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BIOL3017 - Molecular and structural basis for disease > Diabetes and drug targets > Flashcards

Diabetes and drug targets Flashcards

1
Q

Hypoglycemia

A

prolonged low blood sugar levels can result in coma and death

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2
Q

Hyperglycemia

A

recurrent infections, cardiac arrhythmia, stupor, coma, seizures, ketoacidosis, death

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3
Q

How are blood glucose levels controlled

A

a and beta cells in pancrease

High glucose leve;s, B-cells stimulated to release insulin
Low glucose levels, a-cells stimulated to release glucagon

Insulin causes cells and liver to uptake more glucose, sores as glycogen

Glucagon causes liver to break down glycogen and release glucose into blood

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4
Q

How is the pancreas an exocrine and endocrine organ

A

Exocrine: pancreatic duct secretes digestive enzymes and alkaline fluid into small intestine

Endocrine: Hormones from islets of langerhans secreted into blood

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5
Q

Major Cells of the Islets of Langerhans

A

Pancreatic Beta-cells comprise the majority of pancreatic islet cell population; secrete insulin.

Pancreatic Alpha-cells cells in the islet of Langerhans which secrete glucagon.

Pancreatic PP-cells PP cells secrete pancreatic polypeptide.

Pancreatic Delta-cells cells in the islet of Langerhans; known to secrete somatostatin, and vasoactive intestinal peptide

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6
Q

Two types of diabetes

A

Diabetes mellitus (of or pertaining to honey – sweet tasting). The European honey bee is called Apis Mellifera which literally means bee honey-bearing.

Diabetes insipidus (lacking flavour) – a disease in which the pituitary gland does not secrete sufficient vasopressin.

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7
Q

Three main classifications of diabetes mellitus

A

Type 1 – “absolute insulin deficiency” - body is attackingpamcreatic beta cells that make insulin

Type 2 – “insulin resistance”

Gestational - is a condition in which women without previously diagnosed diabetes exhibit high blood glucose levels during pregnancy (esp. during the third trimester due to changes in renal absorption of glucose)

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8
Q

Cellular role of insulin

A

Insulin binds to receptor monomerically

Insulin receptor is a tyrosine kinase

Insulin activates own insulin receptor, undergoes conformational change leading to:

increase in receptor number

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9
Q

What happens to glucose when insulin is secreted

A

Put into glycogen
Or broken to pyruvates
Converted to fatty acids - then triglycerides and adipose tissue

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10
Q

Explain how insulin is exported into secretory vesicles

A

Needs signal sequence, once in ER lumen the signal sequence is cleaved off and insulin is now a soluble protein, now goes into Golgi and into secretory vesicles

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11
Q

Explain the different pHs involved in insulin getting packaged into secretory vesicles

A

ER lumen is 7.2
Golgi 6.7->6.0
Vesicles 5.7

pl of insulin is 5.4, close to buffer of vesicle so protein precipitates, happens in a conterolled way so that insulin is in its stored form in vesicles

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12
Q

What structure does insulin form in the Secretoy vesicles and what is needed for this to occur

A

forms a hexameric structure, zn2+ ions necessary

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13
Q

Explain how insulin develops into a hexameric structure in the secretory vesicle

A

Protease cleavage releases C peptide
Carboxypeptidase produces mature insulin
Packaged with Zn2+ ion that is transported through the Znt8 transporter

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14
Q

What tranporter needed for hexameric stored insulin is a genetic risk for development of both type1/2 diabetes

A

ZnT8

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15
Q

Explain the quaternary structure of insulin

A

at an equlilibrium

Monomers (active form) - Dimers - Hexamers (stored form)

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16
Q

Describe the basic primary structure of insulin

A

See photo

A chain and B chain connected by disulphide bonds

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17
Q

Explain the monomeric structure of insulin

A

A chain - 2 alpha helices
B chain - alpha helix and beta sheet

Contains 3 disulphide bridges (2 inter S-S and 1 intra S-S)

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18
Q

Explain the two different states on monomeric insulin

A

T state - conserved glycine on the B-chain acts as a helix breaker - rest of AAs are just a chain not a helix - elongated structure

R state - point after glycine still an a-helix

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19
Q

Explain the dimeric structure of insulin

A

Dimerisation occurs between the beta sheets

Normally 1 T state and 1 R state, but can be TT or RR

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20
Q

Explain the hexameric structre of insulin

A

Can be T and R states mixed (T3R3) (alternating), All Rs (R6), or all Ts (T6) - these differ in the first N-terminal 8 residues of the B chain, can undergo ligand-mediated interconversion between the states
Central zinc ion

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21
Q

Explain what the molecular size of insulin determines

A

Rate of subcutaneous absorption
e.g. monomeric insulin faster absorption into the capillary membrane than hexameric insulin

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22
Q

what are R6 crystals formed in the presence of?

