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Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
Visa övriga samt affilieringar
2010 (Engelska)Ingår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 16, nr 3, s. 159-164Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors.

Ort, förlag, år, upplaga, sidor
2010. Vol. 16, nr 3, s. 159-164
Nyckelord [en]
Fluorescence polarization, Mycobacterium tuberculosis, Peptide inhibitors, Ribonucleotide reductase
Nationell ämneskategori
Farmaceutiska vetenskaper
Identifikatorer
URN: urn:nbn:se:uu:diva-112344DOI: 10.1002/psc.1214ISI: 000275448300007PubMedID: 20127854OAI: oai:DiVA.org:uu-112344DiVA, id: diva2:290253
Tillgänglig från: 2010-01-26 Skapad: 2010-01-13 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
Ingår i avhandling
1. Peptidomimetic Enzyme Inhibitors: Targeting M. tuberculosis Ribonucleotide Reductase and Hepatitis C Virus NS3 Protease
Öppna denna publikation i ny flik eller fönster >>Peptidomimetic Enzyme Inhibitors: Targeting M. tuberculosis Ribonucleotide Reductase and Hepatitis C Virus NS3 Protease
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis focuses on the design and synthesis of inhibitors targeting Mycobacterium tuberculosis ribonucleotide reductase (RNR) and hepatitis C virus (HCV) NS3 protease; enzymes that have been identified as potential drug targets for the treatment of tuberculosis and hepatitis C, respectively. Small peptides have been recognized as inhibitors of these enzymes. However, the use of peptides as drugs is limited due to their unfavorable properties. These can be circumvented by the development of less peptidic molecules, often referred to as peptidomimetics. When this work was initiated, only a few inhibitors targeting M. tuberculosis RNR had been identified, whereas the HCV NS3 protease was an established drug target. Therefore, early peptidomimetic design strategies were applied to inhibitors of RNR while the NS3 protease inhibitors were subjected to modifications in a later stage of development.

It has previously been shown that peptides derived from the C-terminus of the small subunit of M. tuberculosis RNR can compete for binding to the large subunit, and thus inhibit enzyme activity. To investigate the structural requirements of these inhibitors, different series of peptides were evaluated. First, peptides from an N-terminal truncation, an alanine scan and a designed library were synthesized and evaluated to examine the importance of the individual amino acid residues. Then, a set of N-terminally Fmoc-protected peptides was evaluated, and it was found that the N-terminal group improved the affinity of the peptides even when the length of the compounds was reduced. Furthermore, potential inhibitors of less peptidic character were generated by the introduction of a benzodiazepine-based scaffold.

To further reduce the peptidic character and investigate the binding properties of HCV NS3 protease inhibitors, a series of tripeptides incorporating a β-amino acid was synthesized. Inhibition was evaluated and docking studies were performed to understand how the structural changes affected inhibitory potency. The results illustrated the importance of preserving the hydrogen bonding network and retaining electrostatic interactions in the oxyanion hole between inhibitor and protein.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2010. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 119
Nyckelord
enzyme inhibitor, peptidomimetics, structure-activity relationship, tuberculosis, ribonucleotide reductase, hepatitis C virus, NS3 protease
Nationell ämneskategori
Läkemedelskemi
Forskningsämne
Läkemedelskemi
Identifikatorer
urn:nbn:se:uu:diva-112345 (URN)978-91-554-7716-5 (ISBN)
Disputation
2010-03-12, B21, BMC, Husargatan 3, Uppsala, 09:15 (Svenska)
Opponent
Handledare
Tillgänglig från: 2010-02-18 Skapad: 2010-01-13 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
2. Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes
Öppna denna publikation i ny flik eller fönster >>Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes
2009 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis describes structural and binding studies in enzymes from two different  organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA).

RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors

Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2009. s. 61
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 684
Nyckelord
Mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, fluorescence polarization, lipase, interfacial activation, hydrolase, X-ray structure, substrate specificity
Nationell ämneskategori
Strukturbiologi
Identifikatorer
urn:nbn:se:uu:diva-109697 (URN)978-91-554-7638-0 (ISBN)
Disputation
2009-12-05, B42, Uppsala Biomedical Center (BMC), Husargatan 3, Uppsala, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2009-11-12 Skapad: 2009-10-22 Senast uppdaterad: 2012-09-18Bibliografiskt granskad

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