THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Enzymes

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 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 (http://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 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.14752. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors 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‐2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

couple covalently to the enzyme. It is beyond the scope of the Guide to give mechanistic information about the inhibitors described, although generally this information is available from the indicated literature. Many enzymes require additional entities for functional activity.

L-Arginine turnover
Enzymes → L-Arginine turnover Overview: L-arginine is a basic amino acid with a guanidino sidechain. As an amino acid, metabolism of L-arginine to form L-ornithine, catalysed by arginase, forms the last step of the urea production cycle. L-Ornithine may be utilised as a precursor of polyamines (see Carboxylases and Decarboxylases) or recycled via L-argininosuccinic acid to L-arginine. L-Arginine may itself be decarboxylated to form agmatine, although the prominence of this pathway in human tissues is uncertain. L-Arginine may be used as a precursor for guanidoacetic acid formation in the creatine synthesis pathway under the influence of arginine:glycine amidinotransferase with L-ornithine as a byproduct. Nitric oxide synthase uses L-arginine to generate nitric oxide, with L-citrulline also as a byproduct. L-Arginine in proteins may be subject to post-translational mod-ification through methylation, catalysed by protein arginine methyltransferases. Subsequent proteolysis can liberate asymmetric N G ,N G -dimethyl-L-arginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase activities. ADMA is hydrolysed by dimethylarginine dimethylhydrolase activities to generate L-citrulline and dimethylamine. They generate both mono-methylated and dimethylated products; these may be symmetric (SDMA) or asym-metric (N G ,N G -dimethyl-L-arginine) versions, where both guanidine nitrogens are monomethylated or one of the two is dimethylated, respectively.
Information on members of this family may be found in the online database. Arginase Enzymes → L-Arginine turnover → Arginase Overview: Arginase (EC 3.5.3.1) are manganese-containing isoforms, which appear to show differential distribution, where the ARG1 isoform predominates in the liver and erythrocytes, while ARG2 is associated more with the kidney.
Information on members of this family may be found in the online database.

Nitric oxide synthases
Enzymes → L-Arginine turnover → Nitric oxide synthases Overview: Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [420] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca 2+ /calmodulin (CALM1 CALM2 CALM3, P62158) with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC 50 values in the micromolar range.

Nomenclature
Endothelial

Comments
The presence of a functional ADC activity in human tissues has been questioned [110].
AADC is a homodimer. Inhibited by AMP-activated protein kinase-evoked phosphorylation [515] The activity of ODC is regulated by the presence of an antizyme (ENSG00000104904) and an ODC antizyme inhibitor (ENSG00000155096).

Catecholamine turnover
Enzymes → Catecholamine turnover Overview: Catecholamines are defined by the presence of two adjacent hydroxyls on a benzene ring with a sidechain containing an amine.
These hormone/transmitters are synthesized by sequential metabolism from L-phenylalanine via L-tyrosine. Hydroxylation of L-tyrosine generates levodopa, which is decarboxylated to form dopamine. Hydroxylation of the ethylamine sidechain generates (-)-noradrenaline (norepinephrine), which can be methylated to form (-)-adrenaline (epinephrine). In particular neuronal and adrenal chromaffin cells, the catecholamines dopamine, (-)-noradrenaline and (-)-adrenaline are accumulated into vesicles under the influence of the vesicular monoamine transporters (VMAT1/SLC18A1 and VMAT2/SLC18A2). After release into the synapse or the blood-stream, catecholamines are accumulated through the action cell-surface transporters, primarily the dopamine (DAT/SLC6A3) and norepinephrine transporter (NET/SLC6A2). The primary routes of metabolism of these catecholamines are oxidation via monoamine oxidase activities of methylation via catechol Omethyltransferase. Comments PAH is an iron bound homodimer or -tetramer from the same structural family as tyrosine 3-monooxygenase and the tryptophan hydroxylases. Deficiency or loss-of-function of PAH is associated with phenylketonuria Tyrosine may also be metabolized in the liver by tyrosine transaminase to generate 4-hydroxyphenylpyruvic acid, which can be further metabolized to homogentisic acid. TAT is a homodimer, where loss-of-function mutations are associated with type II tyrosinemia.

Nomenclature
TH is a homotetramer, which is inhibited by dopamine and other catecholamines in a physiological negative feedback pathway [128].
DBH is a homotetramer. A protein structurally-related to DBH (MOXD1, Q6UVY6) has been described and for which a function has yet to be identified [87].

Ceramide turnover
Enzymes → Ceramide turnover Overview: Ceramides are a family of sphingophospholipids synthesized in the endoplasmic reticulum, which mediate cell stress responses, including apoptosis, autophagy and senescence, Serine palmitoyltransferase generates 3-ketosphinganine, which is reduced to sphinganine (dihydrosphingosine). N-Acylation allows the formation of dihydroceramides, which are subsequently re-duced to form ceramides. Once synthesized, ceramides are trafficked from the ER to the Golgi bound to the ceramide transfer protein, CERT (COL4A3BP, Q9Y5P4). Ceramide can be metabolized via multiple routes, ensuring tight regulation of its cellular levels. Addition of phosphocholine generates sphingomyelin while carbohydrate is added to form glucosyl-or galactosylceramides.
Ceramidase re-forms sphingosine or sphinganine from ceramide or dihydroceramide. Phosphorylation of ceramide generates ceramide phosphate. The determination of accurate kinetic parameters for many of the enzymes in the sphingolipid metabolic pathway is complicated by the lipophilic nature of the substrates.

