Medicinal Chemistry of drugs to treat Diabetes

PHAY2003

Dr Andy Wilderspin

Normal control of glucose

The Islets of Langerhans in the Pancreas consist of : 75% β cells - secrete insulin (peptide hormone) when glucose uptake from the intestine is high. INSULIN is a hormone that stimulates certain tissues eg muscle, liver to take up glucose and to promote conversion of glucose to glycogen and fatty acids and triacylglycerol as metabolic fuel stores (ie. In fed state it signals to the body that it should store fuel). In diabetes mellitus this control breaks down.

Insulin 3D Structure

A-chain blue B-chain red

Phe 1

Gly 1

Thr 30

Asn 21

Maturation of pre-proinsulin

Insulin gene expression • Insulin gene located on chromosome 11 present in all cells

•3 exons and 2 introns (one in 5’ untranslated region, one interupts C-peptide)

• Transcription factor pdx-1 activated by a kinase in response to glucose

• NeuroD1 and MafA also key transcription factors in the Islet of Langerhans β cells

• Enhanced transcription produces mRNA 446 nucleotides long

• mRNA is 2.5x more stable when glucose levels are high (half-life 3 days rather than 1 day).

• Pre-proinsulin translation and translocation to RER is quick 5 – 10 minutes

• Signal peptidase cleaves the nascent polypeptide thus removing the pre-sequence

• Proinsulin crosses trans-golgi into the immature β-granules in 20 – 30 minutes

• Multiple proteolytic cleavage occurs as the β-granules mature - takes 1 – 2 hrs

• First PC3 (Pro-hormone convertase 3) cleaves after Arg31-Arg32 (between B and C chain)

• Carboxypeptidase H removes Arg31 and Arg32 to create C-terminus of B-chain

• PC2 cleaves after Lys64-Arg65 to create N-terminus of A-chain and giving mature insulin

• Carboxypeptidase H removes Lys64 and Arg65 giving C-peptide

•The mature insulin is stored in β-granules until ready for exocytosis into the bloodstream

Sequence of human preproinsulin

malwmRllpllallalwgpDpaaa ↓ pre

fvnqHlCgsHlvEalylvCgERgffytpKt ↓RR↓ b-chain

EaEDlqvgqvElgggpgagslqplalEgslq ↓KR↓ c-chain

givEqCCtsiCslyqlEnyCn a-chain

↓ indicates cleavage by signal peptidase, prohormone convertase, or carboxypeptidase H

Human insulin properties

Formula C254H377N65O75S6

Molecular weight 5,733 Number of amino acids 51 Disulphide bridges 3 Positively charged residues 2 Negatively charged residues 4 pI 5.8

Dimeric and hexameric insulin Dimers form by interaction of the 2 B-chains. The two B-chain α-helices interact and the two B-chain β-strands interact to give stable dimers.

Three dimers associate with two zinc ions to form hexameric insulin (via the B-chain His10 sidechains)

Insulin 3D structure

Human Insulin: (2HIU.pdb)

RasMol can be downloaded to display NMR and X-ray structures. From the Protein Data Bank (PDB) simply type human insulin or 2hiu into the search box and download the required “PDB File (text)” file.

Hexameric insulin: (1AIY.pdb) - contains zinc

Milestones 1922: The first patient, Leonard Thompson a 14 year old boy with type 1 diabetes is given the first medical administration of insulin. Previously patients with type 1 diabetes would be put onto starvation diets and would have only months to live. Leonard lived another 13 years before succumbing to pneumonia.

1923: Banting and Macleod are awarded the Nobel Prize in Physiology or Medicine. Banting and Macleod, felt Best and Collip were equally eligible and shared their prize money with the two colleagues.

1955: Insulin is sequenced by Frederick Sanger, and is the first protein to be fully sequenced. In 1958 Sanger receives the Nobel Prize in Chemistry for this research.