A

in the presence of phenol (used in the insulin preparation as an antibacterial agent)

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23
Q

Prolonged acting insulin…

A

Consists of an amorphous or crystalline prep that dissolves slowly

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24
Q

Rapid acting preps can be achieved by…

A

introducing mutations at the dimer interface such as B28

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25
Q

The intrinsic flexibility at the ends of the B chain…

A

plays an important role in governing the physical and chemical stability of insulin

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26
Q

Why is there a need for hexameric formation and crystallisation of insulin within the storage vesicle

A

it stabalises insulin, preventing its degradation

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27
Q

How are hexameric insulin formulations stabalised?

A

binding of allosteric ligands at two loci, the phenolic pockets, and the His B10 Zn2+ sites

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28
Q

Whats the stability order of the three hexameric insulin forms

A

R6 >T3R3 >T6

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29
Q

Explain 3 addatives to insulin preperations

A

Protamine - protein extracted from the nucleus of fish sperm - regulates interactions between dimers and hexamers.

Phenol or metacresol - preservatives

Zinc chloride. Hexamers, made stable by zinc ions, are the predominant quaternary structure of pharmacological insulin

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30
Q

Explain the delayed release of insulin from protamine complexes

A

Protamine holds hexameters together, slows down release of monomeric form

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31
Q

Three general forms of insulin

A

Fast-acting insulin analogues

Long acting insulin analogues

Very long acting insulin analogues

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32
Q

Rapid acting insulin drugs that impair dimerization

A

Lispro (Humalog) ProB28 ➔ Lys
LysB29 ➔ Pro
amino acid positions are essentially reversed

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33
Q

Rapid acting insulin drugs that cause charge repulsion at the dimer interface

A

Aspart (NovoLog)
ProB28 ➔ Asp

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34
Q

Rapid acting insulin drugs that decrease zinc-free self-association

A

hexameric form requires zinc

Glulisine (Apidra) AsnB3 ➔ Lys
LysB29 ➔ Glu

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35
Q

Explain the three mechanisms of action of rapid acting insulin drugs and how they work

A
  1. Impairs dimerization (Lispro)
  2. Charge repulsion at dimer interface (Aspart)
  3. Decreased zinc-free self-association (Glulisine)

All three shift equilibrium from stored form towards the monomeric (active) form, all three analogues act to lower blood glucose

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36
Q

Explain what humalog actually is

A

mixture of:
-insulin lispro solution
- a rapid-acting blood glucose-lowering agent and insulin lispro protamine suspension
- an intermediate-acting blood glucose-lowering agent

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37
Q

Explain what NovoLog actually is

A

(aspart) homologous with regular human insulin with the exception of a single substitution of the amino acid proline by aspartic acid in position B28

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38
Q

Explain what APIDRA actually is

A

glulisine - differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid

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39
Q

Explain the two mechanisms of action for long-term acting insulin drugs

A
  1. Shift in pI to pH 7 leads to isoelectric precipitation on injection (Glargine)
  2. Stabilization of hexamer and binding to serum albumin (Detemir)
  • shift equilibrium to hexameric (stored) form, lowers blood glucose
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40
Q

Explain the long-term acting insulin drug that works by shifting pl of insulin to pH7, leading to isoelectric precipitation on injection

A

Glargine (Lantus) ArgB31-ArgB32 tag
AsnA21 ➔ Gly

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41
Q

Explain the long-term acting insulin drug that works by stabalising the hexamer and binding to serum albumin

A

Detemir (Levemir) - Modification of LysB29 by a tethered fatty acid

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42
Q

Explain what LANTUS is

A
  • a sterile solution of insulin glargine for use as an injection
  • long acting (24h)
    -differs from human insulin in that the amino acid asparagine at position A21 is replaced by glycine and two arginines are added to the C-terminus of the B-chain
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43
Q