Ceramide synthase
Enzymes → Ceramide turnover → Ceramide synthase Overview: This family of enzymes, also known as sphingosine N-acyltransferase, is located in the ER facing the cytosol with an as-yet undefined topology and stoichiometry. Ceramide synthase in vitro is sensitive to inhibition by the fungal derived toxin, fumonisin B1.

Sphingomyelin synthase
Enzymes → Ceramide turnover → Sphingomyelin synthase Overview: Following translocation from the ER to the Golgi under the influence of the ceramide transfer protein, sphingomyelin synthases allow the formation of sphingomyelin by the transfer of phosphocholine from the phospholipid phosphatidylcholine. Sphingomyelin synthase-related protein 1 is structurally related but lacks sphingomyelin synthase activity.

Comments
Glycoceramides are an extended family of sphingolipids, differing in the content and organization of the sugar moieties, as well as the acyl sidechains.

Acid ceramidase
Enzymes → Ceramide turnover → Acid ceramidase Overview: The six human ceramidases may be divided on the basis of pH optimae into acid, neutral and alkaline ceramidases, which also differ in their subcellular location.

Neutral ceramidases
Enzymes → Ceramide turnover → Neutral ceramidases Overview: The six human ceramidases may be divided on the basis of pH optimae into acid, neutral and alkaline ceramidases, which also differ in their subcellular location.

Comments
The enzyme is associated with the plasma membrane [584].

-
Comments: ASAH2B appears to be an enzymatically inactive protein, which may result from gene duplication and truncation.

Alkaline ceramidases
Enzymes → Ceramide turnover → Alkaline ceramidases Overview: The six human ceramidases may be divided on the basis of pH optimae into acid, neutral and alkaline ceramidases, which also differ in their subcellular location. Comments: A ceramide kinase-like protein has been identified in the human genome (CERKL, Q49MI3).

Chromatin modifying enzymes
Enzymes → Chromatin modifying enzymes

Overview:
Chromatin modifying enzymes, and other chromatin-modifying proteins, fall into three broad categories: writers, readers and erasers. The function of these proteins is to dynamically maintain cell identity and regulate processes such as differentiation, development, proliferation and genome integrity via recognition of specific 'marks' (covalent post-translational modifications) on histone proteins and DNA [325]. In normal cells, tissues and organs, precise co-ordination of these proteins ensures expression of only those genes required to specify phenotype or which are required at specific times, for specific functions. Chromatin modifications allow DNA modifications not coded by the DNA sequence to be passed on through the genome and underlies heritable phenomena such as X chromosome inactivation, aging, heterochromatin formation, reprogramming, and gene silencing (epigenetic control). To date at least eight distinct types of modifications are found on histones. These include small covalent modifications such as acetylation, methylation, and phosphorylation, the attachment of larger modifiers such as ubiquitination or sumoylation, and ADP ribosylation, proline isomerization and deimination. Chromatin modifications and the functions they regulate in cells are reviewed by Kouzarides (2007) [325]. Writer proteins include the histone methyltransferases, histone acetyltransferases, some kinases and ubiquitin ligases. Readers include proteins which contain methyl-lysinerecognition motifs such as bromodomains, chromodomains, tudor domains, PHD zinc fingers, PWWP domains and MBT domains. Erasers include the histone demethylases and histone deacetylases (HDACs and sirtuins). Dysregulated epigenetic control can be associated with human diseases such as cancer [161], where a wide variety of cellular and pro-tein abberations are known to perturb chromatin structure, gene transcription and ultimately cellular pathways [35,544]. Due to the reversible nature of epigenetic modifications, chromatin regulators are very tractable targets for drug discovery and the development of novel therapeutics. Indeed, small molecule inhibitors of writers (e.g. azacitidine and decitabine target the DNA methyltransferases DNMT1 and DNMT3 for the treatment of myelodysplastic syndromes [199,637]) and erasers (e.g. the HDAC inhibitors vorinostat, romidepsin and belinostat for the treatment of T-cell lymphomas [177,309]) are already being used in the clinic. The search for the next generation of compounds with improved specificity against chromatin-associated proteins is an area of intense basic and clinical research [71]. Current progress in this field is reviewed by Simó-Riudalbas and Esteller (2015) [ or tetrameric enzymes which use S-adenosyl methionine as a methyl donor, generating S-adenosylhomocysteine as a by-product.
They generate both mono-methylated and dimethylated products; these may be symmetric (SDMA) or asym-metric (N G ,N G -dimethyl-L-arginine) versions, where both guanidine nitrogens are monomethylated or one of the two is dimethylated, respectively.
Information on members of this family may be found in the online database. Overview: Histone deacetylases act as erasers of epigenetic acetylation marks on lysine residues in histones. Removal of the acetyl groups facilitates tighter packing of chromatin (heterochromatin formation) leading to transcriptional repression. The histone deacetylase family has been classified in to five subfamilies based on phylogenetic comparison with yeast homologues: Class I contains HDACs 1, 2, 3 and 8 Class IIa contains HDACs 4, 5, 7 and 9 Class IIb contains HDACs 6 and 10 Class III contains the sirtuins (SIRT1-7) Class IV contains only HDAC11. Classes I, II and IV use Zn + as a co-factor, whereas catalysis by Class III enzymes requires NAD + as a co-factor, and members of this subfamily have ADP-ribosylase activity in addition to protein deacetylase function [521]. HDACs have more general protein deacetylase activity, being able to deacetylate lysine residues in non-histone proteins [104] such as microtubules [270], the hsp90 chaperone [326] and the tumour suppressor p53 [377].
Dysregulated HDAC activity has been identified in cancer cells and tumour tissues [355,509], making HDACs attractive molecular targets in the search for novel mechanisms to treat cancer [639]. Several small molecule HDAC inhibitors are already approved for clinical use: romidepsin, belinostat, vorinostat, panobinostat, belinostat, valproic acid and tucidinostat. HDACs and HDAC inhibitors currently in development as potential anti-cancer therapeutics are reviewed by Simó-Riudalbas and Esteller (2015) [545].

