Pharmacodynamics

1. Pharmacodynamics

2. One of the basic tenets of pharmacology is that drug molecules must exert some chemical influence on one or more constituents of cells in order to produce a pharmacological response.

3. PROTEIN TARGETS FOR DRUG BINDING receptors enzymes carrier molecules ( transporters ) ion channels .

4. DRUG RECEPTORS The term is most often used to describe the target molecules through which soluble physiological mediators-hormones, neurotransmitters, inflammatory mediators, etc.-produce their effects. 'Receptor' is sometimes used to denote any target molecule with which a drug molecule (i.e. a foreign compound rather than an endogenous mediator) has to combine in order to elicit its specific effect.

5. The term receptor is used to describe various cell surface molecules (such as T-cell receptors, integrins , Toll receptors, etc.) involved in the immunological response to foreign proteins and the interaction of cells with each other and with the extracellular matrix.

6. Various carrier proteins are often referred to as receptors, such as the low-density lipoprotein receptor that plays a key role in lipid metabolism and the transferrin receptor involved in iron absorption.

7. DRUG SPECIFICITY it must act selectively on particular cells and tissues. In other words, it must show a high degree of binding site specificity. Conversely, proteins that function as drug targets generally show a high degree of ligand specificity; they will recognise only ligands of a certain precise type and ignore closely related molecules.

8. no drug acts with complete specificity.

9. DRUG-RECEPTOR INTERACTIONS

10. Occupation of a receptor by a drug molecule may or may not result in activation of the receptor. Agonist: Antagonist: affinity : The tendency of a drug to bind to the receptors whereas the tendency for it, once bound, to activate the receptor is denoted by its efficacy .

11. Drugs of high potency will generally have a high affinity for the receptors and thus occupy a significant proportion of the receptors even at low concentrations. Agonists will also possess high efficacy, whereas antagonists will, in the simplest case, have zero efficacy Drugs with intermediate levels of efficacy, such that even when 100% of the receptors are occupied the tissue response is submaximal , are known as partial agonists

12. THE BINDING OF DRUGS TO RECEPTORS The binding of drugs to receptors can often be measured directly by the use of drug molecules labelled with one or more radioactive atoms (usually 3 H, 14 C or 125 I). The main requirements are that the radioactive ligand (which may be an agonist or antagonist) must bind with high affinity and specificity, and that it can be labelled to a sufficient specific radioactivity to enable minute amounts of binding to be measured.

13. The usual procedure is to incubate samples of the tissue (or membrane fragments) with various concentrations of radioactive drug until equilibrium is reached. The tissue is then removed, or the membrane fragments separated by filtration or centrifugation, and dissolved in scintillation fluid for measurement of its radioactive content.

14. TYPES OF RECEPTOR Receptors elicit many different types of cellular effect. Some of them are very rapid, such as those involved in synaptic transmission, operating within milliseconds, whereas other receptor-mediated effects, such as those produced by thyroid hormone or various steroid hormones, occur over hours or days. There are also many examples of intermediate timescales; catecholamines , for example, usually act in a matter of seconds, whereas many peptides take rather longer to produce their effects

15. 4 types of Receptors Ligand Gated ion channels- Type-1 G- Protein Coupled receptors- Type-2 Kinase linked and related receprots - Type-3 Nuclear receptor- Type-4

16. Ligand Gated Ion Channels Also know as ionotropic receptors. These membrane proteins are similar structure to other ion channels but incorporating a ligand binding site in extracellular domain. Molecular Structure It is assembled from four different types of subunit, termed alfa , beta, gama and delta.

17. The oligomeric structure possesses two acetylcholine binding sites, each lying at the interface between one of the two alfa subunits and its neighbour .

18. Gating Mechanism Receptors of this type control the fastest synaptic events in the nervous system

19. G Protein Coupled Receptors (GPCR)

20. Type 2 G-protein-coupled receptors (GPCRs). These are also known as metabotropic receptors or 7-transmembrane-spanning ( heptahelical ) receptors . They are membrane receptors that are coupled to intracellular effector systems via a G-protein. They constitute the largest family, 5 and include receptors for many hormones and slow transmitters, for example the muscarinic acetylcholine receptor, adrenergic receptors and chemokine receptors.

