Structures of Fluoro, Amino, and Thiol Inhibitors Bound to the [Fe<sub>4</sub>S<sub>4</sub>] Protein IspH

Span, Ingrid; Wang, Ke; Wang, Weixue; Jauch, Johann; Eisenreich, Wolfgang; Bacher, Adelbert; Oldfield, Eric; Groll, M.

doi:10.1002/anie.201208469

Cited by 25 publications

(48 citation statements)

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“…In other early work we discovered, based on previous reports that alkynes could bind to and be reduced by 4Fe-4S clusters [9–10] , that alkyne diphosphates such as 5 (propargyl diphosphate, PPP) were low μM inhibitors of IspH (as well as IspG, which also contains a 4Fe-4S cubane-like structure) [1] . Plus, the 1-amino ( 6 ) and 1-thio ( 7 ) analogs of HMBPP have been found to inhibit IspH with K i ~20–50 nM [11–12] , and we [13] and others (PDB ID codes: 3ZGL, 3ZGN) have reported their X-ray structures which are basically the same as found with the HMBPP substrate, with N,S binding to the unique, 4 th Fe. We also reported [14] several other IspH-ligand complex structures with, in each case, a single O-containing ligand (alcoholate or enolate) bound to the unique, 4 th Fe atom with Fe-O bond lengths of ~2 Å.…”

Section: Introductionmentioning

confidence: 69%

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

et al. 2017

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Isoprenoid biosynthesis is an important area for anti-infective drug development and one target is IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase, which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H+/2e− reduction. IspH contains a 4Fe-4S cluster and here, we first investigated how small molecules can bind to the cluster using HYSCORE and NRVS spectroscopies. The results of these as well as other structural and spectroscopic investigations led to the conclusion that in most cases, ligands bind to IspH 4Fe-4S clusters via η1 coordination, forming tetrahedral geometries at the unique 4th Fe, ligand side-chains preventing further ligand (e.g. H2O, O2) binding. Based on these ideas, we sought using in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analog having a Ki ~ 500 nM against Pseudomonas aeruginosa IspH.

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Section: Introductionmentioning

confidence: 69%

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

et al. 2017

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“…31,32 In addition, in order to investigate bonding of the acetylene 5 , we synthesized both [3- 13 C]- 5 and [4- 13 C]- 5 , as described in the Supporting Information, in order to unambiguously determine the hyperfine coupling tensor elements, A ii , for each site.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of the 4Fe–4S proteins IspG and IspH: an EPR, ENDOR and HYSCORE investigation

Guerra

Wang

et al. 2014

Chem. Sci.

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IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e− reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C π-bonding with the olefinic carbons interacting with the unique 4th Fe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such π-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with 17O and 13C labeled samples: the results rule out direct Fe-O bonding and indicate π-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with 13C labeled ligands: the large hyperfine couplings indicate strong Fe-C π-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as “cubane-type” metallo-biochemistry, in general.

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“…[9] Ebenso wurde von zwei Strukturen von LytB mit dem [4Fe-4S]-Cluster gebunden an (E)-4-Amino-3-methylbut-2-en-1-yl diphosphat (4)o der (E)-4-Mercapto-3-methylbut-2-en-1-yl-diphosphat (5), zwei Analoge zu HMBPP,b erichtet. [10] Für diese Amino-und Thiolanaloge von HMPBB wurde gezeigt, dass sie mit K i = 20 nm bzw. K i = 54 nm extrem wirksame LytB-Inhibitoren sind.…”

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“…Abbildung 3e nthält die Resultate der berechneten NISDaten basierend auf den Rçntgenkristallographiedaten. [9,10,20] Es zeigt sich eine angemessene Übereinstimmung zwischen berechneten und experimentellen Daten, die Abweichungen führen wir auf das Vorhandensein von leicht unterschiedlichen Proteinkonformationen in Proteinkristallen und -lç-sungen zurück. Für LytB gebunden an 4 wurde ebenfalls ein NIS-Datensatz, basierend auf der kürzlich von den Gruppen von Groll und Oldfield publizierten Struktur (siehe Abbildung S4 ci nd en Hintergrundinformationen) berechnet.…”

