geometries involving two O-ethyl groups, one C_α atom, and a double bond O atom. The binding interaction of five new α-aminophosphonate N-derivatives (1–5) with calf thymus(CT)-DNA have been investigated by UV–visible and fluorescence emission spectrometry. The apparent binding constant (Kapp) values follows the order: 1 (3.38 × 10⁵ M⁻¹) > 2 (3.04 × 10⁵ M⁻¹) > 4 (2.52 × 10⁵ M⁻¹) > 5 (2.32 × 10⁵ M⁻¹) > 3 (2.10 × 10⁵ M⁻¹), suggesting moderate intercalative binding mode between the compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the compounds 1–5 showed that the quenching mechanism might be a static quenching procedure. For the compounds 1–5, the number of binding sites were about one for BSA and the binding constants follow the order: 1 (2.72 × 10⁴ M⁻¹) > 2 (2.27 × 10⁴ M⁻¹) > 4 (2.08 × 10⁴ M⁻¹) > 5 (1.79 × 10⁴ M⁻¹) > 3 (1.17 × 10⁴ M⁻¹). Moreover, the DNA cleavage abilities of 1 exhibit remarkable changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have been examined by MTT and shown antitumor effect on the tested cells.     

Design of a binuclear Ni(II)–iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag fused proteins

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions under live-cell conditions. In this Letter, we report the design of the binuclear Ni(II)–iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag-fused proteins. We found that the Ni(II)–IDA complex 1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding affinity (K_d = 24 nM), the value of which is 16-fold higher than the conventional Ni(II)–NTA complex (K_d = 390 nM). The strong binding affinity of the Ni(II)–IDA complex was successfully used in the covalent labeling and fluorescence bioimaging of a His-tag fused GPCR (G-protein coupled receptor) located on the surface of living cells.