Cyclic nucleotide turnover/signalling
Enzymes → Cyclic nucleotide turnover/signalling Overview: Cyclic nucleotides are second messengers generated by cyclase enzymes from precursor triphosphates and hydrolysed by phosphodiesterases. The cellular actions of these cyclic nucleotides are mediated through activation of protein kinases (cAMP-and cGMP-dependent protein kinases), ion channels (cyclic nucleotide-gated, CNG, and hyperpolarization and cyclic nucleotide-gated, HCN) and guanine nucleotide exchange factors (GEFs, Epac).

Nomenclature
Rap guanine nucleotide exchange factor 3 Rap guanine nucleotide exchange factor 4   Rank order of affinity cyclic AMP cyclic GMP [409] cyclic AMP cyclic GMP [200] cyclic AMP cyclic GMP [171] cyclic AMP cyclic GMP [249] Inhibitors crisaborole (pIC 50  PDE6 is a membrane-bound tetramer composed of two catalytic chains (PDE6A or PDE6C and PDE6B), an inhibitory chain (PDE6G or PDE6H) and the PDE6D chain. The enzyme is essentially cyclic GMP specific and is activated by the α-subunit of transducin (Gα t ) and inhibited by sildenafil, zaprinast and dipyridamole with potencies lower than those observed for PDE5A. Defects in PDE6B are a cause of retinitis pigmentosa and congenital stationary night blindness.
-.-, were originally defined by their strong absorbance at 450 nm due to the reduced carbon monoxide-complexed haem component of the cytochromes. They are an extensive family of haemcontaining monooxygenases with a huge range of both endogenous and exogenous substrates. These include sterols, fat-soluble vitamins, pesticides and carcinogens as well as drugs. The substrates of some orphan CYP are not known. Listed below are the human enzymes; their relationship with rodent CYP450 enzyme activities is obscure in that the species orthologue may not catalyse the metabolism of the same substrates. Although the majority of CYP450 enzyme activities are concentrated in the liver, the extra-hepatic enzyme activities also contribute to patho/physiological processes. Genetic variation of CYP450 isoforms is widespread and likely underlies a significant proportion of the individual variation to drug administration. Comments CYP1A1 is an extra-hepatic enzyme. It shows a preference for linear planar aromatic molecules [561].

CYP1 family
CYP1A2 is constitutively expressed in liver. It shows a preference for triangular planar aromatic molecules [561].
Mutations have been associated with primary congenitial glaucoma [569]

Comments
Metabolises a vast range of xenobiotics, including antidepressants, benzodiazepines, calcium channel blockers, and chemotherapeutic agents [675]. The active site is plastic, with both homotropic and heterotropic cooperativity observed with some substrates [534]. CYP3A4 catalyses the 25-hydroxylation of trihydroxycholestane in liver microsomes [189].
CYP3A5 is expressed extrahepatically, including in the small intestine. It has overlapping substrate specificity with CYP3A4 [126,641].
Fetal form, rarely expressed in adults. Has overlapping substrate specificity with CYP3A4 [126,641].
Fetal expression only and considered an orphan fCYP [224]. Testosterone may be a substrate [220].

Overview:
The principle endocannabinoids are 2acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (Narachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [232]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [99]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [543]). A transacylation enzyme which forms Nacylphosphatidylethanolamines has been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [443]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [14,179,555]. Comments NAPE-PLD activity appears to be enhanced by polyamines in the physiological range [362], but fails to transphosphatidylate with alcohols [467] unlike phosphatidylcholine-specific phospholipase D.
-The FAAH2 gene is found in many primate genomes, marsupials, and other distantly related vertebrates, but not a variety of lower placental mammals, including mouse and rat [634].
-Comments: Routes for N-acylethanolamine biosynthesis other than through NAPE-PLD activity have been identified [602].