1969: Insulin structure elucidated by Dorothy Hodgkin a Nobel prize winner in 1964 for crystallography

Marketed Pharmaceutical Preparations (from animals)

• Conventional Insulin 1920s Crude acid/alcohol extraction of pancreas • Single Peak Insulin size exclusion chromatography • Sequence differences Porcine insulin has B-chain Threonine 30 changed to Alanine Bovine insulin has 2 further changes in A-chain (Thr8Ala, Ile10Val) C-chains of bovine and porcine have many more differences Immunogenicity arises from sequence differences

Semi-synthetic human insulin (emp) starting from porcine insulin

fvnqHlCgsHlvEalylvCgERgffytpKt human B-chain sequence

fvnqHlCgsHlvEalylvCgERgffytpKa porcine B-chain sequence

fvnqHlCgsHlvEalylvCgER gives des-octapeptide-insulin

Denaturing tryptic cleavage then purify 1-22

Trypsin in reverse, 20 h, 37oC

+ *gffytpK*t (synthetic octapeptide)

Allow reaction to reach equilibrium, then purify from 58% yield of human insulin * Boc protected amino groups

Key drugs

• Insulin - and a variety of modified insulins • Glipizide - and other sulphonylureas • Metformin - other biguanides discontinued

Sulphonylureas for type 2 diabetes

C21H27N5O4S

MWt 445 Bioavailability 100% Protein binding 98%

Blocks K+ channels Cell depolarises Voltage-gated Ca2+ channels open Ca2+ increases Insulin released from granules

Glipizide

Sulphonylureas for type 2 diabetes

Glibenclamide not suitable above 60 years.

Gliclazide is one of two small molecules on the WHO Model List of Essential Medicines

Glipizide

Metformin: for type 2 diabetes

Toluene/HCl

96% MWt 129

Phenformin withdrawn from most markets in the late 1970s due to a higher risk of lactic acidosis (5x)

Buformin also withdrawn from most markets due to a high risk of lactic acidosis

Biguanides: contraindicated in respiratory and kidney disease

Recombinant insulin production: problems to be overcome

• Need the human gene coding for insulin • Bacteria do not switch on genes with enhancers • Bacteria can’t splice out introns • Bacterial ribosomes don’t recognise human mRNA • Bacteria prefer different triplet codons to humans eg CCG • Insulin/proinsulin doesn’t start with Methionine • Bacteria do not have pro-hormone convertases • Bacteria do not oxidise to give disulphide bridges • Bacteria do not normally secrete human proteins Solution was described by David Goeddel (Genentech 1978)

Chemical Gene Synthesis

18 overlapping oligonucleotides for the B-chain and 12 for the A-Chain

DNA Cloning 1. Cut pBR322 plasmid EcoR1 and BamH1 2. Ligate in the synthetic gene 3. Transform competent E. coli cells 4. Recovery in LB broth 5. Grow colonies on agar plates 6. Each colony is a separate clone

2 chain bacterial method (crb)

To maximise transcription and translation: Lac promoter (not pdx-1 enhancer) Shine-Dalgarno RBS (not Kozak) Initiator methionine β-galactosidase fused to Insulin A- or B-chain coding region Rare codons replaced Stop codon Terminator

Processing of A- and B-chains

•Purify fusion proteins •Cleave off β–gal with CNBr •Purify A- and B-chains •Combine and refold chains •Oxidise to form disulphides •Purify active insulin

Eli Lilly and Company introduced Genentech Insulin as Humulin (1982) Sanofi now produce crb (Insuman)

Lilly proinsulin method (prb)

• β-gal fused to methionine and proinsulin

• Purify from bacteria

• Cleave with CNBr to release proinsulin

• Purify, refold and oxidise cysteines

• Cleave with trypsin and carboxypeptidase B

• Separate insulin from C-chain

Required development of cloning methods to get the proinsulin sequence, and extra proteolytic steps after extraction, but gave better yield of refolded intermediate

Novo Nordisk yeast method (pyr) TP1 = triose phosphate isomerase 1 L-IP = Leader-insulin precursor (fusion protein) Leader = pre-pro sequence of yeast α-mating factor IP = Insulin precursor S = EEAEAEAEPK C = AAK N = N-linked carbohydrate sites (NXT/S)

19 aa 66 aa 29 aa 21 aa L-IP

Site-directed Mutagenesis to obtain modified insulins

Recombinant Human Insulins and animal Insulins