Diabetes and drug targets Flashcards
1
Q
Hypoglycemia
A
prolonged low blood sugar levels can result in coma and death
2
Q
Hyperglycemia
A
recurrent infections, cardiac arrhythmia, stupor, coma, seizures, ketoacidosis, death
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
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
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
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.
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)
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
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
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
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
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
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
14
Q
What tranporter needed for hexameric stored insulin is a genetic risk for development of both type1/2 diabetes
A
ZnT8
15
Q
Explain the quaternary structure of insulin
A
at an equlilibrium
Monomers (active form) - Dimers - Hexamers (stored form)
16
Q
Describe the basic primary structure of insulin
A
See photo
A chain and B chain connected by disulphide bonds
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)
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
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
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
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
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)
23
Q
Prolonged acting insulin…
A
Consists of an amorphous or crystalline prep that dissolves slowly
24
Q
Rapid acting preps can be achieved by…
A
introducing mutations at the dimer interface such as B28
25
The intrinsic flexibility at the ends of the B chain...
plays an important role in governing the physical and chemical stability of insulin
26
Why is there a need for hexameric formation and crystallisation of insulin within the storage vesicle
it stabalises insulin, preventing its degradation
27
How are hexameric insulin formulations stabalised?
binding of allosteric ligands at two loci, the phenolic pockets, and the His B10 Zn2+ sites
28
Whats the stability order of the three hexameric insulin forms
R6 >T3R3 >T6
29
Explain 3 addatives to insulin preperations
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
30
Explain the delayed release of insulin from protamine complexes
Protamine holds hexameters together, slows down release of monomeric form
31
Three general forms of insulin
Fast-acting insulin analogues
Long acting insulin analogues
Very long acting insulin analogues
32
Rapid acting insulin drugs that impair dimerization
Lispro (Humalog) ProB28 ➔ Lys
LysB29 ➔ Pro
amino acid positions are essentially reversed
33
Rapid acting insulin drugs that cause charge repulsion at the dimer interface
Aspart (NovoLog)
ProB28 ➔ Asp
34
Rapid acting insulin drugs that decrease zinc-free self-association
hexameric form requires zinc
Glulisine (Apidra) AsnB3 ➔ Lys
LysB29 ➔ Glu
35
Explain the three mechanisms of action of rapid acting insulin drugs and how they work
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
36
Explain what humalog actually is
mixture of:
-insulin lispro solution
- a rapid-acting blood glucose-lowering agent and insulin lispro protamine suspension
- an intermediate-acting blood glucose-lowering agent
37
Explain what NovoLog actually is
(aspart) homologous with regular human insulin with the exception of a single substitution of the amino acid proline by aspartic acid in position B28
38
Explain what APIDRA actually is
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
39
Explain the two mechanisms of action for long-term acting insulin drugs
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
40
Explain the long-term acting insulin drug that works by shifting pl of insulin to pH7, leading to isoelectric precipitation on injection
Glargine (Lantus) ArgB31-ArgB32 tag
AsnA21 ➔ Gly
41
Explain the long-term acting insulin drug that works by stabalising the hexamer and binding to serum albumin
Detemir (Levemir) - Modification of LysB29 by a tethered fatty acid
42
Explain what LANTUS is
- 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
43
Explain what Levemir actually is
- 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
44
Give examples of novel basal insulin analogs undergoing phase 3 clinical trials, and how they act
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
45
Explain features/functions of Insulin Degludec
- 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
46
Explain features/functions of PEGLisPro Insulin (LY2605541)
- long acting blood glucose-lowering agent
- PEGylated version of insulin lispro
Protein PEgylation - increases solubility, Protects from proteolytic degradation, decreases renal clearance
47
Explain way to remember fast-acting insulin analogues
***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)
48
Carbohydrates present in the diet
Polysaccharides - starch, gycogen
Disaccharides - Lactose, maltose, Sucrose
Monosaccharides - Glucose, Fructose, Pentose
49
In the GIT...
all complex carbohydrates are converted to monosaccharides which is the absorbable form
50
Where do enzymes come from?