Nomenclature
Diacylglycerol Comments ---WWL70 has also been suggested to have activity at oxidative metabolic pathways independent of ABHD6 [582].
-Comments on Endocannabinoid turnover: Many of the compounds described as inhibitors are irreversible and so potency estimates will vary with incubation time. FAAH2 is not found in rodents [634] and a limited range of inhibitors have been assessed at this enzyme activity. 2-arachidonoylglycerol has been reported to be hydrolysed by multiple enzyme activities from neural preparations [31], including ABHD2 (P08910) [412] and carboxylesterase 1 (CES1, P23141 [656]). ABHD2 (P08910) has also been described as a triacylglycerol lipase and ester hydrolase [384], while ABHD12 (Q8N2K0) is also able to hydrolyse lysophos-phatidylserine [598]. ABHD12 (Q8N2K0) has been described to be inhibited selectively by pentacyclic triterpenoids, such as oleanolic acid [460].

Further reading on Endocannabinoid turnover
Blankman JL et al.

Eicosanoid turnover
Enzymes → Eicosanoid turnover Overview: Eicosanoids are 20-carbon fatty acids, where the usual focus is the polyunsaturated analogue arachidonic acid and its metabolites. Arachidonic acid is thought primarily to derive from phospholipase A2 action on membrane phosphatidylcholine, and may be re-cycled to form phospholipid through con-jugation with coenzyme A and subsequently glycerol derivatives. Oxidative metabolism of arachidonic acid is conducted through three major enzymatic routes: cyclooxygenases; lipoxygenases and cytochrome P450-like epoxygenases, particularly CYP2J2. Isoprostanes are structural analogues of the prostanoids (hence the nomenclature D-, E-, F-isoprostanes and isothromboxanes), which are produced in the presence of elevated free radicals in a nonenzymatic manner, leading to suggestions for their use as biomarkers of oxidative stress. Molecular targets for their action have yet to be defined.

Leukotriene and lipoxin metabolism
Enzymes → Eicosanoid turnover → Leukotriene and lipoxin metabolism Overview: Leukotriene A 4 (LTA 4 ), produced by 5-LOX activity, and lipoxins may be subject to further oxidative metabolism; ω-hydroxylation is mediated by CYP4F2 and CYP4F3, while βoxidation in mitochondria and peroxisomes proceeds in a manner dependent on coenzyme A conjugation. Conjugation of LTA 4 at the 6 position with reduced glutathione to generate LTC 4 occurs under the influence of leukotriene C 4 synthase, with the subsequent formation of LTD 4 and LTE 4 , all three of which are agonists at CysLT receptors. LTD 4 formation is catalysed by γglutamyltransferase, and subsequently dipeptidase 2 removes the terminal glycine from LTD 4 to generate LTE 4 . Leukotriene A 4 hydrolase converts the 5,6-epoxide LTA 4 to the 5-hydroxylated LTB 4 , an agonist for BLT receptors. LTA 4 is also acted upon by 12S-LOX to produce the trihydroxyeicosatetraenoic acids lipoxins LXA 4 and LXB 4 . Treatment with a LTA 4 hydrolase inhibitor in a murine model of allergic airway inflammation increased LXA 4 levels, in addition to reducing LTB 4 , in lung lavage fluid [491]. LTA 4 hydrolase is also involved in biosynthesis of resolvin Es. Aspirin has been reported to increase endogenous formation of 18S-hydroxyeicosapentaenoate (18S-HEPE) compared with 18R-HEPE, a resolvin precursor. Both enantiomers may be metabolised by human recombinant 5-LOX; recombinant LTA 4 hydrolase converted chiral 5S (6) Comments: LTA4H is a member of a family of arginyl aminopeptidases (ENSFM00250000001675), which also includes aminopep-tidase B (RNPEP, 9H4A4) and aminopeptidase B-like 1 (RNPEPL1, Q9HAU8). Dipeptidase 1 and 2 are members of a family of mem-brane dipeptidases, which also includes (DPEP3, Q9H4B8) for which LTD 4 appears not to be a substrate.

Further reading on Eicosanoid turnover
Ackermann

GABA turnover
Enzymes → GABA turnover Overview: The inhibitory neurotransmitter γ-aminobutyrate (GABA, 4-aminobutyrate) is generated in neurones by glutamic acid decarboxylase. GAD1 and GAD2 are differentially expressed during development, where GAD2 is thought to subserve a trophic role in early life and is distributed throughout the cytoplasm. GAD1 is expressed in later life and is more associated with nerve terminals [160] where GABA is principally accumulated in vesicles through the action of the vesicular inhibitory amino acid transporter SLC32A1. The role of γ-aminobutyraldehyde dehydrogenase (ALDH9A1) in neurotransmitter GABA synthesis is less clear. Following release from neurons, GABA may interact with either GABA A or GABA B receptors and may be accumu-lated in neurones and glia through the action of members of the SLC6 family of transporters. Successive metabolism through GABA transaminase and succinate semialdehyde dehydrogenase generates succinic acid, which may be further metabolized in the mitochondria in the tricarboxylic acid cycle.

