Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 18.104.22.168 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”
Project Title: Structural and kinetic studies of human carbonic anhydrases as multiple drug targets
Project No.: 22.214.171.124/VIAA/3/19/464
Period: 36 months (1 February 2020 – 31 January 2022)
Project costs: 133 806.00 EUR
Project implementer: Dr. biol. Jānis Leitāns
Summary of project:
Carbonic anhydrases (CAs) are widespread enzymes that catalyze the interconversion of a water molecule and carbon dioxide into bicarbonate and hydrogen ions. There are at least 16 known mammalian CA isoforms. Although most of them are responsible for important processes in the organism, some isoform enzymatic activity is associated with different diseases. Human CA IX (hCA IX) is overexpressed in many tumor types and it has been proposed that hCA IX enzymatic activity favors tumor spread. The project consists of multiple hCA IX-ligand complex structural studies and the acquisition of structural knowledge for mitochondrial CA isoforms Va and Vb. These results will notably increase the available structural data of tumor associated hCA IX and de novo structure determination of hCA Va and/or hCA Vb isoforms with further use in anti-obesity drug design.
Information published 03.02.2020.
1 February 2020 – 30 April 2020
Started the optimization of the hCA Va and hCA Vb expression, production and purification systems. hCA Va has been successfully expressed in E.coli, resulting in significant amount of enzymatically active and soluble hCA Va protein. Initial purification protocol has been developed with ongoing optimization steps.
Information published 30.04.2020.
1 May 2020 – 31 July 2020
Purification system of the hCA Va is being optimized and initial crystallization condition screening was started. hCA IX enzyme was co-crystallized with various sulfonamide inhibitors.
Information published 31.07.2020.
1 August 2020 – 31 October 2020
hCA IX enzyme was co-crystallized with sulfonamide class inhibitors, collected difraction data and solved 3D structure. Continued screening of the hCA Va crystallization conditions.
Information published 30.10.2020.
1 November 2020 – 31 January 2021
Optimization of the hCA IX enzyme – benzenesulfonamide derivatives crystallization conditions. Collection of the diffraction data and ligand modeling. hCA Va enzyme crystallization condition optimizing.
Information published 29.01.2021.
1 February 2021 – 30 April 2021
Continued working on hCA IX enzyme co-crystallization experiments in complex with benzensulfonamides. Further crystallization condition screening for hCA Va.
Information published 30.04.2021.
1 May 2021 – 31 July 2021
Collected data from hCA IX – ligand complex crystals and solved 3D structure. Continued screening for hCA Va and hVA Vb crystallization conditions.
Information published 30.07.2021.
1 August 2021 – 31 October 2021
hCA IX was co-crystallized in complex with sulfonamide class inhibitors. Collected diffraction data and solved the 3-D structure.
Information published 29.10.2021.
1 November 2021 – 31 January 2022
Co-crystallization condition optimizing for hCA IX – benzensulfonamides. Collected diffraction data from protein crystals. Solved 3-D structure of the protein and determined ligand binding modes. Scientific manuscript preparation about protein-ligand interactions. Continued hCA Va crystallization condition screening by trying different temperatures.
Information published 31.01.2022.
Progress of the project
1 February 2022 – 30 April 2022
Optimization of the hCA IX – inhibitor complex crystallization condition. Data collection from hCA IX – inhibitor complex crystals. Ligand binding mode determination within the active site of hCA IX. Continued hCA Va crystallization condition screening. Participation in LU 80. conference with project related results.
Information published 29.04.2022.
Progress of the project:
1 May 2022 – 31 July 2022
Preparation of enzymatically active hCA II, hCA Va, hCA IX and hCA XII isoforms for kinetic measurements. Continued hCA Va crystallization condition optimization. hCA IX co-crystallization with inhibitors. Data collection from protein crystals. Structure determination and ligand binding mode determination.
Information published 29.07.2022.
1 August 2022 – 31 October 2022
Kinetic analysis of various carbonic anhydrase isoforms. Sulfonamide-hCA interaction analysis. Co-crystallization of hCA in complex with benzensulfonamide class inhibitors. Poster preparation for the 2nd DrugDiscovery Conference. Participation in the 2nd DrugDiscovery Conference.
Information published 31.10.2022.
1 November 2022 – 31 January 2023
Submitted a patent application to Patent Office of the Republic of Latvia about the method of purification of human mitochondrial carbonic anhydrase Va protein. Co-crystallized hCA with various sulfonamide inhibitors. Processed the protein diffraction data. Successfully determined the 3D structure and identified the binding modes of ligands. Continued to optimize hCA Va crystallization conditions using additives.
Information published 31.01.2023.