Peptides as Drugs

1
Peptides as Drugs
2
Protein Primary Structure and Peptide Bonds

• Proteins primarily exist as L-a amino acids
• Amino acids are linked via peptide bond
• Very stable due to resonance
• Peptide bonds are susceptible to hydrolysis by
  proteases and peptidases
• Aminopeptidase, carboxypeptidase, endopeptidase,
  trypsin, chymotrypsin, pepsin
• Protection of peptide bond gt stability

3
Amino Acids

• Non-Polar, Non-charged side chains
• Gly, Phe, Ala, Val, Pro, Met, Leu, Ile
• Polar, Non-charged
• Ser, Tyr, Cys (participates in disulfide bond
  formation), Trp, Thr, Asn (can be N-glycosylated)
• Polar, Charged
• Basic ( charged) Arg, Lys, His (50 charged at
  physiological pH, what is the pKa?)
• Acidic (- charged) Asp, Glu
• The sequence of a.a. in a peptide determines a
  proteins characteristics and activities.

4
Hierchical Structure of Proteins

• Primary peptide
• Covalently linked aa. through peptide bonds
• Secondary polypeptide fold up according to
  polarity
• Alpha Helix
• Beta sheet
• Also consist of turns, kinks, and loops (i.e.
  hairpin).
• Tertiary 3D, native structure of single
  polypeptide or protein
• Quaternary single polypeptides associate with
  each other
• forming larger protein complexes of geometrically
  specific arrangements

5
Secondary Structures

• Alpha Helix

• Beta Sheets

- Contains mostly hydrophobic residues and the
backbones are fully H-Bonded - The amphipathic
beta sheet contains alternating polar and
non-polar R groups - Antiparallel N-terminus
coincides with the C-terminus of the adjacent
chain H-bond not tilted - Parallel N-terminus
coincides with the N-term of the adjacent chain
H-bond tilted
- The alpha helix repeats every 3.6 residues -
The backbone is about 5Å in diameter - It has a
hydrophobic and non-polar center with hydrophilic
and polar R chains - What drives protein folding
is the hydrophobic effect
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Secondary Structures (cont.)

• Loops
• Can often find pro and gly in this region
• Kinks
• Prolines cannot hydrogen bond gt forms a kink
• Links
• Stabilizes turns via Cys-Cys disulfide bond

7
Post-Translational Modifications

• Pyroglutamyl
• Addition of glutamate to N-term
• Carboxamides
• Amide at the carboxy term
• Slow degradation, prolonged T1/2
• Cyclization
• Ex. Cys-Cys in insulin
• Internal amino acid modifications
• Ex. hydroxylation of proline
• Glycosylation
• O and N-glycosides

8
Biosynthesis of Peptide Hormones and Processing
into Active Forms
Ex. Parathyroid Hormone

• Biosynthesis
• Pre-pro sequence leader sequence, rich in
  methionine
• Pre-pro PTH -gt cleaved -gt Pro PTH -gt cleaved -gt
  PTH (84 residues) -gt post translational
  modification
• PTH is packaged in secretory granule stored at
  cytoplasmic matrix gt they are preformed
• They will be secreted when needed

• Processing
• Pre-pro PTH -31 -7
• Pro PTH -6 -1
• PTH 1 84
• Cleavage of Pre-Pro PTH into Pro PTH and PTH is
  tryptic cleavage (recalling that trypsin can
  cleave the basic residues such as Lys and Arg)

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Address, Message, Potentiator

• PTH must bind to PTH receptor and binding must
  result in conformational change
• Must have sequence to bind to receptor gt address
  sequence
• Another sequence to signal its presence gt
  messenger sequence
• Another sequence that potentiate activity or
  potency gt potentiator sequence

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Address, Message, Potentiator (Cont.)

• Active Sequence 134 aa
• More conserved amongst species with few and minor
  conservative changes
• 2527 aa is very important, loss of activity when
  modified
• Potentiator sequence 1618aa
• Variable region amongst different species
• Differences in activities can be observed by
  digesting the peptide into different lengths
• 134aa full biological activity observed
• 126aa loss of activity due to interruption of
  the 3 important aa sequence (2527)
• 234 and 334aa loss of activity observed gt
  first 2aa may have some importance gt potential
  messenger sequence.
• PTH 1434 is likely to be the address sequence
  since it binds to the receptor and acts as an
  antagonist.