Explain what Levemir actually is

A
  • solution made from insulin detemir
  • long acting (24h)
  • differs from human insulin n that the amino acid threonine in position B30 has been omitted, and a C14 myristic fatty acid chain has been attached to the amino acid B29
  • Fatty acids hydrophobic - bound to albumin, protecting it from being broken down
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44
Q

Give examples of novel basal insulin analogs undergoing phase 3 clinical trials, and how they act

A

Insulin degludec: Modification of LysB29 by a dicarboxylic acid, Allosteric assembly of a linear T6 polymer and albumin binding

PEGLisPro: PEGylation of LysB28 in insulin lispro, Increased hydrodynamic radius
PEG = polyethylene glycol

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45
Q

Explain features/functions of Insulin Degludec

A
  • long acting (40h)
  • phase 3 clinical trials
  • differs from human insulin in that the amino acid threonine in position B30 has been omitted, and a hexadecandioic fatty acid chain has been attached to the amino acid B29
  • forms filaments in subcutaneous tissue in the absence of phenol (TEM showed this Adams et al 2018)- good as has to go though many breakdowns to get to monomeric form
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46
Q

Explain features/functions of PEGLisPro Insulin (LY2605541)

A
  • long acting blood glucose-lowering agent
  • PEGylated version of insulin lispro
    Protein PEgylation - increases solubility, Protects from proteolytic degradation, decreases renal clearance
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47
Q

Explain way to remember fast-acting insulin analogues

A

Destabilise insulin dimer formation
Lis/pro (B28 proline-B29 lysine switched)
Aspart (B28 proline to Aspartate mutation)
Glu/lisine (B3 Asn -> lysine and B29 Lys -> Glutamate mutations)

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48
Q

Carbohydrates present in the diet

A

Polysaccharides - starch, gycogen
Disaccharides - Lactose, maltose, Sucrose
Monosaccharides - Glucose, Fructose, Pentose

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49
Q

In the GIT…

A

all complex carbohydrates are converted to monosaccharides which is the absorbable form

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50
Q

Where do enzymes come from?

A

Saliva
Pancreas
Intestinal brush boarder

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51
Q

What enzymes break di/polysaccharides down into monosaccharides?

A

Saliva and pancreatic a-amylases (poly-di)
lactase (di-mono)
Membrane bound a-glucosidases (di-mono)

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52
Q

Explain the transport of monosaccharides from the intestinal lumen to the blood

A

Microvilli in GIT
between cells = tight junctions to prevent large molecules from crossing
Glucose and galactose go though SGLT1 transporter driven by soidium conc gradient
GLUT2 on basolateral membrane transports Glucose/galactose into bloodsatream

Fructose has seperate transporter called GLUT5, but uses GLUT2 to get to bloodstream

Na+/K+ ATPase maintains sodium gradient within cell

Also important are a-glucosidases - intragel membrane protein (microvilli are covered in these)

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53
Q

Explain features of intestinal alpha-glucosidase

A

Alpha-glucosidase is tethered to the brush border membrane via a transmembrane helix. It contains two catalytic domains (break up larger polysaccharides into glucose)

Two forms MGAM and SI, both have a transmembrane section

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54
Q

Give an example of a competative inhibator of a-glucosidase

A

Acarbose
- a pseudotetrasaccharide
a natural microbial product derived from culture broths of Actinoplanes strain SE 50
- The unsaturated cyclitol component of the molecule has been identified as essential for a-glucosidase inhibitory activity
- binds reversibly, competativly and in a dose-dependant manner to the binding site of a-glucosidase
- consequence is hydrolysis of oligosaccharides is prevented

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55
Q

Name another inhibator of alpha-glucosidase, and how this is different to Acarbose

A

Miglitol
difference as its systemically absorbed, however it is not metabolized and is excreted by the kidneys

56
Q

Name another alpha-glucosidase inhibator (not Miglitol or acarbose)

A

Voglibose

57
Q

Explain two residues of alpha-glucosidase important in catalysis, and how inhibators work,

A

Two aspartic acids D443 (catalytic nucleophile) and D542 (acid/base catalyst)

Inhibitors block access to the active site (surface binding site)