Phosphoinositide-specific phospholipase C
Enzymes → Glycerophospholipid turnover → Phosphoinositide-specific phospholipase C Overview: Phosphoinositide-specific phospholipase C (PLC, EC 3.1.4.11), catalyses the hydrolysis of PIP 2 to IP 3 and 1,2diacylglycerol, each of which have major second messenger functions. Two domains, X and Y, essential for catalytic activity, are conserved in the different forms of PLC. Isoforms of PLC-β are activated primarily by G protein-coupled receptors through members of the G q/11 family of G proteins. The receptor-mediated activation of PLC-γ involves their phosphorylation by receptor tyrosine kinases (RTK) in response to activation of a variety of growth factor receptors and immune system receptors. PLC-1 may represent a point of convergence of signalling via both G protein-coupled and catalytic receptors. Ca 2+ ions are required for catalytic activity of PLC isoforms and have been suggested to be the major physiological form of regulation of PLC-δ activity. PLC has been suggested to be activated non-selectively by the small molecule m3M3FBS [29], although this mechanism of action has been questioned [330]. The aminosteroid U73122 has been described as an inhibitor of phosphoinositide-specific PLC [552], although its selectivity among the isoforms is untested and it has been reported to occupy the H1 histamine receptor [272]. Comments: A series of PLC-like proteins (PLCL1, Q15111; PLCL2, Q9UPR0 and PLCH1, Q4KWH8) form a family with PLCδ and PLCζ 1 isoforms, but appear to lack catalytic activity. PLC-δ2 has been cloned from bovine sources [407].  A binding protein for secretory phospholipase A 2 has been identified which shows modest selectivity for sPLA 2 -1B over sPLA 2 -2A, and also binds snake toxin phospholipase A 2 [16]. The binding protein appears to have clearance function for circulating secretory phospholipase A 2 , as well as signalling functions, and is a candidate antigen for idiopathic membraneous nephropathy [38].

Overview:
Phosphatidylinositol may be phosphorylated at either 3-or 4-positions on the inositol ring by PI 3-kinases or PI 4-kinases, respectively.

Sphingosine kinase
Enzymes → Kinases (EC 2.7.x.x) → Lipid modifying kinases → Sphingosine kinase Overview: SPHK1 and SPHK2 are encoded by different genes with some redundancy of function; genetic deletion of both Sphk1 and Sphk2, but not either alone, is embryonic lethal in mice. There are splice variants of each isoform (SphK1a-c and SphK2a, b), distinguished by their N-terminal sequences. SPHK1 and SPHK2 differ in tissue distribution, sub-cellular localisation, biochemical properties and regulation. They regulate discrete pools of S1P. Receptor stimulation induces SPHK1 translocation from the cytoplasm to the plasma membrane. SPHK1 translocation is regulated by phosphorylation/dephosphorylation, specific protein:protein interactions and interaction with specific lipids at the plasma membrane. SPHK1 is a dimeric protein, as confirmed by its crystal structure which forms a positive cluster, between protomers, essential for interaction with anionic phospholipids in the plasma membrane. SPHK2 is localised to the ER or associated with mitochondria or shuttles in/out of the nucleus, regulated by phos-phorylation. Intracellular targets of nuclear S1P include the catalytic subunit of telomerase (TERT) and regulators of gene expression including histone deacetylases (HDAC 1/2) and peroxisome proliferator-activated receptor gamma (PPARγ). SPHK2 phosphorylates the pro-drug FTY720 (fingolimod, which is used to treat some forms of multiple sclerosis) to a mimic of S1P and that acts as a functional antagonist of S1P 1 receptors. Inhibitors of SPHK1 and SPHK2 have therapeutic potential in many diseases. MP-A08 (pK i 5.2) [474], SKI II (pK i 5.1) [196] Selective inhibitors PF-543 (pK i 8.4) [527] SLC4101431 (pK i 7.1) [100], compound 27d (pIC 50 6.8) [526], opaganib (pK i 5) [181], ROMe (pK i 4.8) [354] Comments SK1 inhibitors induce its proteasomal degradation [373,404]. SK1 crystal structures confirm that it is dimeric [5]; there is no crystal structure available for SK2.
There is no crystal structure available for SK2.

Further reading on Phosphatidylinositol kinases
Raphael J et al.

) [616] -Rat
Comments: The existence of a third non-catalytic version of haem oxygenase, HO3, has been proposed, although this has been suggested to be a pseudogene [250]. The chemical tin protoporphyrin IX acts as a haem oxygenase inhibitor in rat liver with an IC 50 value of 11 nM [152].

Hydrogen sulphide synthesis
Enzymes → Hydrogen sulphide synthesis Overview: Hydrogen sulfide is a gasotransmitter, with similarities to nitric oxide and carbon monoxide. Although the enzymes indicated below have multiple enzymatic activities, the focus here is the generation of hydrogen sulphide (H 2 S) and the enzymatic characteristics are described accordingly. Cystathion-ine β-synthase (CBS) and cystathionine γ-lyase (CSE) are pyridoxal phosphate (PLP)-dependent enzymes. 3-mercaptopyruvate sulfurtransferase (3-MPST) functions to generate H 2 S; only CAT is PLPdependent, while 3-MPST is not. Thus, this third pathway is sometimes referred to as PLP-independent. CBS and CSE are predomi-nantly cytosolic enzymes, while 3-MPST is found both in the cytosol and the mitochondria. For an authoritative review on the pharmacological modulation of H 2 S levels, see Szabo and Papapetropoulos, 2017 [575].

