Experimental Approaches to Identify Selective Picomolar Inhibitors for Carbonic Anhydrase IX

J. Kazokaite-Adomaitiene*, H.M. Becker, J. Smirnoviene, L.J. Dubois, D. Matulis

*Corresponding author for this work

Research output: Contribution to journalReview articlepeer-review

Abstract

Background: Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO2, thereby emerging as essential enzymes for many vital functions. Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant therapeutic target because of its role in cancer progression. Only two CA IX inhibitors have entered clinical trials, mostly due to low affinity and selectivity properties. Objective: The current review presents the design, development, and identification of the selective nano-to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09. Methods and Results: Compounds were selected from our database, composed of over 400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12 human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several biophysical techniques, such as fluorescent thermal shift assay and isothermal titration calorimetry, highlighting their contribution to the analysis of compound affinity and structure-activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor screening, several gene cloning and protein purification strategies are presented, including site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification, and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities of lead inhibitors in intact cancer cells. Conclusions: Novel CA IX inhibitors are promising derivatives for in vivo explorations. Furthermore, the simultaneous targeting of several proteins involved in proton flux upon tumor acidosis and the disruption of transport metabolons might improve cancer management.<comment>Superscript/Subscript Available</comment
Original languageEnglish
Pages (from-to)3361-3384
Number of pages24
JournalCurrent Medicinal Chemistry
Volume28
Issue number17
DOIs
Publication statusPublished - 2021

Keywords

  • Drug development
  • organic chemistry
  • cancer
  • sulfonamide
  • thermal shift assay
  • isothermal titration calorimetry
  • CA IX
  • THERMAL SHIFT ASSAYS
  • INTRINSIC THERMODYNAMICS
  • CATALYTIC-PROPERTIES
  • DRUG DESIGN
  • CA-IX
  • 1-ANILINO-8-NAPHTHALENE SULFONATE
  • TRANSPORT ACTIVITY
  • ENZYME-INHIBITORS
  • BINDING-AFFINITY
  • CO2 PERMEABILITY

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