Stoichiometric Inhibition Research Articles

Discovery of Oral Degraders of the ROS1 Fusion Protein with Potent Activity against Secondary Resistance Mutations.

The development of therapeutic resistance in the majority of patients limits the long-term benefit of ROS1 inhibitor treatment. On-target mutations of the ROS1 kinase domain confer resistance to crizotinib and lorlatinib in more than one-third of acquired resistance cases with no current effective treatment option. As an alternative to stoichiometric inhibition, proteolytic degradation of ROS1 could provide an effective tool to combat resistance generated by these mutations. Our study has identified a potent, orally active ROS1 degrader with an excellent pharmacokinetics profile. The degrader can effectively inhibit ROS1-dependent cell proliferation and tumor growth by degrading the ROS1 kinase, thereby eliminating the active phospho-ROS1. More importantly, the degradation-based therapeutic modality can overcome on-target mutation resistance to tyrosine kinase inhibitors by efficient degradation of the mutated kinase to achieve greater potency than inhibition.

Vaccinia Virus Defective Particles Lacking the F17 Protein Do Not Inhibit Protein Synthesis: F17, a Double-Edged Sword for Protein Synthesis?

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

Exploring cell cycle-mediated regulations of glycolysis in budding yeast.

Coordination of cell cycle with metabolism exists in all cell types that grow by division. It serves to build a new cell, (i) fueling building blocks for the synthesis of proteins, nucleic acids, and membranes, and (ii) producing energy through glycolysis. Cyclin-dependent kinases (Cdks) play an essential role in this coordination, thereby in the regulation of cell division. Cdks are functional homologs across eukaryotes and are the engines that drive cell cycle events and the clocks that time them. Their function is counteracted by stoichiometric inhibitors; specifically, inhibitors of cyclin-cyclin dependent kinase (cyclin/Cdk) complexes allow for their activity at specific times. Here, we provide a new perspective about the yet unknown cell cycle mechanisms impacting on metabolism. We first investigated the effect of the mitotic cyclin/Cdk1 complex Cyclin B/Cdk1-functional homolog in mammalian cells of the budding yeast Clb2/Cdk1-on yeast metabolic enzymes of, or related to, the glycolysis pathway. Six glycolytic enzymes (Glk1, Hxk2, Pgi1, Fba1, Tdh1, and Pgk1) were subjected to in vitro Cdk-mediated phosphorylation assays. Glucose-6-phosphate dehydrogenase (Zwf1), the first enzyme in the pentose phosphate pathway that is important for NADPH production, and 6-phospho-fructo-2-kinase (Pfk27), which catalyzes fructose-2,6-bisphosphate synthesis, a key regulator of glycolysis, were also included in the study. We found that, among these metabolic enzymes, Fba1 and Pgk1 may be phosphorylated by Cdk1, in addition to the known Cdk1-mediated phosphorylation of Gph1. We then investigated the possible effect of Sic1, stoichiometric inhibitor of mitotic cyclin/Cdk1 complexes in budding yeast, on the activities of three most relevant glycolytic enzymes: Hxk2, Glk1, and Tdh1. We found that Sic1 may have a negative effect on Hxk2. Altogether, we reveal possible new routes, to be further explored, through which cell cycle may regulate cellular metabolism. Because of the functional homology of cyclin/Cdk complexes and their stoichiometric inhibitors across evolution, our findings may be relevant for the regulation of cell division in eukaryotes.

