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. 2019 Apr 9;58(14):1918-1930.
doi: 10.1021/acs.biochem.9b00003. Epub 2019 Mar 26.

Structure-Based Design, Synthesis, and Biological Evaluation of Non-Acyl Sulfamate Inhibitors of the Adenylate-Forming Enzyme MenE

Christopher E Evans, Yuanyuan Si, Joe S Matarlo, Yue Yin, Jarrod B French, Peter J Tonge, Derek S Tan

Structure-Based Design, Synthesis, and Biological Evaluation of Non-Acyl Sulfamate Inhibitors of the Adenylate-Forming Enzyme MenE

Christopher E Evans et al. Biochemistry. .

Abstract

N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC50 = 8 μM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future.

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Conflict of interest statement

The authors declare the following competing financial interest(s): D.S.T., P.J.T., C.E.E., and J.S.M. are co-inventors on International Patent Application PCT/US2016/055136; D.S.T., P.J.T., C.E.E., and Y.S. are co-inventors on U.S. Provisional Patent Application 62/802,650.

Figures

Figure 1
Figure 1
Classical menaquinone biosynthesis pathway and structure of MenE inhibitor OSB-AMS (1). At least nine enzymes catalyze the formation of menaquinone from chorismate. The fifth enzyme, MenE, is an acyl-CoA synthetase that catalyzes the ATP-dependent ligation of CoA to o-succinylbenzoate (OSB) via an OSB-AMP intermediate. OSB-AMS (1) is a stable analogue of OSB-AMP in which the acyl phosphate mixed anhydride is replaced with an acyl sulfamate.
Figure 2
Figure 2
Structures of OSB-AMS (1) and linker analogues bound to E. coli MenE (R195K). (a) X-ray co-crystal structure of OSB-AMS (1, gray) bound in the active site (cyan) of MenE with key binding interactions (green) (PDB entry 5C5H). Computationally docked structures of linker analogues (purple) (b) acyl squaramide 3, (c) alkyl sulfamide 4, (d) m-phenyl ether 5, and (e) α-hydroxytetrazole (S)-7, overlaid with OSB-AMS (1, beige) from the co-crystal structure, with predicted active site interactions (light blue).
Figure 3
Figure 3
Synthetic approach to acyl tetrazole analogue 2. Abbreviations: Boc, tert-butoxycarbonyl; DIAD, diisopropyl azodicarboxylate; THF, tetrahydrofuran; TMS, trimethylsilyl.
Figure 4
Figure 4
Synthesis of α-hydroxytetrazole analogue 7. Abbreviations: Boc, tert-butoxycarbonyl; DIAD, diisopropyl azodicarboxylate; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
Figure 5
Figure 5
Synthesis of acyl squaramide analogue 3. Abbreviations: BnOSB (22), 4-{2-[(benzyloxy)carbonyl]phenyl}–4-oxobutanoic acid; DMAP, 4-(dimethylamino)pyridine; DMF, dimethylformamide; EDC, N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide hydrochloride; TASF, tris(dimethylamino)sulfur trimethylsilyl difluoride; TBS, tert-butyldimethylsilyl..
Figure 6
Figure 6
Synthesis of alkyl sulfamide analogue 4. Abbreviations: Boc, tert-butoxycarbonyl; Cbz, carbobenzyloxy; DIAD, diisopropyl azodicarboxylate; TFA, trifluoroacetic acid.
Figure 7
Figure 7
Synthesis of m-phenyl ether analogue 5. p-Phenyl ether analogue 6 and trifluoromethyl-substituted analogue 8 were synthesized by analogous routes from the corresponding Heck coupling partners (cf., 29). Abbreviations: Boc, tert-butoxycarbonyl; Cy = cyclohexyl; DIAD, diisopropyl azodicarboxylate; DMA, dimethylacetamide; dtbpf, 1,1′-bis(di-tert-butylphosphino)ferrocene; NMO, N-methylmorpholine N-oxide; TBAF, tetrabutylammonium fluoride; TBS, tert-butyldimethylsilyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TPAP, tetrapropylammonium perruthenate.
Figure 8
Figure 8
Synthesis of m-3-pyridyl ether analogue 9. Abbreviations: Boc, tert-butoxycarbonyl; Cy, cyclohexyl; DBU, 1,8-diaza-bicyclo[5.4.0]undec-7-ene; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DIAD, diisopropyl azodicarboxylate; DMA, dimethylacetamide; dtbpf, 1,1′-bis(di-tert-butylphosphino)ferrocene; TFA, trifluoroacetic acid; THF tetrahydrofuran.
Figure 9
Figure 9
X-ray co-crystal structure of m-phenyl ether analogue 5 bound to wild-type E. coli MenE. (a) Overlay of structures of E. coli wild-type MenE with m-phenyl ether analogue 5 (gray and purple) bound, E. coli MenE (R195K) with OSB-AMS (1) bound (gray and cyan, PDB entry 5C5H), and S. aureus MenE apo structures (wheat and green or yellow, PDB entry 3IPL chains A and B). Alignment of the large N-terminal domains [E. coli residues 1–351, gray, root-mean-square deviation (rmsd) of 0.49 Å; S. aureus residues 1–396, wheat, rmsd of 1.64 Å relative to 5C5H] reveals a 22° rotation of the small C-terminal domain about the E. coli hinge residue D352 in the structure with analogue 5 (purple) compared to that with OSB-AMS (1) (cyan), while larger 144° and 151° rotations of the C-terminal domains are observed in the S. aureus apo structures (green and yellow). The E. coli linchpin residue K437 (green) is observed in the structure with OSB-AMS (1) but is disordered in the structure with m-phenyl ether analogue 5. (b) Active site of wild-type MenE (cyan) with m-phenyl ether analogue 5 (purple) bound, revealing binding interactions (green) and a 1.5 Å shift of the ribose motif compared to OSB-AMS (1) (gray). (c) Schematic of putative active site interactions (green) of m-phenyl ether analogue 5 (purple) with wild-type MenE (black).
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