Peptide and Protein Therapeutics

Seminar – Bioorganic Chemistry

11.12.2019

•Chun-Chiao Yang

•Diego Garcia

Peptide synthesis

Ahrens, V. M.; Bellmann-Sickert, K.; Beck-Sickinger, A. G. Peptides and PeptideConjugates: Therapeutics on the Upward Path. Future Medicinal Chemistry 2012, 4(12), 1567–1586. https://doi.org/10.4155/fmc.12.76.

Pros & Cons

Small molecules

• Low molecular weight

Cost-effective

Few synthetic steps

Oral availability

Easy crossing of biologicalmembranes

Accumulation

Toxic metabolites

Peptides

• Sequences of ≈ 20 amino acids

High cost

More complex synthetic steps

Intramuscular administration

High selectivity and affinity

Hydrolysis → nontoxic products

Less drug-drug interactions

Stabilization of peptidic drugs

Stabilization of peptidic drugsProblem:

Lack in vivo stability and limitedaccess to the intracellular space.

↓

Peptide stabilization: Anymodification applied increase invivo stability.

Modification of the peptidesequence or attachment ofstabilizing agents to amino acid sidechains.

AMINOPEPTIDASE

PDB ID: 1XJO

DIPEPTIDYL PEPTIDASE

PDB ID: 3JYH

PEGylation (polyethylenglycol)

• Increases water solubility

• Shields from proteases

• Improves bio distribution

• Lowers allergenic properties

• Size 2-40 kDa, increases the mw• ↓ Renal filtration, ↑Half-life

• PEGs for modification arecommercially available, with differentfunctionalities.

Disadvantage:

• No enzyme able to degrade themolecule is known.

• Adagen®, Oncaspar® and Krystexxa®:PEGylate enzymes.

• Cimzia®: TNF-α-blocking antibodyfragment to treat Chron’s disease andrheumatoid arthritis.

• Neulasta®, Mircera® and Somavert®:growth factors.

D-amino acids

• Naturally, only L-amino acids areemployed in peptides andproteins.

• D-peptides are stable towardsproteolysis.

• Binds to the target in an equalmanner as natural all-L-peptides

D-phenylalanine in humancalcitonin derivatives as cell-penetrating peptides in drug-delivery.

Lipidation

Acylation of compounds with fattyacids.

• Higher affinity to albumin.• ↑ time of the drug in the blood, ↓ renal

and hepatic clearance.

Insuline

• C10

• C12

• C14

Levemir®

Lipidated insulin variant is available as along-acting therapeutic

Combinated with D-amino acids, protectpeptides from peptidases.

• Gonadotropinreleasing hormone.

Decapeptide containing a D- and an L-cysteine, cyclized via a thioether bridge.

Cyclization

Integration of non-natural amino acids

N-methyl amino acids Peptoids

Reduced peptidesβ-peptides

(2) Seebach, D.; Matthews, J. L. β-Peptides: A Surprise at Every Turn. Chem. Commun. 1997, No. 21, 2015–2022. https://doi.org/10.1039/A704933A. (3) Automated Synthesis of Peptoids and Peptoid-PeptideHybrids http://cem.com/de/automated-synthesis-of-peptoids-and-peptoid-peptide-hybrids (accessed Dec 4, 2019). (4) Andersson, H.; Hallberg, M. Discovery of Inhibitors of Insulin-Regulated Aminopeptidase asCognitive Enhancers. International journal of hypertension 2012, 2012, 789671. https://doi.org/10.1155/2012/789671.

PASylation®

Conjugation with an at least 100amino acid random sequencecomprising the three amino acidsproline, alanine and serine.

Recombinant vectors encodingthe PAS-sequence.

• Increases resistance againstproteases.

• Ability of renal cleaved.

• Highly soluble.

• Nontoxic and non-immunogenic.

• Easy to purify during synthesis.

HESylation®

Fusion with hydroxyethyl starch (HES)

Linked to cysteine residues, via glycosylated side chains and enzymatically by transglutaminase reactions derivatives.

Better biodegradability than PEG.

Approved peptidic therapeutics

AntibioticsGramicidin D

Linear peptide, which is a mixture of three gramicidines (A–C).

Daptomycin

Val-Gly-Ala-D-

Leu-Ala-D-Val-

Val-Val-Trp-D-

Leu-Trp-D-

Leu-Trp-D-

Leu-Trp-Gly

(5) Andersson, H.; Hallberg, M. Discovery of Inhibitors of Insulin-Regulated Aminopeptidase as CognitiveEnhancers. International journal of hypertension 2012, 2012, 789671. https://doi.org/10.1155/2012/789671.(6) Steenbergen, J.; Alder, J.; Thorne, G.; Tally, F. Daptomycin: A Lipopeptide Antibiotic for the Treatment ofSerious Gram-Positive Infections. The Journal of antimicrobial chemotherapy 2005, 55, 283–288.https://doi.org/10.1093/jac/dkh546.

Gonadotropin-releasing hormone receptor antagonistAbarelix, Cetrorelix, Goserelin and Leuprolide

Suppression of gonadotropinsecretion upon binding in acompetitive manner, blocking theaction of agonists.

Contain single amino acidreplacements in their sequence.