Formulation and Characterization of Hyaluronic Acid Anchored Polycaprolactone Nanoparticles for Delivery of Anticancer Drugs

Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase, is a chemotherapeutic agent for treating a variety of neoplasms. The objective of the present study was to synthesize nanoparticles of MTX using hyaluronic acid-polyethyleneglycol-polycaprolactone (HA-PEG-PCL) copolymer for tumor targeting. Targeting efficiency of HA-PEG-PCL nanoparticles was compared with non-HA-containing nanoparticles polycaprolactone. The copolymers were chemically synthesized and characterized by IR and Nuclear Magnetic Resonance (NMR) spectroscopes. The nanoparticles were characterized for shape and morphology by transmission electron microscopy (TEM), particle size, percentage of drug entrapment and in-vitro drug release profile. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies were also performed to appraise the crystalline or amorphous nature of MTX inside the polymer matrix. Formulations were prepared using different MTX: polymer ratios (1:0.05-3:0.15 w/w) and the optimum formulation with the drug: polymer ratio of 1:0.05 showed the mean particle size of 185 ± 2.21 nm and entrapment efficiency of 93.16 ± 2.1% in the case of HA-PEG-PCL nanoparticles, while the values were 140 ± 1.24 nm and 90.12 ± 1.3%, respectively, in the case of PCL nanoparticles. The nanoparticles prepared using copolymers HA-PEG-PCL showed their potential to sustain the MTX release. MTX-HA-PEG-PCL nanoparticles showed 95.08 ± 2.50% release of MTX in 96 hours while MTX-PCL nanoparticles released almost 100% of the drug in the same duration. The stability was evaluated in terms of particle size and residual drug content, the nanoparticles formulations were found to be more stable at 5 ± 3°C and then 40 ± 2°C. These results suggest that HA-PEG-PCL nanoparticles could be an efficient delivery system for MTX for targeted therapy of cancer.

Protein therapeutics and their lessons: Expect the unexpected when inhibiting the multi-protein cascade of the complement system.

Over a century after the discovery of the complement system, the first complement therapeutic was approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). It was a long-acting monoclonal antibody (aka 5G1-1, 5G1.1, h5G1.1, and now known as eculizumab) that targets C5, specifically preventing the generation of C5a, a potent anaphylatoxin, and C5b, the first step in the eventual formation of membrane attack complex. The enormous clinical and financial success of eculizumab across four diseases (PNH, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), and anti-aquaporin-4 (AQP4) antibody-positive neuromyelitis optica spectrum disorder (NMOSD)) has fueled a surge in complement therapeutics, especially targeting diseases with an underlying complement pathophysiology for which anti-C5 therapy is ineffective. Intensive research has also uncovered challenges that arise from C5 blockade. For example, PNH patients can still face extravascular hemolysis or pharmacodynamic breakthrough of complement suppression during complement-amplifying conditions. These "side" effects of a stoichiometric inhibitor like eculizumab were unexpected and are incompatible with some of our accepted knowledge of the complement cascade. And they are not unique to C5 inhibition. Indeed, "exceptions" to the rules of complement biology abound and have led to unprecedented and surprising insights. In this review, we will describe initial, present and future aspects of protein inhibitors of the complement cascade, highlighting unexpected findings that are redefining some of the mechanistic foundations upon which the complement cascade is organized.

PCNA degradation exhibits superior pharmacological effects over stoichiometric inhibition

In this issue of Cell Chemical Biology, Chang etal. report that a PCNA-targeting bioPROTAC, an engineered E3 ligase linked to a PCNA-binding peptide, has superior pharmacological effects over inhibition of PCNA. Their findings demonstrate the feasibility of translating bioPROTACs from a concept to a candidate therapeutic modality.

Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death

Biodegraders are targeted protein degradation constructs composed of mini-proteins/peptides linked to E3 ligase receptors. We gained deeper insights into their utility by studying Con1-SPOP, a biodegrader against proliferating cell nuclear antigen (PCNA), an oncology target. Con1-SPOP proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut) as it had more potent anti-proliferative effects and uniquely induced DNA damage, cell apoptosis, and necrosis. Proteomics showed that PCNA degradation gave impaired mitotic division and mitochondria dysfunction, effects not seen with the stoichiometric inhibitor. We further showed that doxycycline-induced Con1-SPOP achieved complete tumor growth inhibition invivo. Intracellular delivery of mRNA encoding Con1-SPOP via lipid nanoparticles (LNPs) depleted endogenous PCNA within hours of application with nanomolar potency. Our results demonstrate the utility of biodegraders as biological tools and highlight target degradation as a more efficacious approach versus stoichiometric inhibition. Once invivo delivery is optimized, biodegraders may be leveraged as an exciting therapeutic modality.