Hydrolases
Enzymes → Hydrolases Overview: Listed in this section are hydrolases not accumulated in other parts of the Concise Guide, such as monoacylglycerol lipase and acetylcholinesterase. Pancreatic lipase is the predominant mechanism of fat digestion in the alimentary system; its inhibition is associated with decreased fat absorption. CES1 is present at lower levels in the gut than CES2 (P23141), but predominates in the liver, where it is responsible for the hydrolysis of many aliphatic, aromatic and steroid esters. Hormone-sensitive lipase is also a relatively non-selective esterase associated with steroid ester hydrolysis and triglyceride metabolism, particularly in adipose tissue. Endothelial lipase is secreted from endothelial cells and regulates circulating cholesterol in high density lipoproteins.

Inositol phosphate turnover
Enzymes → Inositol phosphate turnover Overview: The sugar alcohol D-myo-inositol is a component of the phosphatidylinositol signalling cycle, where the principal second messenger is inositol 1,4,5-trisphosphate, IP 3 , which acts at intracellular ligand-gated ion channels, IP 3 receptors to elevate intracellular calcium. IP 3 is recycled to inositol by phosphatases or phosphorylated to form other active inositol polyphosphates. Inositol produced from dephosphorylation of IP 3 is recycled into membrane phospholipid under the influence of phos-phatidyinositol synthase activity (CDP-diacylglycerol-inositol 3phosphatidyltransferase [EC 2.7.8.11]).
Information on members of this family may be found in the online database.

Inositol polyphosphate phosphatases
Enzymes → Inositol phosphate turnover → Inositol polyphosphate phosphatases Overview: Members of this family exhibit phosphatase activity towards IP 3 , as well as towards other inositol derivatives, in-cluding the phospholipids PIP 2 and PIP 3 . With IP 3 as substrate, 1-phosphatase (EC 3. Information on members of this family may be found in the online database.

Comments:
In vitro analysis suggested IP 3 and IP 4 were poor substrates for SKIP, synaptojanin 1 and synaptojanin 2, but suggested that PIP 2 and PIP 3 were more efficiently hydrolysed [523].

Nomenclature
Most inhibitors of these enzymes have been assessed in cell-free investigations and so may appear to 'lose' potency and selectiv-ity in intact cell assays. In particular, ambient ATP concentrations may be influential in responses to inhibitors, since the majority are directed at the ATP binding site [128] .

Nomenclature
Rho associated coiled-coil containing protein kinase 1 Rho associated coiled-coil containing protein kinase 2 Systematic nomenclature ROCK1 ROCK2

Cyclin-dependent kinase (CDK) family
Enzymes → Kinases (EC 2.7.x.x) → CMGC: Containing CDK, MAPK, GSK3, CLK families → Cyclin-dependent kinase (CDK) family Overview: Five of the cyclin-dependent kinases (CDKs: 7, 8, 9, 12, and 13) are involved in the phosphorylation of serine residues in the C-terminal domain of RNA polymerase II, the enzyme that is responsible for the transcription of protein-coding genes into mRNA in eukaryotes. Phosphorylation of RNA polymerase II at Ser5 is essential for transcriptional initiation, and phosphorylation of Ser 2 contributes to transcriptional elongation and termination. All five of the C-terminal domain kinases can phosphorylate Ser5, but only CDK9, CDK12, and CDK13 can phosphorylate at Ser2 [59,321,352]. Comments on Cyclin-dependent kinase (CDK) family: The development of CDK inhibitors as anticancer drugs is reviewed in [576], with detailed content covering CDK4 and CDK6 inhibitors that are under clinical evaluation. Data produced by Jorda et al. (2018) highlights the caution that must be used when deploying commercially available CDK inhibitors as pharmacological probes [296], as most of them are more promiscuous in their selectivity than indicated. To make their findings easily accessible the Jorda data is hosted on the cyclin-dependent kinase inhibitor database (CDKiDB). Comments Due to its Tau phosphorylating activity, small molecule inhibitors of GSK-3β are being investigated as potential treatments for Alzheimer's disease (AD) [41]. GSK-3β also plays a role in canonical Wnt pathway signalling, the normal activity of which is crucial for the maintenance of normal bone mass. It is hypothesised that small molecule inhibitors of GSK-3β may provide effective therapeutics for the treatment of diseases characterised by low bone mass [393].

Comments -
The JAK2 V617F mutation, which causes constitutive activation, plays an oncogenic role in the pathogenesis of the myeloproliferative disorders, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis [74,133]. Small molecule compounds which inhibit aberrant JAK2 activity are being developed as novel anti-cancer pharmaceuticals. --

Src family
Enzymes → Kinases (EC 2.7. x.x) → TK: Tyrosine kinase → Non-receptor tyrosine kinases (nRTKs) → Src family Overview: Activation of Src-family kinases leads to both stimulatory and inhibitory signaling responses, with cell-specific and signaling pathway-specific outcomes and redundancy of kinase function.

Lanosterol biosynthesis pathway
Enzymes → Lanosterol biosynthesis pathway Overview: Lanosterol is a precursor for cholesterol, which is synthesized primarily in the liver in a pathway often described as the mevalonate or HMG-CoA reductase pathway. The first two steps (formation of acetoacetyl CoA and the mitochondrial generation of (S)-3-hydroxy-3-methylglutaryl-CoA) are also associated with oxidation of fatty acids. PON2 forms heterotrimers [150]. PON3 likely forms heterodimers in vivo [150].

