Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 μM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.
- MeSH
- antibakteriální látky chemie metabolismus farmakologie terapeutické užití MeSH
- bakteriální proteiny chemie metabolismus MeSH
- gastrointestinální nemoci farmakoterapie mikrobiologie patologie MeSH
- Helicobacter pylori účinky léků enzymologie MeSH
- hypoxanthinfosforibosyltransferasa chemie metabolismus MeSH
- hypoxanthiny chemie metabolismus farmakologie terapeutické užití MeSH
- infekce vyvolané Helicobacter pylori farmakoterapie patologie MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- organofosfonáty chemie metabolismus farmakologie terapeutické užití MeSH
- pentosyltransferasy chemie metabolismus MeSH
- prekurzory léčiv chemie metabolismus farmakologie terapeutické užití MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Therapeutic treatment of tuberculosis (TB) is becoming increasingly problematic due to the emergence of drug resistant Mycobacterium tuberculosis (Mt). Thus, new targets for anti-TB drug discovery need to be identified to combat and eradicate this disease. One such target is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) which synthesises the 6-oxopurine nucleoside monophosphates essential for DNA/RNA production. [3R,4R]-4-Hypoxanthin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine and [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine (compound 6) are the most potent inhibitors of MtHGPRT yet discovered having Ki values of 60 nM. The crystal structure of the MtHGPRT.6 complex was obtained and compared with that of human HGPRT in complex with the same inhibitor. These structures provide explanations for the 60-fold difference in the inhibition constants between these two enzymes and a foundation for the design of next generation inhibitors. In addition, crystal structures of MtHGPRT in complex with two pyrrolidine nucleoside phosphosphonate inhibitors plus pyrophosphate provide insights into the final stage of the catalytic reaction. As the first step in ascertaining if such compounds have the potential to be developed as anti-TB therapeutics, the tetra-(ethyl L-phenylalanine) tetraamide prodrug of 6 was tested in cell based assays. This compound arrested the growth of virulent Mt not only in its replicating phase (IC50 of 14 μΜ) but also in its latent phase (IC50 of 29 μΜ). Furthermore, it arrested the growth of Mt in infected macrophages (MIC50 of 85 μΜ) and has a low cytotoxicity in mammalian cells (CC50 of 132 ± 20 μM). These inhibitors are therefore viewed as forerunners of new anti-TB chemotherapeutics.
- MeSH
- antituberkulotika chemická syntéza chemie farmakologie MeSH
- bisfosfonáty chemie farmakologie MeSH
- hypoxanthinfosforibosyltransferasa antagonisté a inhibitory metabolismus MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- Mycobacterium tuberculosis cytologie účinky léků metabolismus MeSH
- nukleosidy chemie farmakologie MeSH
- pyrrolidiny chemie farmakologie MeSH
- THP-1 buňky MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Sedimentation equilibrium and size-exclusion chromatography experiments on Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT) have established the existence of this enzyme as a reversibly associating mixture of dimeric and tetrameric species in 0.1 M Tris-HCl-0.012 M MgCl2, pH 7.4. Displacement of the equilibrium position towards the larger oligomer by phosphate signifies the probable existence of MtHGPRT as a tetramer in the biological environment. These data thus add credibility to the relevance of considering enzyme function in the light of a published tetrameric structure deduced from X-ray crystallography. Failure of 5-phospho-α-d-ribosyl-1-pyrophosphate (PRib-PP) to perturb the dimer-tetramer equilibrium position indicates the equivalence and independence of binding for this substrate (the first to bind in an ordered sequential mechanism) to the two oligomers. By virtue of the displacement of the equilibrium position towards dimer that is affected by removing MgCl2 from the Tris-HCl buffer, it can be concluded that divalent metal ions, as well as phosphate, can affect the oligomerization. These characteristics of MtHGPRT in solution are correlated with published crystal structures of four enzyme-ligand complexes.
Systematic structure-activity studies on a diverse family of nucleoside phosphonic acids has led to the development of potent antiviral drugs such as HPMPC (CidofovirTM), PMEA (AdefovirTM), and PMPA (TenofovirTM), which are used in the treatment of CMV-induced retinitis, hepatitis B, and HIV, respectively. Here, we present the synthesis of a novel class of acyclic phosphonate nucleotides that have a locked conformation via a pyrrolidine ring. NMR analysis of these compounds revealed that the pyrrolidine ring has a constrained conformation when in the cis-form at pD < 10 via hydrogen bonding. Four of these compounds were tested as inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases. The most potent has a Ki of 0.6 μM for Plasmodium falciparum HGXPRT.
- MeSH
- adenin chemie MeSH
- antimalarika chemická syntéza MeSH
- chemie farmaceutická MeSH
- hypoxanthin chemie MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- kyslík chemie MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- organofosfonáty chemie MeSH
- pentosyltransferasy antagonisté a inhibitory MeSH
- Plasmodium falciparum účinky léků MeSH
- pyrrolidiny chemie MeSH
- racionální návrh léčiv MeSH
- vodíková vazba MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human tuberculosis is a chronic infectious disease affecting millions of lives. Because of emerging resistance to current medications, new therapeutic drugs are needed. One potential new target is hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT), a key enzyme of the purine salvage pathway. Here, newly synthesized acyclic nucleoside phosphonates (ANPs) have been shown to be competitive inhibitors of MtHGPRT with Ki values as low as 0.69 μM. Prodrugs of these compounds arrest the growth of a virulent strain of M. tuberculosis with MIC50 values as low as 4.5 μM and possess low cytotoxicity in mammalian cells (CC50 values as high as >300 μM). In addition, the first crystal structures of MtHGPRT (2.03-2.76 Å resolution) have been determined, three of these in complex with novel ANPs and one with GMP and pyrophosphate. These data provide a solid foundation for the further development of ANPs as selective inhibitors of MtHGPRT and as antituberculosis agents.
- MeSH
- antituberkulotika chemie farmakologie MeSH
- antitumorózní látky chemie farmakologie MeSH
- difosfáty chemie metabolismus MeSH
- hypoxanthinfosforibosyltransferasa chemie metabolismus MeSH
- inhibitory enzymů chemie farmakologie MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kyselina 5'-guanylová chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- molekulární struktura MeSH
- Mycobacterium tuberculosis účinky léků enzymologie MeSH
- nádorové buňky kultivované MeSH
- nádory plic farmakoterapie patologie MeSH
- organofosfonáty chemie metabolismus MeSH
- prekurzory léčiv chemie farmakologie MeSH
- proliferace buněk účinky léků MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- tuberkulóza farmakoterapie mikrobiologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
