The Concise Guide to PHARMACOLOGY 2023/24: Introduction and Other Protein Targets.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Structure Guided Design of Bacteriophage Qß Mutants as Next Generation Carriers for Conjugate Vaccines.

Vaccines are critical tools to treat and prevent diseases. For an effective conjugate vaccine, the carrier is crucial, but few carriers are available for clinical applications. In addition, a drawback of current protein carriers is that high levels of antibodies against the carrier are induced by the conjugate vaccine, which are known to interfere with the immune responses against the target antigen. To overcome these challenges, we obtained the near atomic resolution crystal structure of an emerging protein carrier, i.e., the bacteriophage Qß virus like particle. On the basis of the detailed structural information, novel mutants of bacteriophage Qß (mQß) have been designed, which upon conjugation with tumor associated carbohydrate antigens (TACAs), a class of important tumor antigens, elicited powerful anti-TACA IgG responses and yet produced lower levels of anticarrier antibodies as compared to those from the wild type Qß-TACA conjugates. In a therapeutic model against an aggressive breast cancer in mice, 100% unimmunized mice succumbed to tumors in just 12 days even with chemotherapy. In contrast, 80% of mice immunized with the mQß-TACA conjugate were completely free from tumors. Besides TACAs, to aid in the development of vaccines to protect against COVID-19, the mQß based conjugate vaccine has been shown to induce high levels of IgG antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating its generality. Thus, mQß is a promising next-generation carrier platform for conjugate vaccines, and structure-based rational design is a powerful strategy to develop new vaccine carriers.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Introduction and Other Protein Targets.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15537. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Mice with GNAO1 R209H Movement Disorder Variant Display Hyperlocomotion Alleviated by Risperidone.

Neurodevelopmental disorder with involuntary movements (Online Mendelian Inheritance in Man: 617493) is a severe, early onset neurologic condition characterized by a delay in psychomotor development, hypotonia, and hyperkinetic involuntary movements. Heterozygous de novo mutations in the GNAO1 gene cause neurodevelopmental disorder with involuntary movements. Gα o, the gene product of GNAO1, is the alpha subunit of Go, a member of the heterotrimeric Gi/o family of G proteins. Go is found abundantly throughout the brain, but the pathophysiological mechanisms linking Gα o functions to clinical manifestations of GNAO1-related disorders are still poorly understood. One of the most common mutant alleles among the GNAO1 encephalopathies is the c.626G>A or p.Arg209His (R209H) mutation. We developed heterozygous knock-in Gnao1 +/R209H mutant mice using CRISPR/Cas9 methodology to assess whether a mouse model could replicate aspects of the neurodevelopmental disorder with involuntary movements clinical pattern. Mice carrying the R209H mutation exhibited increased locomotor activity and a modest gait abnormality at 8-12 weeks. In contrast to mice carrying other mutations in Gnao1, the Gnao1 +/R209H mice did not show enhanced seizure susceptibility. Levels of protein expression in multiple brain regions were unchanged from wild-type (WT) mice, but the nucleotide exchange rate of mutant R209H Gα o was 6.2× faster than WT. The atypical neuroleptic risperidone has shown efficacy in a patient with the R209H mutation. It also alleviated the hyperlocomotion phenotype observed in our mouse model but suppressed locomotion in WT mice as well. In this study, we show that Gnao1 +/R209H mice mirror elements of the patient phenotype and respond to an approved pharmacological agent. SIGNIFICANCE STATEMENT: Children with de novo mutations in the GNAO1 gene may present with movement disorders with limited effective therapeutic options. The most common mutant variant seen in children with GNAO1-associated movement disorder is R209H. Here we show, using a novel Gnao1 +/R209H mouse, that there is a clear behavioral phenotype that is suppressed by risperidone. However, risperidone also affects wild-type mouse activity, so its effects are not selective for the GNAO1-associated movement disorder.

Loss-of-Function Mutations in Human Regulator of G Protein Signaling RGS2 Differentially Regulate Pharmacological Reactivity of Resistance Vasculature.