A22: Presenilin
Enzymes → Peptidases and proteinases → AD: Aspartic (A) Peptidases → A22: Presenilin Overview: Presenilin (PS)-1 or -2 act as the catalytic component/essential co-factor of the γ-secretase complex responsible for the final carboxy-terminal cleavage of amyloid precursor protein (APP) [302] in the generation of amyloid beta (Aβ) [9,579]. Given that the accumulation and aggregation of Aβ in the brain is piv-otal in the development of Alzheimer's disease (AD), inhibition of PS activity is one mechanism being investigated as a therapeutic option for AD [211]. Several small molecule inhibitors of PS-1 have been investigated, with some reaching early clinical trials, but none have been formally approved. Dewji et al. (2015) have reported that small peptide fragments of human PS-1 can significantly inhibit Aβ production (total Aβ, Aβ40 and Aβ42) both in vitro and when infused in to the brains of APP transgenic mice [139]. The most active small peptides in this report were P4 and P8, from the amino-terminal domain of PS-1.
Information on members of this family may be found in the online database. -) which derive their name from Cysteine ASPartate-specific proteASES, include at least two families; initiator caspases (caspases 2, 8, 9 and 10), which are able to hydrolyse and activate a second family of effector caspases (cas-pases 3, 6 and 7), which themselves are able to hydrolyse further cellular proteins to bring about programmed cell death. Caspases are heterotetrameric, being made up of two pairs of subunits, generated by a single gene product, which is proteolysed to form the mature protein. Members of the mammalian inhibitors of apoptosis proteins (IAP) are able to bind the procaspases, thereby preventing maturation to active proteinases.
Information on members of this family may be found in the online database.

M1: Aminopeptidase N
Enzymes → Peptidases and proteinases → MA: Metallo (M) Peptidases → M1: Aminopeptidase N Overview: Aminopeptidases catalyze the cleavage of amino acids from the amino (N) terminus of protein or peptide substrates, and are involved in many essential cellular functions. Members of this enzyme family may be monomeric or multi-subunit complexes, and many are zinc metalloenzymes [590].

Nomenclature
Leukotriene  Information on members of this family may be found in the online database.

Comments
Folate hydrolase is also known as NAALADase as it is responsible for the hydrolysis of N-acetaspartylglutamate to form N-acetylaspartate and L-glutamate (L-glutamic acid). In the gut, the enzyme assists in the assimilation of folate by hydrolysing dietary poly-gamma-glutamylfolate. The enzyme is highly expressed in the prostate, and its expression is up-regulated in cancerous tissue. A tagged version of the antibody capromab has been used for imaging purposes.
Comments: Folate hydrolase is also known as NAALADase as it is responsible for the hydrolysis of N-acetaspartylglutamate to form N-acetylaspartate and L-glutamate. In the gut, the enzyme assists in the assimilation of folate by hydrolysing dietary poly-gamma-glutamylfolate. The enzyme is highly expressed in the prostate, and its expression is up-regulated in cancerous tissue. A tagged version of the antibody capromab has been used for imaging purposes.

T1: Proteasome
Enzymes → Peptidases and proteinases → PB: Threonine (T) Peptidases → T1: Proteasome Overview: The T1 macropain beta subunits form the catalytic proteinase core of the 20S proteasome complex [106]. This catalytic core enables the degradation of peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the cleavage site. The β5 subunit is the principal target of the approved drug proteasome inhibitor bortezomib.

Overview:
Hypoxia-inducible factors (HIFs) are rapidlyresponding sensors of reductions in local oxygen tensions, prompting changes in gene transcription. Listed here are the 4-prolyl hydroxylase family, members of which have been iden-tified to hydroxylate proline residues in HIF1α (HIF1A; Q16665) leading to an increased degradation through proteasomal hydrolysis. This action requires molecular oxygen and 2-oxoglutarate, and so reduced oxygen tensions prevents HIF1α hydroxylation, allowing its translocation to the nucleus and dimerisation with HIF1β (also known as ARNT; P27540), thereby allowing interaction with the genome as a transcription factor.

Sphingosine kinase
Enzymes → Sphingosine 1-phosphate turnover → Sphingosine kinase Overview: SPHK1 and SPHK2 are encoded by different genes with some redundancy of function; genetic deletion of both Sphk1 and Sphk2, but not either alone, is embryonic lethal in mice. There are splice variants of each isoform (SphK1a-c and SphK2a, b), distinguished by their N-terminal sequences. SPHK1 and SPHK2 differ in tissue distribution, sub-cellular localisation, biochemical properties and regulation. They regulate discrete pools of S1P. Receptor stimulation induces SPHK1 translocation from the cytoplasm to the plasma membrane. SPHK1 translocation is regulated by phosphorylation/dephosphorylation, specific protein:protein interactions and interaction with specific lipids at the plasma membrane. SPHK1 is a dimeric protein, as confirmed by its crystal structure which forms a positive cluster, between protomers, essential for interaction with anionic phospholipids in the plasma membrane. SPHK2 is localised to the ER or associated with mitochondria or shuttles in/out of the nucleus, regulated by phos-phorylation. Intracellular targets of nuclear S1P include the catalytic subunit of telomerase (TERT) and regulators of gene expression including histone deacetylases (HDAC 1/2) and peroxisome proliferator-activated receptor gamma (PPARγ). SPHK2 phosphorylates the pro-drug FTY720 (fingolimod, which is used to treat some forms of multiple sclerosis) to a mimic of S1P and that acts as a functional antagonist of S1P 1 receptors. Inhibitors of SPHK1 and SPHK2 have therapeutic potential in many diseases. MP-A08 (pK i 5.2) [474], SKI II (pK i 5.1) [196] Selective inhibitors PF-543 (pK i 8.4) [537] SLC4101431 (pK i 7.1) [100], compound 27d (pIC 50 6.8) [526], opaganib (pK i 5) [181], ROMe (pK i 4.8) [354] Comments SK1 inhibitors induce its proteasomal degradation [373,404]. SK1 crystal structures confirm that it is dimeric [5]; there is no crystal structure available for SK2.