21. MOLECULAR STRUCTURE G-protein-coupled receptors consist of a single polypeptide chain of up to 1100 residues. Their characteristic structure comprises seven transmembrane α helices, similar to those of the ion channels discussed above, with an extracellular N-terminal domain of varying length, and an intracellular C-terminal domain. GPCRs are divided into three distinct families. There is considerable sequence homology between the members of one family, but none between different families. They share the same seven-helix ( heptahelical ) structure, but differ in other respects, principally in the length of the extracellular N terminus and the location of the agonist binding domain.

22. G-protein-coupled receptor families

23. Gating Mechanism

24. TARGETS FOR G-PROTEINS adenylyl cyclase , the enzyme responsible for cAMP formation phospholipase C , the enzyme responsible for inositol phosphate and diacylglycerol (DAG) formation ion channels , particularly calcium and potassium channels Rho A/Rho kinase , a system that controls the activity of many signalling pathways controlling cell growth and proliferation, smooth muscle contraction, etc.

25. The adenylyl cyclase / cAMP system cAMP (cyclic 3´,5´-adenosine monophosphate ) as an intracellular mediator cAMP is a nucleotide synthesised within the cell from ATP by the action of a membrane-bound enzyme, adenylyl cyclase . It is produced continuously and inactivated by hydrolysis to 5´-AMP, by the action of a family of enzymes known as phosphodiesterases ( PDEs ).

26. Many different drugs, hormones and neurotransmitters act on GPCRs and produce their effects by increasing or decreasing the catalytic activity of adenylyl cyclase , thus raising or lowering the concentration of cAMP within the cell. There are several different molecular isoforms of the enzyme, some of which respond selectively to Gα s or Gα i

27. The phospholipase C/ inositol phosphate system The phosphoinositide system, an important intracellular second messenger system, was first discovered in the 1950s by Hokin and Hokin . They found that secretion was accompanied by increased turnover of a minor class of membrane phospholipids known as phosphoinositides (collectively known as Pis ). Subsequently , Michell and Berridge found that many hormones that produce an increase in free intracellular Ca 2+ s concentration (which include, for example, muscarinic agonists and α- adrenoceptor agonists acting on smooth muscle and salivary glands, and vasopressin acting on liver cells) also increase PI turnover.

28. Subsequently, it was found that one particular member of the PI family, namely phosphatidylinositol (4,5) bisphosphate (PIP 2 ), which has additional phosphate groups attached to the inositol ring, plays a key role. PIP 2 is the substrate for a membrane-bound enzyme, phospholipase Cβ ( PLCβ ), which splits it into DAG and inositol (1,4,5) trisphosphate ( IP 3 ), both of which function as second messengers as discussed below. The activation of PLCβ by various agonists is mediated through a G-protein ( G q ). After cleavage of PIP 2 ,, DAG being phosphorylated to form phosphatidic acid (PA), while the IP 3 is dephosphorylated and then recoupled with PA to form PIP 2 once again.

29. Inositol phosphates and intracellular calcium Inositol (1,4,5) trisphosphate is a water-soluble mediator that is released into the cytosol and acts on a specific receptor-the IP 3 receptor-which is a ligand -gated calcium channel present on the membrane of the endoplasmic reticulum. The main role of IP 3 is to control the release of Ca 2+ from intracellular stores. Because many drug and hormone effects involve intracellular Ca 2+ , this pathway is particularly important. IP 3 is converted inside the cell to the (1,3,4,5) tetraphosphate , IP 4 , by a specific kinase . The exact role of IP 4 remains unclear, but there is evidence that it too is involved in Ca 2+ signalling .

30. Diacylglycerol and protein kinase C Diacylglycerol is produced as well as IP 3 whenever receptor-induced PI hydrolysis occurs. The main effect of DAG is to activate a membrane-bound protein kinase , protein kinase C (PKC), which catalyses the phosphorylation of a variety of intracellular proteins. DAG , unlike the inositol phosphates, is highly lipophilic and remains within the membrane. It binds to a specific site on the PKC molecule, which migrates from the cytosol to the cell membrane in the presence of DAG, thereby becoming activated. There are 10 different mammalian PKC subtypes, which have distinct cellular distributions and phosphorylate different proteins. Most are activated by DAG and raised intracellular Ca 2+ , both of which are produced by activation of GPCRs.