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Isoprenoidbiosynthese in pathogenen Bakterien: Nukleare inelastische Streuung ermöglicht Einblicke in den ungewöhnlichen [4Fe‐4S]‐Cluster vom E.‐coli‐Protein LytB/IspH

et al. 2015

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Krankheitsverursachende Mikroorganismen sind zusehends resistent gegen Antibiotikatherapien geworden, während Krankheiten, die als ausgerottet galten, verstärkt wieder auftauchen. Tu berkulose beispielweise ist sogar in den Industrieländern wieder auf dem Vormarsch und verursacht weltweit mehr als 1.1 Millionen Tote pro Jahr.D er Methylerythritolphosphat(MEP)-Weg [1] stellt eine Alternative zum Mevalonatweg [2] dar und wird bei der Biosynthese von Isopentenyldiphosphat (IPP, 1)u nd Dimethylallyldiphosphat (DMAPP, 2)b eschritten. Diese entscheidenden Bausteine sind an der Bildung von essenziellen Te rpenoiden der meisten pathogenen Bakterien (unter anderem Mycobacterium tuberculosis)und Pflanzenplastiden beteiligt. Deswegen gilt der MEP-Weg als Angriffspunkt bei der Entwicklung von neuen antimikrobiellen Stoffen, da er essenziell für Mikroorganismen ist, beim Menschen jedoch nicht vorkommt.[3] Im letzten Schritt dieser Biosyntheseroute wird (E)-4-Hydroxy-3-methylbut-2-en-1-yl-diphosphat (HMBPP, 3) in eine Mischung von IPP (1)u nd DMAPP (2) )a ufweist. [4,5] Dieser Wert ist innerhalb des experimentellen Fehlers identisch mit dem ungewçhnlichen vierten Eisenplatz in der citratgebundenen Form von Aconitase.[6] Ausd iesem Grund wurde vorgeschlagen, dass die Koordinationssphäre dieses speziellen Eisenplatzes drei anorganische Schwefel vom Eisen-Schwefel-Cluster umfasst, sowie zusätzlich drei oder zwei Nicht-Schwefel-Liganden (O und/oder N) in einer Bindungsart, die ähnlich zu der von substratgebundener Aconitase ist. [4,6] Erste Rçntgenstrukturanalysen von substratfreiem LytB von Aquifex aeolicus [7] und Escherichia coli [8]

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Structures of Fluoro, Amino, and Thiol Inhibitors Bound to the [Fe₄S₄] Protein IspH

Abstract: The iron–sulfur protein IspH catalyzes a key step in isoprenoid biosynthesis in bacteria and malaria parasites. Crystal structures of IspH complexed with three substrate analogues reveal their mode of binding and suggest new routes to inhibitor design.

Cited by 25 publications

References 26 publications

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

Inhibition of the 4Fe–4S proteins IspG and IspH: an EPR, ENDOR and HYSCORE investigation

Isoprenoidbiosynthese in pathogenen Bakterien: Nukleare inelastische Streuung ermöglicht Einblicke in den ungewöhnlichen [4Fe‐4S]‐Cluster vom E.‐coli‐Protein LytB/IspH

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Structures of Fluoro, Amino, and Thiol Inhibitors Bound to the [Fe4S4] Protein IspH

Abstract: The iron–sulfur protein IspH catalyzes a key step in isoprenoid biosynthesis in bacteria and malaria parasites. Crystal structures of IspH complexed with three substrate analogues reveal their mode of binding and suggest new routes to inhibitor design.

Cited by 25 publications

References 26 publications

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

Inhibition of the 4Fe–4S proteins IspG and IspH: an EPR, ENDOR and HYSCORE investigation

Isoprenoidbiosynthese in pathogenen Bakterien: Nukleare inelastische Streuung ermöglicht Einblicke in den ungewöhnlichen [4Fe‐4S]‐Cluster vom E.‐coli‐Protein LytB/IspH

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Structures of Fluoro, Amino, and Thiol Inhibitors Bound to the [Fe₄S₄] Protein IspH