58
Q

Placement of the Cyclitol group within intestinal alpha-glucosidase

A

Intestinal - cyclitol group buried

59
Q

Out of Miglitol and Acrabose, which cannot bind to pancreatic alpha-amylase and why

A

Miglitol cannot bind due to deep groove of carbohydrate binding site

60
Q

Role of amylase vs glucosidase

A

alpha-amylase - breaks break down complex carbohydrates in the gut
alpha-glucosidase - breaks down smaller polysaccharide units to monosaccharides for absorption

61
Q

Depolarization

A

a decrease in the measured voltage potential between the inside and the outside of the cell, i.e., the cell cytoplasm becomes less negative than the resting potential

62
Q

Factors responsible for creation of resting membrane potential

A

(1) primarily - the continuous action of the Na+/K+ ATPase pumps using the energy from the hydrolysis of ATP.
(2) differences in Na+ and K+ (and, to a lesser extent, Cl-) concentrations inside and outside the cell create the resting membrane potential (approx -70 mV)

63
Q

Explain the role of ion channels in insulin secretion

A
  1. Glucose is taken up by the cells via the Na+-glucose transporter GLUT2
  2. Energy released from breakdown of glucose via glycolysis and mitochondrial TCA cycle.
  3. Energy in form of ATP. As this increases the ATP/ADP ratio increases leads to ATP binding to the KATP.
  4. KATP = channel complex consisting of 4 Kir6.2 subunits and 4 sulphonylurea receptors (SURs). Binding of ATP to the Kir6.2 shuts the channel.
  5. As a result - membrane potential increases (depolarises from a resting potential of -70 mV)
  6. Causes activation (opening) of voltage-dependent Ca2+-channels
  7. Calcium ions enter and stimulate the fusion of the insulin-containing secretory vesicles thus releasing insulin
64
Q

What is KATP and when does it shut?

A

K+ channel complex consisting of 4 Kir6.2 subunits and 4 sulphonylurea receptors (SURs). Binding of ATP to the Kir6.2 shuts the channel

65
Q

Explain what Sulfonylureas do

A

Sulfonylureas reduce KATP channel activity by binding to the regulatory SUR subunits

compete with the action of Mg2+-ADP on the SUR.

66
Q

Name some sulfonylureas

A

glibenclamide (GBC)
tolbutamide
acetohexamide
glipzide
glimepiride

67
Q

Explain SUR1 topology

A

17 transmembrane helices, split between 3 TMDs (0-2)
2 intracellular Nucleotide binding domains (ADP binding sites)

SUR1 present in the pancreatic beta-cells – higher affinity for sufonylureas than SUR2 present in heart and skeletal muscle

68
Q

Explain the KATP structure in terms of experimental techniques

A

Large protein
Cryo-EM structure highlighted the mechanims of assembly of subunits and the gating (Martin 2017)

69
Q

Where is the ATP binding site on KATP located

A

Kir6.2 (intracellularly)

70
Q

Explain the mechanistic details of KATP

A
  1. KATP is open (no insulin secretion) when ADP is bound to NBDs of TMD1 and TMD2
  2. Opens up a site for lipid PIP2 to bind between TMD0 and Kir6.2 channel
  3. After feeding, ADP levels drop so NBDs are free and ATP levels are high
  4. The resulting conformational change causes loss of the PIP2 binding site, while ATP able to bind to the Kir6.2 channel, locking it in the closed state
  5. results in more insulin secretion.
71
Q

Explain how the Sulfonylurea GBC (Glibenclamide) works

A

Wedges itself between TMD0 and TMD1&2, blocking the PIP2 binding site and mimicking the ATP-bound closed state, resulting in more insulin secretion

72
Q

When it comes to KATP function, what is SUR1 doing ariuynd the Kir6.2 channel

A

Regulation - the ratio of ATP:ADP that defines the state of the channel i.e., whether the channel is in the closed or open state. This is because the nucleotide binding domains of the SUR bind ADP which results in channel opening.

