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

Abstract

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.

The Reductive Dehydroxylation Catalyzed by IspH, a Source of Inspiration for the Development of Novel Anti-Infectives

“…Co-crystallographic structures of 48 with oxidized IspH were in accordance with the unexpected Mössbauer spectroscopy results and demonstrated that the binding between the alkyne residue and the fourth iron is not direct, indeed a water molecule is in between [ 46 ]. This result was further validated using NRVS measurements of the IspH- 48 complex [ 84 ]. NRVS studies of the free-substrate IspH had previously shown that the apical iron was bound to three sulfide ions of the cluster and to three molecules of water [ 38 ].…”

“…On the other hand, due to the impossibility of studying the oxidized IspH by EPR, the inhibitor 48 was examined by EPR in complex with the reduced IspH. The results obtained by O’Dowd et al [ 84 ] revealed plausible π-interactions between the inhibitor 48 and the reduced [4Fe-4S] cluster.…”

“…O’Dowd et al used an in silico approach to screen a series of compounds from ZINC and NCI libraries and after having tested some hits for IspH inhibition, they identified 52 and 53 as two new IspH inhibitors ( Figure 10 ) [ 84 ]. In both compounds, it is possible to see a common barbituric acid sub-structure which makes the hits more drug-like, similarly to antibacterial agents developed by Pharmacia Corporation [ 85 ].…”

“…The compound 52 resulted to be a good inhibitor of IspH from P. aeruginosa , with an IC 50 = 22 µM. On the other hand, the analog 53 inhibits IspH from E. coli , showing as result an IC 50 = 23 µM [ 84 ]. After docking experiments of 53 into the A. aeolicus IspH structure (PDB ID: 3DNF), the authors proposed a plausible binding mode where the barbiturate might chelate the apical iron of the [4Fe-4S] 2+ cluster, similarly to how barbiturate enolates do with Zn 2+ in other metalloenzymes [ 86 , 87 ].…”

“…After docking experiments of 53 into the A. aeolicus IspH structure (PDB ID: 3DNF), the authors proposed a plausible binding mode where the barbiturate might chelate the apical iron of the [4Fe-4S] 2+ cluster, similarly to how barbiturate enolates do with Zn 2+ in other metalloenzymes [ 86 , 87 ]. However, EPR studies for 53 in complex with the reduced IspH from P. aeruginosa did show a very low signal intensity, probably due to a change of the redox potential upon ligand binding or to a different relaxation mode [ 84 ]. These compounds could be developed as potential drugs for several reasons: they solved the problem related to the lability of the diphosphate moiety present in other inhibitors in vivo [ 82 ]; they respect the Lipinski’s rule [ 88 ] and they are not PAINS compounds [ 89 ].…”