An MDM2 degrader for treatment of acute leukemias.

In acute myeloid leukemia (AML), p53 tumor suppressor activity can be reduced due to enhanced expression of MDM2 which promotes the degradation of p53. In TP53 wild-type malignancies, therapy with small molecule antagonists of MDM2 results in antileukemic activity. Current treatment strategies, however, have been limited by poor tolerability and incomplete clinical activity. We have developed a proteolysis-targeting chimera (PROTAC) MS3227 that targets MDM2 by recruiting the E3 ligase Von Hippel-Lindau, resulting in proteasome-dependent degradation of MDM2. In WT TP53 leukemia cell lines, MS3227 led to activation of p53 targets p21, PUMA, and MDM2 and resulted in cell-cycle arrest, apoptosis, and decreased viability. The catalytic PROTAC MS3227 led to more potent activation when compared to a stoichiometric inhibitor, in part by dampening the negative feedback mechanism in the p53 - MDM2 circuit. The effectiveness of MS3227 was also observed in primary patient specimens with selectivity towards leukemic blasts. The addition of MS3227 enhanced the activity of other anti-leukemic agents including azacytidine, cytarabine, and venetoclax. In particular, MS3227 treatment was shown to downregulate MCL-1, a known mediator of resistance to venetoclax. A PROTAC-based approach may provide a means of improving MDM2 inhibition to gain greater therapeutic potential in AML.

Estimation of the efficiency of the C60 and C70 fullerenes as inhibitors of the radical chain oxidation of сumene

In the present work, the antioxidant efficiency of the C60 and C70 fullerenes has been quantitatively assessed with two methods: the chemiluminescence method and semilogarithmic method for processing kinetic curves based on the transformation of kinetic absorption curves at AIBN-induced cumene oxidation. As found, the measured values of the stoichiometric inhibition coefficient for C70 fullerene (f = 5.1 and 3.2) are higher than for C60 (f = 1.1 and 1.1), which indicates a higher inhibitory activity of the C70 fullerene as compared with C60. At the same time, both fullerenes demonstrate lower reactivity toward peroxyl radicals generated upon the hydrocarbon oxidation in comparison with well-known antioxidants, ionol and α-tocopherol.

Targeted Degradation of PCNA Outperforms Stoichiometric Inhibition to Result in Programed Cell Death

Targeted Degradation of PCNA Outperforms Stoichiometric Inhibition to Result in Programed Cell Death

Константа скорости реакции пероксильного радикала 1,4-диоксана с α-токоферолом и тролоксом

The study of the properties of water-soluble antioxidants is of practical interest. Research is hindered by the fact that such substances are poorly soluble in substrates, which are most widespread in determining the quantitative characteristics of antioxidants. Usually it is cumene, ethylbenzene, styrene. In this work, we used a method based on a model reaction of the radical chain 1,4-dioxane oxidation. This substrate is the most suitable solvent for evaluating the water-soluble substances antioxidant activity. The inhibitory activity of α-tocopherol (vitamin E) and trolox (a water-soluble analogue of α-tocopherol) was studied in a model system of radical chain 1,4-dioxane oxidation. Air oxygen served as the oxidizing agent. The experiments were carried out at 333 K. Initiation was carried out with 2,2'-azo-bis-isobutyronitrile. The reaction kinetics was monitored by the rate of oxygen uptake using a manometric setup. In this work to measure the initiation rate, the inhibitors method is used. The standard α-tocopherol inhibitor was used for this purpose. α-Tocopherol is an effective inhibitor with a known stoichiometric inhibition ratio of two. The method used in our study makes it possible to measure correct the length of the induction period using the kinetics of oxygen absorption. The induction period was determined by graphical and integral methods. The initiation rate was calculated based on the induction period values. The resulting value is in satisfactory agreement with the data based on the literary value of the decay rate constant 2,2'-azo-bis-isobutyronitrile. The rate constant of the reaction of the 1,4-dioxane peroxyl radical with α-tocopherol and trolox (fk7, L mol-1s-1, 333 K) was measured: (1.6 ± 0.1)∙106, (1.2 ± 0.1)∙106. The stoichiometric inhibition coefficient for trolox in the system of radical chain 1,4-dioxane oxidation was determined: f = 2.4 ± 0.2.

EIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

Structures and Strategies of Anti-CRISPR-Mediated Immune Suppression.

More than 50 protein families have been identified that inhibit CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-mediated adaptive immune systems. Here, we analyze the available anti-CRISPR (Acr) structures and describe common themes and unique mechanisms of stoichiometric and enzymatic suppressors of CRISPR-Cas. Stoichiometric inhibitors often function as molecular decoys of protein-binding partners or nucleic acid targets, while enzymatic suppressors covalently modify Cas ribonucleoprotein complexes or degrade immune signaling molecules. We review mechanistic insights that have been revealed by structures of Acrs, discuss some of the trade-offs associated with each of these strategies, and highlight how Acrs are regulated and deployed in the race to overcome adaptive immunity.

Competitive tight-binding inhibition of VKORC1 underlies warfarin dosage variation and antidotal efficacy

AbstractDose control of warfarin is a major complication in anticoagulation therapy and overdose is reversed by the vitamin K antidote. Improving the dosage management and antidotal efficacy requires mechanistic understanding. Here we find that effects of the major predictor of warfarin dosage, SNP −1639 G>A, follow a general correlation that warfarin 50% inhibitory concentration decreases with cellular level of vitamin K epoxide reductase (VKORC1), suggesting stoichiometric inhibition. Characterization of the inhibition kinetics required the use of microsomal VKORC1 with a native reductant, glutathione, that enables effective warfarin inhibition in vitro. The kinetics data can be fitted with the Morrison equation, giving a nanomolar inhibition constant and demonstrating that warfarin is a tight-binding inhibitor. However, warfarin is released from purified VKORC1-warfarin complex with increasing amount of vitamin K, indicating competitive inhibition. The competition occurs also in cells, resulting in rescued VKORC1 activity that augments the antidotal effects of vitamin K. Taken together, warfarin is a competitive inhibitor that binds VKORC1 tightly and inhibits at a stoichiometric (1:1) concentration, whereas exceeding the VKORC1 level results in warfarin overdose. Thus, warfarin dosage control should use VKORC1 level as a major indicator, and improved antidotes may be designed based on their competition with warfarin.

Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase.

γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here, we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembrane-mimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.

Synthesis and antioxidant properties of (dodecylsulfanyl)methyl quercetin derivatives

Mono-, bis-, and tris[(dodecylsulfanyl)methyl]-substituted quercetin derivatives were obtained by the reaction of quercetin with (N,N-diethylaminomethyl) dodecyl sulfide. These compounds under the conditions of AIBN-initiated oxidation of styrene (37 °C) terminated oxidation chains with a stoichiometric inhibition coefficients of 2.0±0.1 and rate constants of 5.1 · 105, 4.8 · 105, and 2.9 · 105 L mol-1 s-1, respectively. Based on the rate constants, the O-H bond energy values in the molecules of quercetin and its mono-, bis-, and tris- [(dodecylsulfanyl) methyl]-substituted derivatives were calculated and found to be equal to 334.4, 341.4, 341.7, and 344.7 kJ mol-1, respectively. It was established that the ability of the synthesized thio derivatives of quercetin to inhibit thermal autooxidation of lard (133 °C) is 1.3-2.7 times higher than that of the reference antioxidants (a-tocopherol, BHT, and BHA). According to the in silico assessment of acute toxicity for rats, the synthesized derivatives are low-toxic and non-toxic compounds.