Regulator of G protein signaling 2 (RGS2) plays a role in reducing vascular contraction and promoting relaxation due to its GTPase accelerating protein activity toward Gαq. Previously, we identified four human loss-of-function (LOF) mutations in RGS2 (Q2L, D40Y, R44H, and R188H). This study aimed to investigate whether those RGS2 LOF mutations disrupt the ability of RGS2 to regulate vascular reactivity. Isolated mesenteric arteries (MAs) from RGS2-/- mice showed an elevated contractile response to 5 nM angiotensin II and a loss of acetylcholine (ACh)-mediated vasodilation. Reintroduction of a wild-type (WT) RGS2-GFP plasmid into RGS2-/- MAs suppressed the vasoconstrictor response to angiotensin II. RGS2 LOF mutants failed to suppress the angiotensin II constriction response compared with RGS2 WT. In contrast, ACh-mediated vasoconstriction was restored by expression of RGS2 WT, D40Y, and R44H but not by RGS2 Q2L or R188H. Phosphorylation of RGS2 D40Y and R44H by protein kinase G (PKG) may explain their maintained function to support relaxation in MAs. This is supported by phosphomimetic mutants and suppression of vasorelaxation mediated by RGS2 D40Y by a PKG inhibitor. These results demonstrate that RGS2 attenuates vasoconstriction in MAs and that RGS2 LOF mutations cannot carry out this effect. Among them, the Q2L and R188H mutants supported less relaxation to ACh, whereas relaxation mediated by the D40Y and R44H mutant proteins was equal to that with WT protein. Phosphorylation of RGS2 by PKG appears to contribute to this vasorelaxation. These results provide insights for precision medicine targeting the rare individuals carrying these RGS2 mutations. SIGNIFICANCE STATEMENT: Regulator of G protein signaling 2 (RGS2) has been implicated in the control of blood pressure; rare mutations in the RGS2 gene have been identified in large-scale human gene sequencing studies. Four human mutations in RGS2 that cause loss of function (LOF) in cell-based assays were examined in isolated mouse arteries for effects on both vasoconstriction and vasodilation. All mutants showed the expected LOF effects in suppressing vasoconstriction. Surprisingly, the D40Y and R44H mutant RGS2 showed normal control of vasodilation. We propose that this is due to rescue of the mislocalization phenotype of these two mutants by nitric oxide-mediated/protein kinase G-dependent phosphorylation. These mechanisms may guide drug discovery or drug repurposing efforts for hypertension by enhancing RGS2 function.

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein α-subunits, accelerating GTP hydrolysis. Selective inhibition of RGS proteins increases G-protein activity and may provide unique tissue specificity. Thiadiazolidinones (TDZDs) are covalent inhibitors that act on cysteine residues to inhibit RGS4, RGS8, and RGS19. There is a correlation between protein flexibility and potency of inhibition by the TDZD 4-[(4- fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione (CCG-50014). In the context of a single conserved cysteine residue on the α 4 helix, RGS19 is the most flexible and most potently inhibited by CCG-50014, followed by RGS4 and RGS8. In this work, we identify residues responsible for differences in both flexibility and potency of inhibition among RGS isoforms. RGS19 lacks a charged residue on the α 4 helix that is present in RGS4 and RGS8. Introducing a negative charge at this position (L118D) increased the thermal stability of RGS19 and decreased the potency of inhibition of CCG-50014 by 8-fold. Mutations eliminating salt bridge formation in RGS8 and RGS4 decreased thermal stability in RGS8 and increased potency of inhibition of both RGS4 and RGS8 by 4- and 2-fold, respectively. Molecular dynamics simulations with an added salt bridge in RGS19 (L118D) showed reduced RGS19 flexibility. Hydrogen-deuterium exchange studies showed striking differences in flexibility in the α 4 helix of RGS4, 8, and 19 with salt bridge-modifying mutations. These results show that the α 4 salt bridge-forming residue controls flexibility in several RGS isoforms and supports a causal relationship between RGS flexibility and the potency of TDZD inhibitors. SIGNIFICANCE STATEMENT: Inhibitor potency is often viewed in relation to the static structure of a target protein binding pocket. Using both experimental and computation studies we assess determinants of dynamics and inhibitor potency for three different RGS proteins. A single salt bridge-forming residue determines differences in flexibility between RGS isoforms; mutations either increase or decrease protein motion with correlated alterations in inhibitor potency. This strongly suggests a causal relationship between RGS protein flexibility and covalent inhibitor potency.