31. One of the subtypes is activated by the lipid mediator arachidonic acid generated by the action of phospholipase A 2 on membrane phospholipids, so PKC activation can also occur with agonists that activate this enzyme. The various PKC isoforms , like the tyrosine kinases discussed below act on many different functional proteins, such as ion channels, receptors, enzymes (including other kinases ) and cytoskeletal proteins. Kinases in general play a central role in signal transduction, and control many different aspects of cell function. The DAG-PKC link provides a channel whereby GPCRs can mobilise this army of control freaks.

32. Ion channels as targets for G-proteins G-protein-coupled receptors can control ion channel function directly by mechanisms that do not involve second messengers such as cAMP or inositol phosphates. Early examples came from studies on potassium channels. In cardiac muscle, for example, mAChRs are known to enhance K + permeability (thus hyperpolarising the cells and inhibiting electrical activity). Similar mechanisms operate in neurons, where many inhibitory drugs such as opiate analgesics reduce excitability by opening potassium channels. These actions are produced by direct interaction between the βγ subunit of G and the channel, without the involvement of second messengers

33. The Rho/Rho kinase system This recently discovered signal transduction pathway is activated by certain GPCRs (and also by non-GPCR mechanisms), which couple to G-proteins of the G 12/13 type. The free G-protein α subunit interacts with a guanosine nucleotide exchange factor , which facilitates GDP-GTP exchange at another GTPase , Rho. Rho-GDP , the resting form, is inactive, but when GDP-GTP exchange occurs, Rho is activated, and in turn activates Rho kinase .

34. Rho kinase phosphorylates many substrate proteins and controls a wide variety of cellular functions, including smooth muscle contraction and proliferation, angiogenesis and synaptic remodelling . By enhancing hypoxia-induced pulmonary artery vasoconstriction, activation of Rho kinase is thought to be important in the pathogenesis of pulmonary hypertension. Specific Rho kinase inhibitors are in development for a wide range of clinical indications-an area to watch

35. Family A is by far the largest, comprising most monoamine, neuropeptide and chemokine receptors. Family B includes receptors for some other peptides, such as calcitonin and glucagon. Family C is the smallest, its main members being the metabotropic glutamate and GABA receptors ( Ch. 33 ) and the Ca 2+ -sensing receptors 8

36. A G-Protein-Coupled Receptor Or G Protein-linked Receptor 7 transmembrane domains

37. The disassembly of G-Protein upon stimulation Spontaneous deactivation is very fast, in minutes. However, with the help of RGS (regulator of G protein signaling, a GAP for a unit), signals can be shut off even faster

38. The Activation cycle of G-Protein

39. GPCR Signaling: cAMP

40. The visualization of cAMP in nerve cells GPCR->Gs->adenylyl cyclase->cAMP Gi

41. cAMP cycle: GPCR->Gs->adenylyl cyclase->cAMP Cyclic AMP phosphodiesterase breaks down cAMP to 5 ’ -AMP

42. The function of cAMP Targeting PKA (cyclic-AMP-dependent protein kinase A)

43. The Whole Signaling Network related to cAMP

44. Terminology: CRE(cyclic AMP response element); CREB: CRE binding protein; CBP: CREB binding protein

45. GPCR Signaling: Calcium

46. Three Types of Inositol phospholipids PI, PI(4)P, PI(4,5)P2

47. Phospholipase C- b (PLC- b ) Produces DAG (diacylglycerol) and IP3 (inositol 1,4,5-trisphosphate (IP3)) Gq->PLC- b

48. Gq signaling pathways and Calcium

49. Fertilization of an egg by a sperm triggering an increase in cytosolic Calcium 3 major types of calcium channels: Voltage dependent Ca channels on plasma membrane IP3-gated Ca release channels on ER membrane Ryanodine receptor on ER membrane

50. Calcium uptake and deprivation Na/Ca exchanger on plasma membrane, 2. Ca pump on ER membrane, 3. Ca binding molecules, 4. Ca pump on Mitochondia

51. Calcium Frequency encoding signaling strength Local Ca blips, sparks, puffs, reflecting local opening of individual channels in ER, strong local signal induces global activity, the elevated Calcium trigger calcium deprivation system

52. Targeting molecules for Calcium Calcium binding protein Calmodulin

53. Ca 2+ /calmodulin dependent protein kinase (CaM-kinase) Memory function: 1. calmodulin dissociate after 10 sec of low calcium level; 2. remain active after calmodulin dissociation