Inhibition by polyphenols containing OH groups at different positions in the presence of lecithin

An inhibition by the semisynthetic polyphenols bearing bicyclic isobornyl substituents and OH groups arranged variously relative to each other was investigated in the presence of lecithin. A decreased efficiency of the inhibition by the phenolic antioxidants in the presence of soy bean lecithin, which manifests itself as the increased initial oxidation rate, has been revealed. The most significant changes in the inhibitory effect were found for a polyphenol containing the OH groups at the meta position, while those the smallest ones were observed for the para position relative to each other. In the case of meta-substituted polyphenol, the decreased stoichiometric inhibition coefficient was observed along with the increased initial oxidation rate. The formation of lecithin complexes with polyphenols was detected by UV-Vis and IR spectroscopy, which leads to a decrease in their inhibitory effect.

NRF2-regulated cell cycle arrest at early stage of oxidative stress response mechanism.

Oxidative stress results in activation of several signal transduction pathways controlled by the PERK-substrate NRF2 (nuclear factor erythroid 2-related factor 2); meanwhile the ongoing cell division cycle has to be blocked. It has been recently shown that Cyclin D1 got immediately down-regulated via PERK pathway in response to oxidative stress leading to cell cycle arrest. However, the effect of NRF2 on cell cycle regulation has not been explored yet. We aimed to reveal a crosstalk between PERK-substrate NRF2 and the key elements of cell cycle regulatory network upon oxidative stress using molecular biological techniques- Although Cyclin D1 level remained constant, its activity was blocked by various stoichiometric inhibitors (such as p15, p21 and p27) even at low level of oxidative stress. The activity of these CDK inhibitors completely disappeared, when the addition of oxidative agent was combined with silencing of either PERK or NRF2.This further confirms the important role of NRF2 in blocking Cyclin D1 with stoichiometric inhibitors at early stage of oxidative stress.

Antiradical Activity of Porphyrins with a Diisobornylphenol Fragment at the Macrocycle Periphery.

This article focuses on the antiradical activity of a number of 2,6-diisobornylphenol-porphyrin conjugates with various spacers between the porphyrin and phenolic fragments in the model reaction of ethylbenzene oxidation initiated by azoisobutyric acid dinitrile. The study has shown that the electronic effects of the groups directly related to the 2,6-diisobornylphenol fragment exert the predominant influence both on the reactivity of the phenolic hydroxyl group in interaction with free radicals and on the antiradical activity of the molecule as a whole. The antiradical activity of the molecule is generally less affected by the nature of the substituents in the porphyrin macrocycle, mainly due to a change in the stoichiometric inhibition coefficient in the presence of relatively easily oxidizable groups. It was found that the length of the spacer between the porphyrin and phenolic fragments does not affect the antiradical activity of the conjugate.

Methotrexate-conjugated L-lysine coated iron oxide magnetic nanoparticles for inhibition of MCF-7 breast cancer cells

Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase enzyme, is a chemotherapeutic agent for treating a diversity of neoplasms. In this study, we design and developed a new formulation of MTX that serves as drug carrier and examined its cytotoxic effect in vitro. This target drug delivery system is dependent on the release of the MTX within the lysosomal compartment. The iron oxide magnetic nanoparticles (IONPs) were first surface-coated with L-lysine and subsequently conjugated with MTX through amidation between the carboxylic acid end groups on MTX and the amine groups on the IONPs surface. MTX-conjugated L-lysine coated IONPs (F-Lys-MTX NPs) was characterized by X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, vibrating sample magnetometer, and transmission electron microscopy techniques. The cytotoxicity of the void of MTX and F-Lys-MTX NPs were compared to each other by MTT assay of the treated MCF-7 cell lines. The results showed that the ζ-potential of F-Lys-MTX NPs was about −5.49 mV and the average size was 43.72 ± 4.73 nm. Model studies exhibited the release of MTX via peptide bond cleavage in the presence of proteinase K and at low pH. These studies specify that F-Lys-MTX NPs have a very remarkable anticancer effect, for breast cancer cell lines.