73
Q

ATP binding _____ KATP, ADP binding _____ KATP

A

Closes
Opens

74
Q

Trick to remember long-term acting insulin drugs

A

GLAD = Glargine is Long-Acting like Detemir

75
Q

Trick to remember fast acting insulin analogues

A

LAG = Lispro, Aspart, Glulisine

76
Q

If glucose is the only factor influencing the release of insulin

A
  • then administration of the same amount of glucose orally or intravenously should be equivalent
  • It’s not only factor. 50 – 70 % of the total insulin secreted is due to the incretin effect
77
Q

Explain the incretin effect

A

The difference between Isoglycemic IV Glucose infusion and Oral glucose

In diabetes this is seen to be much lower (Nauck 1986)

78
Q

What are incretins

A
  • hormones secreted from the GIT into circulation in response to nutrient ingestion
  • enhance glucose-stimulated insulin secretion
79
Q

What two hormones account for the incretin effect in humans

A

Gastric Inhibitory Peptide (GIP)
Glucagon-Like Peptide-1 (GLP-1)

80
Q

Features of GIP

A
  • An incretin responsible for the incretin effect in humans
  • Derived from 153 AA polyprotein encoded by GIP gene
  • Circulates as biologically active 42AA peptide
  • Synthesised. by K cells found in mucose of duodenum and jejunum of GIT
  • induses insulin secretion and lipolysis
  • no significant effect on food intake and BW
81
Q

Out of GIP and GLP-1, which causes insulin secretion in a diabetic state?

A

GLP-1

82
Q

Features of GLP-1

A

-Released from L cells in ileum and colon
- Potent inhibition of gastric emptying and glucagon secretion
- reduces food intake and body weight
-increases B-cell growth and survival

83
Q

Explain synthesis of Glucagon and GLP-1

A

pro-glucagon in intestinal L-cells
Glucagon and GLP-1 both present on this
In GIT and brain - Prohormone convertase 1 causes productiobn of GLP-1 by splicing
In pancreas - production of glucagon and MPGF by splicing differently

84
Q

Structure of Glucagon vs GLP-1

A

Similar AA sequence
Alpha helices
both bind to GCPRs in similar (but not same) ways
N-terminus homologous (as activates receptor)
C-terminus different (as its for recognition)

85
Q

Which class of GPCRs are we talking about when referring to binding of GLP-1 and glucagon?

A

Class B/2 (secretin receptor family)

86
Q

GEF stand for

A

Guanine nucleotide exchange factors

87
Q

Explain the GEF action of GPCRs

A

Ga- bound tp GDP = inactive
ligand binds, causes conformational change - GDP converts to GTP (GEF action) and alpha+GTP falls off, goes onto signal

88
Q

Structure of the extracellular domain of GLP-1R (a GPCR)

A
  • consists of mostly beta sheet and one alpha helix
  • groove in the ECD provides a binding site for the C-terminal section of the GLP-1 helix
89
Q

Model for GLP-1 binding to GLP-1R

A

C-terminal part of the ligand binds the ECD of the receptor, followed by binding of the N-terminal part of the ligand to the transmembrane (7TM) receptor domain

90
Q

What do the downstream effects of stimulating GPCRs depend on

A

Which G protein type(s) it couples to - in this case Ga-s

91
Q

What are the two mechanisms of activating intracellular calcium stores?

A

PKA dependent
PKA independent
Both result of a further increase in insulin

92
Q

Explain the activation of intracellular calcium stores

A

ER is a major intracellular calcium ion store
1. Binding of GLP to its receptor activates (GDP is replaced with GTP) the Gαs signalling pathway
2. Activated Gαs is released from G-protein, binds and activates adenylyl cyclase – increasing cAMP level
3. cAMP activates the Protein Kinase A (PKA) which (1) phosphorylates KATP resulting in channel closure and (2) phosphorylates IP3 receptors resulting in channel opening (↑intracellular Ca2+)
4. cAMP binds Epac2 which activates ryanodine receptors resulting in channel opening (↑intracellular Ca2+)
5. Ca2+ ions cause secretory vesicles to fuse and release their contents

93
Q

What are the prolonged effects of GLP-1R activation

A

As well as initial boost in insulin
also

Decreases ER stress
Increase insulin biosynthesis
β-cell proliferation & neogenesis
Inhibition of Apoptosis

94
Q

Drug targeting of GLP-1R

A

Substrate for GLP-1R is a peptide therefore cannot be taken orally – like insulin must be inject.
Half life of GLP-1 peptide is minutes hence the natural peptide is not appropriate.
Use our knowledge of insulin analogues and apply the same approach:

Taspoglutide is the 8-(2-methylalanine)-35-(2-methylalanine)-36-L-argininamide derivative of the amino acid sequence 7–36 of human glucagon-like peptide I (IN 2010 PHASE III HALTED due to serious hypersensitivity reactions and GIT side effects)

Exenatide (Byetta) is a synthetic version of the exendin-4 peptide from the Glia monster. Exenatide bears a 50% amino acid homology to GLP-1 and it has a longer half-life in vivo

95
Q

Why does exendin-4 (Exenatide) last longer than GLP-1 and other analogues

A

Not cleaved by DPP4 due to presence of glycine residue instead of proline or arganine, as well as this its 9 residues extra N-terminal region is thought to play a part in it not being cleaved (M Doyle 2003)

96
Q

Explain the structure of the drug semaglutide

A

chemically similar to human GLP-1( 94% similarity) The only differences are two AA substitutions at 8 and 34, where alanine and lysine are replaced by 2-aminoisobutyric acid and arginine, respectively

AA substitution at position 8 prevents chemical breakdown by dipeptidyl peptidase-4
lysine at position 26 is in its derivative form (acylated with stearic diacid)

Long tale contains PEG group

97
Q

Explain function of drug semaglutide

A

forms a series of aggregates (concentration dependent) - highest concentrations forms branched tree-like aggregates

fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor potency

98
Q

Why is the half-life of GLP-1 so low?

A

DPP4 cleaves off the 2 AAs on the N terminus, making it unable to activate the GCPR (recognition no activation)

Once this is cleaved it will be broken down further

99
Q

DPP4 features

A
  • serine protease family
  • homodimeric membrane bound and soluble forms
  • diverse range of substrates are known
  • plays a major role in glucose metabolism
  • responsible for degradation of incretins e.g. GLP-1
  • DPP4 also functions in restricting the inflammatory actions of the chemokine CCL11/eotaxin, so that inhibiting DPP4 might unleash the recruitment of inflammatory cells
100
Q

Substrates for CD26/DPP4(IV)

A

proline(or alanine)-containing peptides including:

growth factors, chemokines, neuropeptides, and vasoactive peptides.

101
Q

Explain DPP4 inhibators

A
  • After secretion, GLP-1 is rapidly metabolized by the enzyme DPP-4 in the liver
    DPP-IV inhibitors:
  • such as sitagliptin inhibit the breakdown of GLP-1 by DPP-4
  • only produce an effect when blood sugar is elevated, causing the release of GLP-1 from the small intestine.
  • Don’t inhibit gastic emptying, while GLP-1 agonist do.
  • been observed to increase satiety (feeling full), decreased food intake.
  • found to be weight neutral in clinical trials, compared to weight loss seen with GLP-1 receptor agonists
102
Q

DPP4 inhibitor features

A

Also called Gliptins
- Standard small molecular inhibitors.
- First generation inhibitors were directed against the DPP family.DPP8 & 9 are intracellular – adverse side effects observed in animal models but none in human clinical trials (chemistry v pharmacology).
- Second generation focused on generating DPP4 specific drugs.

103
Q

DPP4 inhibators approved for use in the US

A

Saxagliptin and sitagliptin

104
Q

DPP4 inhibators approved for use, commercially available outside US only

A

Vildagliptin

105
Q

Trick for remembering GLP-1 agonists

A

end in ‘tide’
Exendatide – Glia monster’s exendin-4 peptide
Lixisendatide – exendatide with six (xis) L(i)ysine residues attached
Taspoglutide (off market)
Liraglutide – fatty acid lipid attached via a glutamyl spacer to GLP-1
Albglutide – Albumin fused to Gluagon-like peptide 1
Semaglutide – ? fused to Gluagon-like peptide 1

106
Q

What type of Diabetes drug was withdrawn from use

A

PPAR agonists

107
Q

Where is glucose re-absorbed

A

Proximal tubule of the nephron in the kidneys:
There’s an S1 segment and and S2/S3 segment

S1 close to bowman capsule
S2/3 both have transporters that will reabsorb the glucose

Transporters = SGLT (sodium glucose linked transporter) - same as in intestines
This time transports from proximal tubule into bloodstream
2 isoforms, SGLT1 (10% second) and SGLT2 (90% first)
Normally - all the glucose is reabsorbed, in diabetes there’s so much that it overwhelms the system and glucose gets into urine