Saliva
Pancreas
Intestinal brush boarder
51
What enzymes break di/polysaccharides down into monosaccharides?
Saliva and pancreatic a-amylases (poly-di)
lactase (di-mono)
Membrane bound a-glucosidases (di-mono)
52
Explain the transport of monosaccharides from the intestinal lumen to the blood
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)
53
Explain features of intestinal alpha-glucosidase
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
54
Give an example of a competative inhibator of a-glucosidase
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
55
Name another inhibator of alpha-glucosidase, and how this is different to Acarbose
Miglitol
difference as its systemically absorbed, however it is not metabolized and is excreted by the kidneys
56
Name another alpha-glucosidase inhibator (not Miglitol or acarbose)
Voglibose
57
Explain two residues of alpha-glucosidase important in catalysis, and how inhibators work,
Two aspartic acids D443 (catalytic nucleophile) and D542 (acid/base catalyst)
Inhibitors block access to the active site (surface binding site)
58
Placement of the Cyclitol group within intestinal alpha-glucosidase
Intestinal - cyclitol group buried
59
Out of Miglitol and Acrabose, which cannot bind to pancreatic alpha-amylase and why
Miglitol cannot bind due to deep groove of carbohydrate binding site
60
Role of amylase vs glucosidase
alpha-amylase - breaks break down complex carbohydrates in the gut
alpha-glucosidase - breaks down smaller polysaccharide units to monosaccharides for absorption
61
Depolarization
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
Factors responsible for creation of resting membrane potential
(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
Explain the role of ion channels in insulin secretion
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
What is KATP and when does it shut?
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
Explain what Sulfonylureas do
Sulfonylureas reduce KATP channel activity by binding to the regulatory SUR subunits
compete with the action of Mg2+-ADP on the SUR.
66
Name some sulfonylureas
glibenclamide (GBC)
tolbutamide
acetohexamide
glipzide
glimepiride
67
Explain SUR1 topology
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
Explain the KATP structure in terms of experimental techniques
Large protein
Cryo-EM structure highlighted the mechanims of assembly of subunits and the gating (Martin 2017)
69
Where is the ATP binding site on KATP located
Kir6.2 (intracellularly)
70
Explain the mechanistic details of KATP
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
Explain how the Sulfonylurea GBC (Glibenclamide) works
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
When it comes to KATP function, what is SUR1 doing ariuynd the Kir6.2 channel
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
ATP binding _____ KATP, ADP binding _____ KATP
Closes
Opens
74
Trick to remember long-term acting insulin drugs
GLAD = Glargine is Long-Acting like Detemir
75
Trick to remember fast acting insulin analogues
LAG = Lispro, Aspart, Glulisine
76
If glucose is the only factor influencing the release of insulin
- 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
Explain the incretin effect
The difference between Isoglycemic IV Glucose infusion and Oral glucose
In diabetes this is seen to be much lower (Nauck 1986)
78
What are incretins
- hormones secreted from the GIT into circulation in response to nutrient ingestion
- enhance glucose-stimulated insulin secretion
79
What two hormones account for the incretin effect in humans
Gastric Inhibitory Peptide (GIP)
Glucagon-Like Peptide-1 (GLP-1)
80
Features of GIP
- 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
Out of GIP and GLP-1, which causes insulin secretion in a diabetic state?
GLP-1
82
Features of GLP-1
-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
Explain synthesis of Glucagon and GLP-1
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
Structure of Glucagon vs GLP-1
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
Which class of GPCRs are we talking about when referring to binding of GLP-1 and glucagon?
Class B/2 (secretin receptor family)
86
GEF stand for
Guanine nucleotide exchange factors
87
Explain the GEF action of GPCRs
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
Structure of the extracellular domain of GLP-1R (a GPCR)
- 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
Model for GLP-1 binding to GLP-1R
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
What do the downstream effects of stimulating GPCRs depend on
Which G protein type(s) it couples to - in this case Ga-s
91
What are the two mechanisms of activating intracellular calcium stores?