54. Ca 2+ /calmodulin dependent protein kinase (CaM-kinase) Frequency decoder of Calcium oscillation High frequence, CaM-kinase does not return to basal level before the second wave of activation starts

55. Desensitization of GPCR 1. Inhibitory structural alteration of receptor; 2. receptor internalization; 3. receptor degration GRK (G protein-linked receptor kinase) Arrestin takes to clathrin-coated pits and degradation

56. GPCR Signaling Summary 1. G-protein types 2. cAMP and Calcium signaling pathways 3. desensitization

57. En z yme- linked Cell Surface Receptors Receptor Tyrosine kinases: phosphorylate specific tyrosines Tyrosine kinase associated receptors: associate with intracellular proteins that have tyrosine kinase activity. Receptorlike tyrosine phosphatases: remove phosphate group Receptor Serine/ Threonine kinases: phosphorylate specific Serine/ Threonine Receptor guanylyl cyclases: directly catalyzes the production of cGMP Histidine kinase associated receptors: kinase phoshorylates itself on histidine and then transfers the phosphate to a second intracellular signaling protein.

58. Receptor Tyrosine Kinases (RTKs) Intrinsic tyrosine kinase activity Soluble or membrane-bound ligands: Nerve growth factor, NGF Platelet-derived growth factor, PDGF Fibroblast growth factor, EGF Epidermal growt factor, EGF Insulin Downstream pathway activation: Ras-MAP kinase pathway

59. TYROSINE KINASE RECEPTORS these receptors traverse the membrane only once respond exclusively to protein stimuli cytokines mitogenic growth factors: platelet derived growth factor epidermal growth factor

60. Functions include: Cell proliferation, differentiation Cell survival Cellular metabolism Some RTKs have been discovered in cancer research Her2, constitutively active form in breast cancer EGF-R overexpression in breast cancer Other RTKs have been uncovered in studies of developmental mutations that block differentiation

61. Outline Activated RTKs transmit signal to Ras protein Ras transduces signal to downstream serine-threonine kinases Ultimate activation of MAP kinase Activation of transcription factors

62. Ligand binding to RTKs Most RTKs are monomeric ligand binding to EC domain induces dimerization FGF binds to heparan sulfate enhancing its binding to receptor: dimeric receptor-ligand complex Some ligands are dimeric: direct dimerization of receptors Insulin receptors occur naturally as a dimer Activation is due to the conformational change of the receptor upon ligand binding

63. Substrate + ATP Substrate -P + ADP Protein Tyrosine Kinase Protein Tyrosine Phosphatase (PTP)

64. Tyrosine Protein Phosphorylation Eukaryotic cells coordinate functions through environmental signals - soluble factors, extracellular matrix, neighboring cells. Membrane receptors receive these cues and transduce signals into the cell for appropriate response. Tyrosine kinase signalling is the major mechanism for receptor signal transduction. Tyrosine protein phosphorylation is rare (1%) relative to serine/threonine phosphorylation. TK pathways mediate cell growth, differentiation, host defense, and metabolic regulation. Protein tyrosine phosphorylation is the net effect of protein tyrosine kinases (TKs) and protein tyrosine phosphatases (PTPs).

65. Tyrosine kinase linked receptors Bi-functional receptor / enzyme Activated by hormones Over-expression can result in cancer

66. 4.1 Structure N H 2 C O 2 H Cell membrane Catalytic binding region (closed in resting state) Ligand binding region Extracellular N -terminal chain Intracellular C -terminal chain Hydrophilic transmembrane region ( a -helix) 4. Tyrosine kinase linked receptors

67. 4.2 Reaction catalysed by tyrosine kinase N C O Protein Protein OH Tyrosine residue Tyrosine kinase Mg ++ ATP ADP N C O Protein Protein O Phosphorylated tyrosine residue P 4. Tyrosine kinase linked receptors

68. 4.3 Epidermal growth factor receptor (EGF- R) Inactive EGF-R monomers Cell membrane Binding site for EGF EGF - protein hormone - bivalent ligand Active site of tyrosine kinase Induced fit opens tyrosine kinase active sites Ligand binding and dimerisation OH OH OH HO Phosphorylation ATP ADP OP OP OP PO EGF 4. Tyrosine kinase linked receptors