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Insulin, Diabetes, and the Antidiabetic Drugs
12
Glucagon

• Secreted by the a pancreatic cells
• Function
• Elevation of blood glucose via mobilizing glucose
  stores
• 29 aa. Peptide
• Pro-glucagon 150 a.a. long
• Processed in pancreatic a cells to generate the
  active peptide
• Use
• Acts at GPCR in the liver to facilitate
  gluconeogenesis and mobilization of glycogen
  stores
• Can be used to treat severe hypoglycemia in Type
  1 Diabetics

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Incretins GLP-1

• Differential processing of glucagon in small
  intestine yields GLP-1 (truncated GLP)
• Released upon food ingestion and sensing of sugar
• Function
• Promotes insulin synthesis/secretion, glucagon
  inhibition, increase in satiety, and slows
  gastric emptying
• Inactivated by DPP IV via cleavage of GLP-1
  peptide
• Byetta (Exenatide) BID SC Inection
• Modified form of Exentin-4 (GLP-1 analogue
  produced by Gila Monster)
• GLP-1 Agonist
• Extended half life and DPP IV resistance
• Help regenerate ß Cells and inhibit apoptosis of
  ß cells
• Associated with weight loss without association
  with hypoglycemia
• Liraglutide QD SC Injection
• Modified analogue of GLP-1
• Have increased T1/2 due to increased
  lipophilicity of FA gt binds to albumin

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Incretins DPP IV Inhibitors

• DPP IV readily inactivates GLP-1 gt regulate
  glucose homeostasis
• Diabetic patients have an increase in DPP IV
  inactivation of GLP-1
• DPP IV Inhibitors Gliptins
• Inhibits DPP IV gt Increase in T1/2 of GLP-1
• Approved Sitalgliptin (Januvia)
• In the pipline Vildagliptin (Galvus),
  Saxagliptin

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Insulin

• Produced by pancreatic ß cells
• Produced initially as Pro-insulin C-peptide A
  chain B chain
• Processed intracellularly to removed the
  connecting peptide and 4 additional a.a. gt
  Yielding A Chain and B Chain linked by disulfide
  bonds
• Processing machineryfor pro-insulin is
  promiscuous
• Insulin is preformed -gt packaged into golgi gt
  stored in ß cells
• Sequence conserved in human, bovine, and porcine
  with minimal a.a. changes
• Changes mostly occur in a.a. 810 of A Chain and
  amine (a.a. 15) and carboxy (a.a. 28 29)
  terminus of B Chain
• Both bovine and porcine insulin have been used
  for diabetic patients
• Patients using human insulin show fewer warning
  signs of hypoglycemia due to excess insulin

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Insulin (Cont.)

• Secondary and Tertiary Structure
• A Chain 2 alpha helices that are antiparallel
• B Chain Helix Elbow Beta Turn
• Arm region hydrophobic interaction between
  monomers -gt H-bond form at interface gt
  dimerization
• Dimer-Dimer association gt Hexamer
• Insulin is stored as hexamer in storage cells
• 2 Zn stabilizes the hexameric form
• Only monomer can interact with receptor
• Inversion of B29 Lys and B28 Pro -gt Change in
  configuration -gt dimer formation of insulin
  sterically inhibited gt exist as monomeric form
  (Lispro)
• Lispro is acting insulin because theres no dimer
  dissociation step. It also does not associate
  with Zn in the formulation

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Insulin (Cont.)

• On Receptor
• When insulin binds to insulin receptor -gt
  autophosphorylation of receptor and
  phosphorylation of IR1, IR2gt transduction
  cascade
• Receptor is a dimeric tyrosine kinase receptor
• Cortisol lowers affinity of insulin for receptor
  gt potential risk of diabetes
• Control of Insulin Release of ß Cells
• At rest low ATP -gt Kchannel remains open and
  responsive
• Cell is fully polarized gt negative cell
  potential and low insuling release
• High glucose glucose causes an increase in
  intracelluar ATP -gt K channel closes -gt
  depolarization -gt Ca2 influx gt insulin release
• Sulfonylurea antagonizes K Channel -gt maintains
  depoloarization gt increase in insulin release

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Insulin (Cont.)