PKA dependent
PKA independent
Both result of a further increase in insulin
92
Explain the activation of intracellular calcium stores
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
What are the prolonged effects of GLP-1R activation
As well as initial boost in insulin
also
Decreases ER stress
Increase insulin biosynthesis
β-cell proliferation & neogenesis
Inhibition of Apoptosis
94
Drug targeting of GLP-1R
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
Why does exendin-4 (Exenatide) last longer than GLP-1 and other analogues
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
Explain the structure of the drug semaglutide
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
Explain function of drug semaglutide
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
Why is the half-life of GLP-1 so low?
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
DPP4 features
- 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
Substrates for CD26/DPP4(IV)
proline(or alanine)-containing peptides including:
growth factors, chemokines, neuropeptides, and vasoactive peptides.
101
Explain DPP4 inhibators
- 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
DPP4 inhibitor features
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
DPP4 inhibators approved for use in the US
Saxagliptin and sitagliptin
104
DPP4 inhibators approved for use, commercially available outside US only
Vildagliptin
105
Trick for remembering GLP-1 agonists
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
What type of Diabetes drug was withdrawn from use
PPAR agonists
107
Where is glucose re-absorbed
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
Explain glucose uptake at S1 of the tubule lumen
The SGLT2 trabnsprter has high capacity and low affinity (90% glucose re-absorbed)
Fructose transported by SGLT5 instead of GLUTE5
109
Explain glucose uptake at S2/3 of the tubule lumen
S2/3 same apart from changed to SGLT1 instead of 2
SGLT1 is low capacity high affinity (absorbs last 10%)
110
Why does SGLT1 need to be high affinity
the glucose concentrations will have dropped, this allows it got absorb the glucose left
111
When it comes to glucose re-absorbtion, what do we want to do when it comes to diabetes
lower the amount of glucose being re-absorbed, as this is too high
112
Out of SGLT1 and SGLT2 which is better for a drug target and why?
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
SGLT2 inhibitors (origin)
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
SGLT2 inhibators (current)
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
Structurally, what are SGLT2 inhibators?
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
What are the advantages of inhibiting SGLT2 in the kidney
- 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
Side effects of SGLT2 inhibitors
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
Metformin features
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
What drug is recommended as the initial drug for treatment of type 2 diabetes
Metformin
120
How does metformin work at a molecular level
Still unclear how it works after 60 years
121
How does metformin suppress hepatic glucose output
decreases liver glycogenolysis but and gluconeogenesis (probable primary effect) and increases glucose uptake in muscle (to be stored as glycogen)
122
explain briefly the 4 mechanisms of metformin
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
Explain how metformin works through inhibiting mitochondrial respiratory complex 1
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
Explain how metformin works through inhibition of AMP deaminase
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
Explain how metformin works by inhibiting mitochondrial glycerophosphate dehydrogenase (G3DPH)
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
Explain amylin
- 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
Amylin structure
N terminus a bit wavey - slightly helical (dynamic struture so sometimes forms a helix sometimes doesn’t
C-terminal part binds to GPCR
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How does amylin work
Stimulates cAMP accumulation via the calcitonin/RAMP (Receptor activity modifying protein) receptor
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Explain the different amylin receptors and the role RAMP plays in the receptor
AMY1 - Calcetonin receptore (CR) + RAMP 1
AMY2 - CR + RAMP2
AMY3 - CR + RAMP 3
RAMP required to associate CR to transport receptor to PM
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Amylin slows rate of glucose appearance down by:
Inhibition of gastric emptying
Inhibition of digestive secretion [gastric acid, pancreatic enzymes, and bile ejection]
Reduction in food intake by increasing sensations of satiety
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How does Amylin slow appearance of new glucose down
Inhibiting secretion of the gluconeogenic hormone glucagon
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What part of the brain carries out the actions that amylin have
area postrema (glucose sensitive part of the brain stem)
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Explain the presence of amyloid plaques in diabetes
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.
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Amylin fibril features
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
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Pramlintide features
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.
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Why can pramlintide induce nausa
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.
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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?
PEGylate it - (Guterres 2013)