69. Active site on one half of dimer catalyses phosphorylation of Tyr residues on other half Dimerisation of receptor is crucial Phosphorylated regions act as binding sites for further proteins and enzymes Results in activation of signalling proteins and enzymes Message carried into cell 4.3 Epidermal growth factor receptor (EGF- R) 4. Tyrosine kinase linked receptors

70. 4.4 Insulin receptor (tetrameric complex) Insulin Cell membrane Insulin binding site Kinase active site OH OH OH HO OP Phosphorylation ATP ADP OP OP PO Kinase active site opened by induced fit 4. Tyrosine kinase linked receptors

71. 4.5 Growth hormone receptor Tetrameric complex constructed in presence of growth hormone Growth hormone binding site Kinase active site Kinase active site opened by induced fit GH OH OH OH HO kinases GH receptors (no kinase activity) GH binding & dimerisation OP OP OP PO ATP ADP Activation and phosphorylation OH Binding of kinases OH OH HO 4. Tyrosine kinase linked receptors

72. P P P P P P P P P P Ligand signalling protein Ligand 4. Tyrosine kinase linked receptors 4.6 Signalling pathways

73. 1-TM Receptors Tyrosine kinase inherent or associated Guanylate cyclase Signalling proteins cGMP PLC g IP 3 kinase GAP Grb2 Others PIP 3 Ca ++ PKC IP 3 DG 4. Tyrosine kinase linked receptors 4.6 Signalling pathways

74. Tyrosine kinase active site (inactive) Receptor binding site OH HO HO OH GROWTH FACTOR RECEPTOR 4. Tyrosine kinase linked receptors 4.6 Signalling pathways

75. Growth factor OH HO HO OH 1) Binding of growth factor 2) Conformational change OH HO HO OH Dimerisation OH HO HO OH OH HO HO OH Phosphorylation OP PO PO OP OP PO PO OP Grb2 OP PO PO OP OP PO PO OP OP OH Binding and phosphorylation of Grb2 Grb2 Binding Ras and GTP/GDP exchange OP PO PO OP OP PO PO OP OP GDP GTP Ras 4. Tyrosine kinase linked receptors 4.6 Signalling pathways

76. 4.6 Signalling pathways Raf (inactive) Raf (active) Mek (inactive) Mek (active) Map kinase (inactive) Map kinase (active) Transcription factor (inactive) Transcription factor (active) Gene transcription OP PO PO OP OP PO PO OP OP Ras 4. Tyrosine kinase linked receptors

77. Ras Monomeric GTPase switch protein Its activation is enhanced by GEF GDP-GTP exchange Deactivation of Ras-GTP complex requires GAP, which increases intrinsic GTPase activity 100 fold Lifetime of Ras-GTP is higher than that of G  Ras is a small protein (170 aa. Vs 300 aa of G  ) G  has a domain that functions like GAP

78. Mutant ras proteins are associated with many cancers Mutant ras can bind GTP but can not hydrolyze it, and thus remain constitutively in “on” state Most oncogenic ras proteins contain a mutation in codon 12 (Gly) This blocks the binding of GAP to ras, and prevents GTP hydrolysis.

79. Linking ras to RTKs Experimental evidences Fibroblasts were induced to proliferate with FGF and EGF Anti-ras antibody microinjected: cell proliferation arrest Injection of mutant ras proteins allows cell to proliferate in the absence of growth factors. Ligand-bound RTKs activate ras! How?

80. Two cytosolic proteins are involved: GRB2, Sos SH2 domain in GRB2 binds to a P*-tyrosine residue in the activated receptor Two SH3 domains of GRB2 bind to and activate Sos Sos is GEF protein and convert inactive GDP-ras into active GTP-ras Developmental studies elucidated the role of GRB2 and Sos in linking RTKs to ras activation

81. TYPE 4: NUCLEAR RECEPTORS Receptors for steroid hormones such as oestrogen and the glucocorticoids were present in the cytoplasm of cells and translocated into the nucleus after binding with their steroid partner. Other hormones, such as the thyroid hormone T 3 fat-soluble vitamins D and A (retinoic acid) and their derivatives that regulate growth and development, were found to act in a similar fashion.