Interplay of cysteine exposure and global protein dynamics in small-molecule recognition by a regulator of G-protein signaling protein.

Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein-protein interaction assay on a single-cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo-state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side-chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations in the RGS/Gα protein-protein interface.

Interpreting Hydrogen-Deuterium Exchange Events in Proteins Using Atomistic Simulations: Case Studies on Regulators of G-Protein Signaling Proteins.

Hydrogen-deuterium exchange (HDX) experiments are widely used in studies of protein dynamics. To predict the propensity of amide hydrogens for exchange with deuterium, several models have been reported in which computations of amide-hydrogen protection factors are carried out using molecular dynamics (MD) simulations. Given significant variation in the criteria used in different models, the robustness and broader applicability of these models to other proteins, especially homologous proteins showing distinct amide-exchange patterns, remains unknown. The sensitivity of the predictions when MD simulations are conducted with different force-fields is yet to tested and quantified. Using MD simulations and experimental HDX data on three homologous signaling proteins, we report detailed studies quantifying the performance of seven previously reported models (M1-M7) of two general types: empirical and fractional-population models. We find that the empirical models show inconsistent predictions but predictions of the fractional population models are robust. Contrary to previously reported work, we find that the solvent-accessible surface area of amide hydrogens is a useful metric when combined with a new metric defining the distances of amide hydrogens from the first polar atoms in proteins. On the basis of this, we report two new models, one empirical (M8) and one population-based (M9). We find strong protection of amide hydrogens from solvent exchange both within the stable helical motifs and also in the interhelical loops. We further observe that the exchange-competent states of amide hydrogens occur on the sub 100 ps time-scale via localized fluctuations, and such states among amides of a given protein do not appear to show any cooperativity or allosteric coupling.

Designing for scale: optimising the health information system architecture for mobile maternal health messaging in South Africa (MomConnect).

MomConnect is a national initiative coordinated by the South African National Department of Health that sends text-based mobile phone messages free of charge to pregnant women who voluntarily register at any public healthcare facility in South Africa. We describe the system design and architecture of the MomConnect technical platform, planned as a nationally scalable and extensible initiative. It uses a health information exchange that can connect any standards-compliant electronic front-end application to any standards-compliant electronic back-end database. The implementation of the MomConnect technical platform, in turn, is a national reference application for electronic interoperability in line with the South African National Health Normative Standards Framework. The use of open content and messaging standards enables the architecture to include any application adhering to the selected standards. Its national implementation at scale demonstrates both the use of this technology and a key objective of global health information systems, which is to achieve implementation scale. The system's limited clinical information, initially, allowed the architecture to focus on the base standards and profiles for interoperability in a resource-constrained environment with limited connectivity and infrastructural capacity. Maintenance of the system requires mobilisation of national resources. Future work aims to use the standard interfaces to include data from additional applications as well as to extend and interface the framework with other public health information systems in South Africa. The development of this platform has also shown the benefits of interoperability at both an organisational and technical level in South Africa.

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors.

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations.

OBJECTIVE: To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene. METHODS: We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor. RESULTS: Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; <90% maximal inhibition). Six other mutations show variable levels of expression but exhibit normal or even gain-of-function (GOF) behavior, as demonstrated by significantly lower EC50 values for α2A adrenergic receptor-mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures. CONCLUSIONS: Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.