108
Q

Explain glucose uptake at S1 of the tubule lumen

A

The SGLT2 trabnsprter has high capacity and low affinity (90% glucose re-absorbed)

Fructose transported by SGLT5 instead of GLUTE5

109
Q

Explain glucose uptake at S2/3 of the tubule lumen

A

S2/3 same apart from changed to SGLT1 instead of 2

SGLT1 is low capacity high affinity (absorbs last 10%)

110
Q

Why does SGLT1 need to be high affinity

A

the glucose concentrations will have dropped, this allows it got absorb the glucose left

111
Q

When it comes to glucose re-absorbtion, what do we want to do when it comes to diabetes

A

lower the amount of glucose being re-absorbed, as this is too high

112
Q

Out of SGLT1 and SGLT2 which is better for a drug target and why?

A

If we targeted SGL1 it would also affects small intestine and partially heart (not so good)
Probably better for drugs to target SGLT2 as found exclusively in kidney - also advantage as most of the reabsorption is through this (90%)

113
Q

SGLT2 inhibitors (origin)

A

Phlorizin has played a vital role in understanding the mechanism of renal glucose reabsorption and the role of hyperglycaemia in diabetes.

This agent was first isolated in 1835 by French chemists from the root bark of the apple tree.

Subsequently, it found to be a potent but relatively non-selective inhibitor of both SGLT2 and SGLT1

But based on this you can modify to to find something that works

114
Q

SGLT2 inhibators (current)

A

Dapagliflozin is a selective SGLT2 inhibitor with 1000x selectivity over SGLT1.

The FDA approved dapagliflozin on January 8, 2014 for glycemic control, along with diet and exercise, in adults with type 2 diabetes.

The SGLT2 inhibitors in human clinical trials have good efficacy, but they do not inhibit >30–50% of the filtered glucose load - means there’s potentially another pathway we don’t understand yet that could be targeted in terms of glucose reabsorption

canagliflozin

115
Q

Structurally, what are SGLT2 inhibators?

A

taken glucose and attached extra things onto it (glucose part determines specificity)
Affinity mostly though hydrophobic interactions - high affinity necessary to prevent off target effects
Specificity comes through hydroxyl groups (found on glucose and therefore the inhibitors)

116
Q

What are the advantages of inhibiting SGLT2 in the kidney

A
  • glucose is cleared from the circulation without the need for insulin- lowering the demands on pancreatic β-cells;
  • glucose excretion decreases as blood glucose levels decrease, thereby limiting the risk of severe hypoglycemia;
    -urinary glucose excretion (UGE) may lower blood pressure and may lead to weight loss.
  • Novel mechanism is compatible with other glucose-lowering therapies.
117
Q

Side effects of SGLT2 inhibitors

A

Repeated urinary tract infections due to increased glucose in urinary tract system

Genital infections

Increased haematocrit. - blood becomes thicker - problems with strokes so blood thinners needed to be taken also

Decreased blood pressure

Possible increase in bladder cancer

118
Q

Metformin features

A

approved by the British National Formulary in 1958 but only approved in the USA in 1995

only antidiabetic drug that has been conclusively shown to prevent the cardiovascular complications of diabetes.

It helps reduce LDL cholesterol and triglyceride levels.

It is not associated with weight gain.

Low risk of hypoglycemia.

Metformin works by suppressing glucose production by the liver

Cheap and safe, most used drug for diabetesa treatment in world

119
Q

What drug is recommended as the initial drug for treatment of type 2 diabetes

A

Metformin

120
Q

How does metformin work at a molecular level

A

Still unclear how it works after 60 years

121
Q

How does metformin suppress hepatic glucose output

A

decreases liver glycogenolysis but and gluconeogenesis (probable primary effect) and increases glucose uptake in muscle (to be stored as glycogen)

122
Q

explain briefly the 4 mechanisms of metformin

A
  1. inhibition of mitochondrial respiratory complex 1
  2. inhibition of AMP deaminase
  3. Inhibition of mitochondrial G3PDH
  4. altering gut microbiota and altering insulin release
123
Q

Explain how metformin works through inhibiting mitochondrial respiratory complex 1

A

Inhibition of respiratory complex 1 -> decreased ATP synthesis and a rise in the cellular AMP:ATP ratio ->AMPK activation -> inhibition of hepatic gluconeogenesis, increased glucose uptake in skeletal muscle

124
Q

Explain how metformin works through inhibition of AMP deaminase

A

AMP Kinase (AMPK) is considered to be a master controller of cellular metabolism.