82. Structure Structural design comprised of four modules . The N-terminal domain displays the most heterogeneity. It harbours the AF1 (activation function 1) site that binds to other cell-specific transcription factors in a ligand -independent way and modifies the binding or activity of the receptor itself. Alternative splicing of genes may yield several receptor isoforms each with slightly different N-terminal regions. The core domain of the receptor is highly conserved and consists of the structure responsible for DNA recognition and binding.

83. At the molecular level, this comprises two zinc fingers - cysteine - (or cystine / histidine ) rich loops in the amino acid chain that are held in a particular conformation by zinc ions. The main function of this portion of the molecule is to recognise and bind to the hormone response elements located in genes that are sensitive to regulation by this family of receptors, but it plays a part in regulating receptor dimerisation as well.

84. ER, oestrogen receptor; FXR, farnesoid receptor; GR, glucocorticoid receptor; LXR, liver oxysterol receptor; MR, mineralocorticoid receptor; PPAR, peroxisome proliferator receptor; PR, prolactin receptor; RXR, retinoid receptor; TR, thyroid receptor; VDR, vitamin D receptor

85. Steroid receptors, become mobile in the presence of their ligand and can translocate from the cytoplasm to the nucleus, while others such as the RXR probably dwell mainly within the nuclear compartment. They regulate many drug metabolic enzymes and transporters and are responsible for the biological effects of approximately 10% of all prescription drugs.

86. There are also many illnesses associated with malfunctioning of the nuclear receptor system, including inflammation, cancer, diabetes, cardiovascular disease, obesity and reproductive disorders

87. CLASSIFICATION OF NUCLEAR RECEPTORS Class I consists largely of receptors for the steroid hormones, including the glucocorticoid and mineralocorticoid receptors (GR and MR, respectively), as well as the oestrogen , progesterone and androgen receptors (ER, PR, and AR, respectively). In the absence of their ligand , these receptors are predominantly located in the cytoplasm, complexed with heat shock and other proteins and possibly reversibly attached to the cytoskeleton or other structures.

88. Following diffusion (or possibly transportation) of their ligand partner into the cell and high-affinity binding, these receptors generally form homodimers and translocate to the nucleus. Here they can transactivate or transrepress genes by binding to 'positive' or 'negative' hormone response elements . Large numbers of genes can be regulated in this way by a single ligand . Class I receptors generally recognise hormones that act in a negative feedback fashion to control biological events.

89. Class II nuclear receptors Their ligands are generally lipids already present to some extent within the cell. This group includes peroxisome proliferator -activated receptor ( PPAR ) that recognises fatty acids; the liver oxysterol ( LXR ) receptor that recognises and acts as a cholesterol sensor, the farnesoid ( bile acid ) receptor ( FXR ), a xenobiotic receptor ( SXR ; in rodents the PXR) that recognises a great many foreign substances, including therapeutic drugs the constitutive androstane receptor ( CAR ), which not only recognises the steroid androstane but also some drugs such as phenobarbital .

90. They induce drug- metabolising enzymes such as CYP3A (which is responsible for metabolising about 60% of all prescription drugs), They also bind some prostaglandins and non-steroidal drugs, as well as the antidiabetic thiazolidinediones and fibrates . Unlike the receptors in class I, these receptors almost always operate as heterodimers together with the retinoid receptor (RXR). They tend to mediate positive feedback effects (e.g. occupation of the receptor amplifies rather than inhibits a particular biological event).

91. A third group of nuclear receptors is really a subgroup of class II in the sense that they form obligate heterodimers with RXR They too play a part in endocrine signalling . The group includes the thyroid hormone receptor ( TR ), the vitamin D receptor ( VDR ) and the retinoic acid receptor ( RAR ).

92. CONTROL OF GENE TRANSCRIPTION Hormone response elements are the short (four or five base pairs) sequences of DNA to which the nuclear receptors bind to modify gene transcription. They are usually present symmetrically in pairs or half sites , although these may be arranged together in different ways. Each nuclear receptor exhibits a preference for a particular sequence.

93. In the nucleus, the ligand -bound receptor recruits further proteins including coactivators or corepressors to modify gene expression through its AF1 and AF2 domains. Some of these coactivators are enzymes involved in chromatin remodelling such as histone acetylase which, together with other enzymes, regulates the unravelling of the DNA to facilitate access by polymerase enzymes and hence gene transcription.