The DRY motif and the four corners of the cubic ternary complex model.

Recent structural data on GPCRs using a variety of spectroscopic approaches suggest that GPCRs adopt a dynamic conformational landscape, with ligands stabilizing subsets of these states to activate one or more downstream signaling effectors. A key outstanding question posed by this emerging dynamic structural model of GPCRs is what states, active, inactive, or intermediate are captured by the numerous crystal structures of GPCRs complexed with a variety of agonists, partial agonists, and antagonists. In the early nineties the discovery of inverse agonists and constitutive activity led to the idea that the active receptor state (R⁎) is an intrinsic property of the receptor itself rather than of the RG complex, eventually leading to the formulation of the cubic ternary complex model (CTC). Here, by a careful analysis of a series of data obtained with a number of mutants of the highly conserved E/DRY motif, we show evidences for the existence of all the receptor states theorized by the CTC, four 'uncoupled (R, R⁎ and HR and HR⁎), and, consequently four 'coupled' (RG, R⁎G, HRG and HR⁎G). The E/DRY motif located at the cytosolic end of transmembrane helix III of Class A GPCRs has been widely studied and analyzed because it forms a network of interactions believed to lock receptors in the inactive conformation (R), and, thus, to play a key role in receptor activation. Our conclusions are supported by recent crystal and NMR spectra, as well as by results obtained with two prototypical GPCRs using a new FRET technology that de-couples G protein binding to the receptor from signal transduction. Thus, despite its complexity and limitations, we propose that the CTC is a useful framework to reconcile pharmacological, biochemical and structural data.

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value.

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild-type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl-TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl-triacylglycerols (acetyl-TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl-TAG levels to up to 85 mol% in field-grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl-TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn-3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl-TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl-TAG oils were reduced, enabling use of this oil in several nonfood and food applications.

An annotated database of Arabidopsis mutants of acyl lipid metabolism.

KEY MESSAGE: We have constructed and annotated a web-based database of over 280 Arabidopsis genes that have characterized mutants associated with Arabidopsis acyl lipid metabolism. Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. In addition, the database of mutants is integrated within the ARALIP plant acyl lipid metabolism website ( http://aralip.plantbiology.msu.edu ) so that information on mutants is displayed on and can be accessed from metabolic pathway maps. Mutants for at least 30% of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. The database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.

Clinical inquiries. Which drugs are best when aggressive Alzheimer's patients need medication?

Atypical antipsychotics are effective; so are selective serotonin reuptake inhibitors (SSRIs), and they may be safer. Atypical antipsychotics are an effective short-term (6-12 weeks) treatment for aggressive behavior in patients with Alzheimer's disease because they consistently decrease aggression scores (strength of recommendation [SOR]: A, multiple randomized controlled trials [RCTs]). However, evidence of drug-related deaths in patients taking these drugs mandates weighing the benefits against the risks. SSRIs may be a safer, effective alternative (SOR: B, limited studies).

Health information system reform in South Africa: developing an essential data set.

Health services are increasingly under pressure to develop information systems that are responsive to changing health needs and appropriate to service objectives. Developing an essential data set provides managers with a clearly defined set of indicators for monitoring and evaluating services. This article describes a process that resulted in the creation of an essential data set at district level. This had a significant impact on neighbouring districts and resulted in the development of a regional essential data set, which in turn helped to influence the creation of a provincial and then national essential data set. Four key lessons may be drawn from the process. The development of an essential data set both requires and can contribute to a process that allows the reporting requirements to be adjusted over time in response to changing circumstances. In addition, it contributes to (and requires) the integration of programme reporting requirements into a coherent information system. While the case study describes a bottom-up approach, a top-down consultative process is advocated because it establishes a framework within which information needs can be reviewed. Lastly, the use of surveys can aid efforts to keep the essential elements to a minimum. In conclusion, the development of an essential data set contributes to strengthening health services because it necessitates dialogue between programme managers and defines indicators to be monitored by them.