• Function
• In Liver
• Inhibits glycogenolysis, conversion of fats into
  keto acids, and conversion of a. a. to glucose
• Promotess glucose storage as glycogens, increase
  TG synthesis and VLDL formation
• In Muscle
• Increase protein and glycogen synthesis
• In Fat
• Increases TG storage

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Sulfonylurea

• 1st Generation

MOA Antagonizes ATP depedend K Channels gt
induce insulin secretion, decrease in
gluconeogenesis All have similar
efficacy Duration of action differ because of
the way drugs are metabolized
Name?
Tolbutamide
Acetohexamide
Tolazamide
Chlorpropamide
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Sulfonylurea

• 2nd Generation

• Meglinides

Most Potent
Both classes have same MOA as 1st Generation
Sulfonylurea Longer acting and rapid onset
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Biguanides
MOA Decrease in hepatic glucose output, decrease
in insulin resistance (thought to be due to
inhibition of gluconeogenesis) Excreted from
kidney, Highly bioavailable No Hypoglycemia SE
GI
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Acarbose and Amylin

• Acarbose

MOA delays carbohydrate digestion and slows
glucose absorption in small intestine SE GI
(bloating and gas

• Amylin (Pramlintide)

MOA Amylin analogue, act as an antagonist that
inhibits glucagon release by liver, reduces
appeitite. Combo with Insulin
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Thiazolidenediones

• MOA PPAR? agonist
• Regulate glucose via nuclear interaction with
  PPAR?
• Increase expression of genes responsible for
  insulin release, f.a. oxidation, and insulin
  sensitivity
• Decrease expression of genes responsible for
  lipid production
• Combo with Insulin

Troglitazone
Rosiglitazone
Pioglitazone
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Thyroid
25
Thyroid Hormone
Outer Ring
Inner Ring (Tyr)

• All thyroids are produced from Tyr by iodination
  and dimerization
• Thyroglobulin Iodinates and acts as a storage
  of thyroid in colloids
• Initially made as T4, the inactive form -gt
  deiondination from the periphery gt T3 (active
  form)
• T4T3 51 gt most thyroids in body are not
  active
• rT3 is dominant in placenta of fetus, active
  form in developing fetus
• No biological activity in adults (notice the
  different iodination sites?)
• About 1/3 of T4 are converted to T3 and 40 of
  T4 are converted to rT3

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Thyroid Hormone Plasma Binding and Transport

• Thyroid Binding Protein
• Carries 7080 of secreted thyroid hormone
• 75 of T4 binding
• tight binding
• Thyroxine-Binding Prealbumin
• Carries 1015 of thyroid hormone
• 1015 of T4 binding
• Low binding affinity
• Serum Albumin
• Carries remainder of secreted thyroid hormones
• 510 of T4 binding, 1015 of T3 binding
• lowest binding affinity for the hormones
• Note once thyroid binds to the binding proteins,
  it cannot be deiondinated or metabolized

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SAR

• L-Tyr is the active form gt stereoselective (at
  the chiral carbon)
• Phenol gp is essential
• Provides H-bonding to the receptor
• Rings must be perpendicular to each other
• I3 and I5 interact with receptor
• Also prevents rotation of the outer ring gt
  required for binding of receptor
• Perpendicular orientation sensitive to bridging
  atom linking rings (O gt S, CH2)
• Substitution at C3 and C5 with halogen or
  nonpolar methyl gt can cross placenta
• 4 requires OH, NH2
• T4 contains I in this position -gt cannot fit into
  receptor gt inactive
• Most substitutions at C3 and C5 that is isoteric
  to I (halogen, methyl) and nonpolar will have
  some activity

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Thyroid Hormone Antagonists and Agonists

• Receptor specific
• TRa cardiac stimulation
• TRß cholesterol and energy metabolism
• GC-1 TRß specific gt decrease in plama
  cholesterol level
• T3 Non-specific
• Agonists
• Levothyroxine (T4) slower onset than T3 gt
  longer T1/2
• Liothyronin (T3) rapid onset of action, not
  used
• Liotrix mixture of T4 and T3 (41)gt resemble
  natural form in body
• Thyroid dessicated Theyroid gland from animal

Both Regulate Developement
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Antithyroid Agents
A.
D.
C.
B.
E.
Propylthiouracil
Perchlorate
Propranolol
Methimazole
Ipodate

• Match the molecule above to the MOA
• Thyroid peroxidase inhibitor
• Inhibit Thyroid cardiac response
• Inhibit iodine uptake
• Inhibit peripheral deiondonases

• Answer
• Thioamides A, D
• Beta Blocker C
• Anion Inhibitors E
• Iodinated contrast Media B