Metformin indirectly activates AMP kinase by inhibiting AMP deaminase, thereby increasing cellular AMP.

High cellular levels of AMP, activate AMPK resulting in a decrease in gluconeogenesis ect.

125
Q

Explain how metformin works by inhibiting mitochondrial glycerophosphate dehydrogenase (G3DPH)

A
  1. Metformin inhibits a mitochondrial glycerophosphate dehydrogenase (mGPD).
  2. This decelerates the dihydroxyacetone phosphate (DHAP)–glycerol-3-phosphate (G3P) shuttle
  3. As a result, the ratios of G3P to DHAP, NADH to NAD, and lactate to pyruvate all increase
  4. Decreased glucose formation and export
    (Madiraju 2014)

Overall activation altered metabolic state of hepatic cells

126
Q

Explain amylin

A
  • also known as Islet Amyloid PolyPeptide (IAPP)
    -37-residue peptide hormone.
  • co-secreted with insulin from the pancreatic β-cells in the ratio of approximately 100:1 (insulin:amylin)
  • synthesised as a larger protein that is cleaved (including signal sequence) to produce the mature active peptide
  • plays a role in glycemic regulation by slowing gastric emptying and promoting satiety, thereby preventing post-prandial spikes in blood glucose levels.
127
Q

Amylin structure

A

N terminus a bit wavey - slightly helical (dynamic struture so sometimes forms a helix sometimes doesn’t

C-terminal part binds to GPCR

128
Q

How does amylin work

A

Stimulates cAMP accumulation via the calcitonin/RAMP (Receptor activity modifying protein) receptor

129
Q

Explain the different amylin receptors and the role RAMP plays in the receptor

A

AMY1 - Calcetonin receptore (CR) + RAMP 1
AMY2 - CR + RAMP2
AMY3 - CR + RAMP 3

RAMP required to associate CR to transport receptor to PM

130
Q

Amylin slows rate of glucose appearance down by:

A

Inhibition of gastric emptying
Inhibition of digestive secretion [gastric acid, pancreatic enzymes, and bile ejection]
Reduction in food intake by increasing sensations of satiety

131
Q

How does Amylin slow appearance of new glucose down

A

Inhibiting secretion of the gluconeogenic hormone glucagon

132
Q

What part of the brain carries out the actions that amylin have

A

area postrema (glucose sensitive part of the brain stem)

133
Q

Explain the presence of amyloid plaques in diabetes

A

Pancreatic amyloid plaques formed by amylin are present in > 95% T2DM patients.

Plaque abundance correlates with severity of disease.

amylin does not form a compact globular structure but residues numbered 5 – 20 do, transiently, sample helical conformations.

134
Q

Amylin fibril features

A

Cross-β architecture in which the β-strands run perpendicular to the fibril long axis with the inter-strand hydrogen bonds oriented parallel to the long axis.

1st 7 residues of amylin may not be part of the β-structure core due to conformational restrictions imposed by the disulfide bridge.

How these fibrils cause cell death is still unclear – ER stress, localised inflammation, defects in autophagy, membrane pore formation - Death of beta cells main reason for diabetes, some transplants of beta cells have been done

135
Q

Pramlintide features

A

Proline amylin peptide

esembles the hormone, amylin.

Designed to avoid fibril formation.

Pramlintide is a 37-amino acid polypeptide that differs structurally from human amylin by the replacement of alanine, serine, and serine at positions 25, 28, and 29 respectively with proline.

Green tea contains a flavinoid that inhibits fibril formation – potential new target.

136
Q

Why can pramlintide induce nausa

A

The area postrema detects toxins in the blood and acts as a vomit-inducing centre.

The most frequent and severe adverse effect of pramlintide is nausea, which occurs mostly at the beginning of treatment and gradually reduces.

137
Q

Based on the modifications of the protein insulin and the peptide GLP-1, can you think of a way of altering amylin that would increase the life time of amylin in the blood?

A

PEGylate it - (Guterres 2013)

BIOL3017 - Molecular and structural basis for disease (6 decks)

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