• During fertility treatment,prevent the premature surge ofluteinizing hormone.

• Lead to a decrease oftestosterone levels

(7) National Center for Biotechnology Information. PubChem Database. Abarelix, CID=16131215,https://pubchem.ncbi.nlm.nih.gov/compound/Abarelix (accessed on Dec. 4, 2019)

Anticoagulants

Bivalirudin and Lepirudin

Derivatives of peptides producedin medicinal leeches’ saliva.

Treatment of heparin inducedthrombocytopenia by inhibition ofthrombin.

(8) Steenbergen, J.; Alder, J.; Thorne, G.; Tally, F. Daptomycin: A Lipopeptide Antibiotic for the Treatment of Serious Gram-Positive Infections. The Journal of antimicrobial chemotherapy 2005, 55, 283–288.https://doi.org/10.1093/jac/dkh546. (9) Warkentin, T. E. . CHAPTER 44 - The Diagnosis and Management of Heparin-Induced Thrombocytopenia. In The Vein Book; Bergan, J. J., Ed.; Academic Press: Burlington, 2007; pp 395–403. https://doi.org/10.1016/B978-012369515-4/50047-8.

DiabetesExenatide

Glucagon-like peptide 1(GLP 1), resistant to dipeptidyl peptidase IV resistant.Agonist on the GLP1-receptor. • Stimulation of glucose-dependent

insulin secretion.• Suppression of glucagon secretion.• Prolonged residence times of food

in the stomach.• Reduction in food intake.

Liraglutide

• Lipidated lysine residue.• Attaches to serum albumin to

increase the half-life.• After binding to the glucagon

receptor, leads to an activation of glycogen phosphorylase and a release of glucose-1-phosphate into the bloodstream.

• It is used in diabetes patients in the treatment of hypoglycemia after insulin overdosing.

(10) Chia, C.; Egan, J. Role and Development of GLP-1 ReceptorAgonists in the Management of Diabetes. Diabetes, metabolicsyndrome and obesity : targets and therapy 2009, 2, 37.https://doi.org/10.2147/DMSOTT.S4283.

Terapeutic peptides in clinical trailsBioorganic Chemistry

Clinical phases Phase I (20-100 People)

Pharmacokinetics, pharmacodynamics and general safety of drug.

Phase II (100-300 People) Small-scale study to investigate the drug effectiveness and side effect.

Phase III (300-3000 People) Large-scale study to investigate the drug effectiveness and side effect.

Phase IV (anyone seeking for treatment from physician) Post Marketing Surveillance.

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Examples of peptidic vaccines andimmunotherapeutics Melanomas

Glycoprotein 100-derived peptide

Cytostatic temozolomide with Telomerase peptide vaccine GV1001

Breast Cancer Peptide AE37

Peptide GP2

Obesity Obinepitide

TM30339

Pramlintide

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Temozolomide with telomerase peptide vaccine Combination therapy

Try to enhance the immunologic response

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Kyte JA, Gaudernack G, Dueland S et al. Clin. Cancer Res. 17(13), 4568–4580 (2011)

25 Collected patients

T-cell responses were detected

Not immunotherapeutics approach and what next…?

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Liu Z, Wang F, Chen X. Integrin alpha(v)beta(3)-Targeted Cancer Therapy. Drug Dev Res. 2008

Intergrin targeting may be an alternative.

Basic research on future therapeuticpeptides

Bioorganic Chemistry

What can we do more with peptides?

targetGPCR

agonists

inverse agonists

antagonists

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https://www.nature.com/scitable/topicpage/gpcr-14047471/

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G-protein-coupled receptor (GPCR)

Signal molecule

Intelligent linker strategies

Functions of peptides

peptidetherapeutics

As drug

As a carrier

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Intelligent linkers Acetal linker

Disufide linker

Ester linker

Hydrazone linker

GFLG sequence

Valine-citrulline sequence

Furin cleavage site

Dipeptidyl peptidase IV cleavage site

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Acetal linker

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Easy to synthesis

PH-dependent hydrolysis

Bifunction

Ex: mesoporous silica withmelittin

Hydrazone linker Acid labile linkers

Peptides amphiphiles

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Ester linker Can be cleavage by enzyme or pH change

Easy to degrade in blood

Ex: nanotubes fused to therapeutic molecule

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Liu Z, Chen K, Davis C, et al. Drug delivery with carbon nanotubes for in vivo cancer treatment. Cancer Res. 2008

Linker is cleavaged by special enzyme

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GFLG Sequence Valine-citrulline SequenceFurin cleavage site

Cathepsin B

Dipepeptidyl peptidase IV (Dppiv)

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Diez-Torrubia A, Balzarini J, Andrei G et al. Dipeptidyl peptidase IV dependent water-soluble prodrugs of highly lipophilic bicyclicnucleoside analogues. J. Med. Chem.(2011)

Peptidic therapeutics can do more…

Conclusion

• Due to the limit of small-molecules drugs and their toxicity, a newway of treatment has been developed with peptide drugs.

• Stabilization techniques and linkers are crucial for the growth of thisfield.

• In last decade there are more and more new approved peptide-drugs.

• Stability and the cost of peptide drugs are still having challenges.

Thank you!