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High-affinity recognition of the human C-reactive protein independent of phosphocholine
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.ORCID iD: 0000-0002-3585-0289
Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, CBCS, Stockholm, Sweden..
Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, CBCS, Stockholm, Sweden..
Gothenburg Univ, Swedish NMR Ctr, Gothenburg, Sweden..
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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.

Place, publisher, year, edition, pages
2017. Vol. 15, no 21, p. 4644-4654
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-326501DOI: 10.1039/c7ob00684eISI: 000402742300021PubMedID: 28513744OAI: oai:DiVA.org:uu-326501DiVA, id: diva2:1131487
Funder
Swedish Research Council, 2014-4057Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2017-10-05Bibliographically approved
In thesis
1. Development of Peptide Binders: Applied to Human CRP, Carbonic Anhydrase (II, IX) and Lysine Demethylase 1
Open this publication in new window or tab >>Development of Peptide Binders: Applied to Human CRP, Carbonic Anhydrase (II, IX) and Lysine Demethylase 1
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
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
polypeptide conjugates, molecular recognition, peptide cyclization, protein targets
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-330489 (URN)978-91-513-0086-3 (ISBN)
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

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Yang, JieNorberg, ThomasBaltzer, Lars

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