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Development of Peptide Binders: Applied to Human CRP, Carbonic Anhydrase (II, IX) and Lysine Demethylase 1
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.ORCID iD: 0000-0002-3585-0289
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

In this thesis, a polypeptide binder concept is illustrated. By conjugation to a set of sixteen polypeptides, a small binding molecule can evolve into a polypeptide binder with increased affinity and selectivity. The concept was applied to 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) and acetazolamide (AZM) for development of high affinity binders targeting human C-reactive protein (CRP) and human carbonic anhydrase (HCA) II and IX respectively. In addition, peptididic macrocycles were developed as inhibitors of lysine specific demethylase 1 (LSD1).

CRP is a well-known biomarker of inflammation in humans and binders recognizing it are therefore of large interest as medical diagnostics. Until now, phosphocholine (PCh) and derivatives are the only known small molecule binders for CRP, but they have low μM affinity and bind CRP in a Ca2+ dependent manner. The small molecule DQ was designed as a CRP binder that is structurally unrelated to PCh. Its polypeptide conjugate, 4-C25l22-DQ, was demonstrated as a strong, Ca2+ independent binder for CRP, and had an affinity approximately three orders of magnitude higher than DQ itself.

HCA IX is a protein that is interesting for diagnosis of cancer. AZM is a small molecule inhibitor of HCAs with a dissociation constant of 38 nM for HCA II and 3 nM for HCA IX. Interestingly, polypeptide conjugate 4-C10L17-AZM displayed stronger binding to both HCA II (KD 4 nM) and HCA IX (KD 90 pM). This result provided evidence that the binder concept can be applied also for small molecules which already have high affinity for their protein receptors.

LSD1 is an enzyme that regulates the methylation of Lys 4 of histone 3 via a PPI-like interaction and which is of therapeutic interest in certain cancers. Based on the structures of two peptidic ligands bound to LSD1, we sequentially prepared truncated, mono-substituted and macroyclic peptides in order to develop reversible inhibitors of LSD1. Some stapled cyclic peptides bound to LSD1 with 10-fold higher affinity than the corresponding linear parent peptide. Changing the staple into a lactam further improved the binding potency and the best lactams inhibited the enzymatic activity of LSD1 at low μM Ki values.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1568
Keywords [en]
polypeptide conjugates, molecular recognition, peptide cyclization, protein targets
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-330489ISBN: 978-91-513-0086-3 (print)OAI: oai:DiVA.org:uu-330489DiVA, id: diva2:1145942
Public defence
2017-11-16, B/C2:305, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-10-24 Created: 2017-10-01 Last updated: 2017-10-24
List of papers
1. High-affinity recognition of the human C-reactive protein independent of phosphocholine
Open this publication in new window or tab >>High-affinity recognition of the human C-reactive protein independent of phosphocholine
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2017 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 15, no 21, p. 4644-4654Article in journal (Refereed) Published
Abstract [en]

A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the μM to nM range for which no small molecule ligands are known.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-326501 (URN)10.1039/c7ob00684e (DOI)000402742300021 ()28513744 (PubMedID)
Funder
Swedish Research Council, 2014-4057
Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2017-10-05Bibliographically approved
2. Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide
Open this publication in new window or tab >>Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide
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2017 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 20, p. 5838-5848Article in journal (Refereed) Published
Keywords
Human Carbonic Anhydrase, Acetazolamide, Recognition, Surface plasmon resonance, Tumor
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-330361 (URN)10.1016/j.bmc.2017.09.017 (DOI)000413402100064 ()
Note

The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38 nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4 nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3 nM, whereas that of the 4-C10L17-AZM conjugate was 90 pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to μM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX.

Available from: 2017-09-28 Created: 2017-09-28 Last updated: 2018-02-08Bibliographically approved
3. Macrocyclic peptides as inhibitors of human LSD1
Open this publication in new window or tab >>Macrocyclic peptides as inhibitors of human LSD1
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lysine demethylase 1 (LSD1) regulates the degree of methylation of Lys4 of histone 3 in the nucleosome core particle. As LSD1 is overexpressed in certain cancers,  inhibitors have potential for use as drugs. Guided by the structures of two peptidic ligands bound to LSD1 we prepared truncated, mono-substituted and macrocyclic peptides to find leads for development of specific and revserible inhibitors. Surface plasmon resonance biosensor analysis revealed that some stapled, macrocyclic peptides had up to 10-fold higher affinity for LSD1 than the corresponding linear native peptide. Furthermore, peptides cyclized by lactamization were low mM inhibitors of LSD1, with the most effective one being >25-fold more potent than the linear native reference.

National Category
Chemical Sciences Biological Sciences
Identifiers
urn:nbn:se:uu:diva-330378 (URN)
Funder
Swedish Research Council
Available from: 2017-09-28 Created: 2017-09-28 Last updated: 2017-10-05

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