Overview:
The thyroid hormones triiodothyronine and thyroxine, usually abbreviated as triiodothyronine and T 4 , respectively, are synthesized in the thyroid gland by sequential metabolism of tyrosine residues in the glycosylated homodimeric protein thyroglobulin (TG, P01266) under the influence of the haem-containing protein iodide peroxidase. Iodide peroxidase/TPO is a haem-containing enzyme, from the same structural family as eosinophil peroxidase (EPX, P11678), lactoperoxidase (LPO, P22079) and myeloperoxidase (MPO, P05164). Circulating thyroid hormone is bound to thyroxine-binding globulin (SERPINA7, P05543).
Substrates of the prenyltransferases include Ras, Rho, Rab, other Ras-related small GTP-binding proteins, G-protein γ-subunits, nuclear lamins, centromeric proteins and many proteins involved in visual signal transduction.
In relation to the causative association between oncogenic Ras proteins and cancer, farnesyltransferase has become an important mechanistic drug discovery target.
Information on members of this family may be found in the online database. Overview: Histone deacetylases act as erasers of epigenetic acetylation marks on lysine residues in histones. Removal of the acetyl groups facilitates tighter packing of chromatin (heterochromatin formation) leading to transcriptional repression. The histone deacetylase family has been classified in to five subfamilies based on phylogenetic comparison with yeast homologues: Class I contains HDACs 1, 2, 3 and 8 Class IIa contains HDACs 4, 5, 7 and 9 Class IIb contains HDACs 6 and 10 Class III contains the sirtuins (SIRT1-7) Class IV contains only HDAC11. Classes I, II and IV use Zn + as a co-factor, whereas catalysis by Class III enzymes requires NAD + as a co-factor, and members of this subfamily have ADP-ribosylase activity in addition to protein deacetylase function [521].
Dysregulated HDAC activity has been identified in cancer cells and tumour tissues [355,509] Overview: In humans, the peptidyl arginine deiminases (PADIs; HGNC family link) are a family of five enzymes, PADI1-4 and PADI6. PADIs catalyze the deimination of protein L-arginine residues to L-citrulline and ammonia, generating peptidyl-citrulline on histones, fibrinogen, and other biologically relevant proteins. The human isozymes exhibit tissue-specific expression patterns [294]. Overexpression and/or increased PADI activity is observed in several diseases, including rheumatoid arthri-tis, Alzheimer's disease, multiple sclerosis, lupus, Parkinson's disease, and cancer [47]. Pharmacological PADI inhibition reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis [423].
Information on members of this family may be found in the online database.

Small monomeric GTPases
Overview: small G-proteins, are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP). They are a type of G-protein found in the cytosol that are homologous to the alpha subunit of heterotrimeric G-proteins, but unlike the alpha subunit of G proteins, a small GTPase can function independently as a hydrolase enzyme to bind to and hydrolyze a guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). The best-known members are the Ras GTPases and hence they are sometimes called Ras subfamily GTPases.

RAS subfamily
Enzymes → 3.6.5.2 Small monomeric GTPases → RAS subfamily Overview: The RAS proteins (HRAS, NRAS and KRAS) are small membrane-localised G protein-like molecules of 21 kd. They act as an on/off switch linking receptor and non-receptor tyrosine kinase activation to downstream cytoplasmic or nuclear events. Binding of GTP activates the switch, and hydrolysis of the GTP to GDP inactivates the switch. The RAS proto-oncogenes are the most frequently mutated class of proteins in human cancers. Common mutations compromise the GTP-hydrolysing ability of the proteins causing constitutive activation [564], which leads to increased cell proliferation and decreased apoptosis [674]. Because of their importance in oncogenic transformation these proteins have become the targets of intense drug discovery effort [33].
Information on members of this family may be found in the online database.

RAB subfamily
Enzymes → 3.6.5.2 Small monomeric GTPases → RAB subfamily Overview: The Rab family of proteins is a member of the Ras superfamily of monomeric G proteins. Rab GTPases regulate many steps of membrane traffic, including vesicle formation, vesicle movement along actin and tubulin networks, and membrane fu-sion. These processes make up the route through which cell surface proteins are trafficked from the Golgi to the plasma membrane and are recycled. Surface protein recycling returns proteins to the surface whose function involves carrying another protein or substance inside the cell, such as the transferrin receptor, or serves as a means of regulating the number of a certain type of protein molecules on the surface ( see HGNC RAB, 65 genes ).
Information on members of this family may be found in the online database.