Identification of a Series of Tricyclic Natural Products as Potent Broad-Spectrum Inhibitors of Metallo-β-Lactamases

Abstract

This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-beta-lactamase inhibitors. Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-beta-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed K(i) values of 79, 17, and 3.4 microM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-beta-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-beta-lactamase (50% inhibitory concentration > 1,000 microM). The lack of activity against angiotensin-converting enzyme and serine beta-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 A. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B. fragilis producing CfiA, making these strains sensitive to meropenem (MIC < or = 4 microg/ml). Consequently, this series of metallo-beta-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-beta-lactamases.

The 1.5-Å Structure of Chryseobacterium meningosepticum Zinc β-Lactamase in Complex with the Inhibitor, D-Captopril

“…In this context, the analysis of the three-dimensional structures of MBLs in complex with potential inhibitors becomes a powerful tool for rational drug design. At present, the structures of Bacteroides fragilis CcrA complexed with Mes, biphenyl tetrazole, or a tricyclic natural product (32) and of (23,31) Pseudomonas aeruginosa IMP-1 complexed with mercaptocarboxylate (30) or with succinic acid derivatives (33) have been reported.…”

“…Because of the great interest in MBL inhibition, several classes of inhibitors have been described (17,18); these include phenazines (19), ketone derivatives of L-and D-alanine and trifluoromethyl alcohol (20), thioesters (21,22), biphenyl tetrazoles (23,24), amino acid-derived hydroxamates (25), thiols (26 -30), and tricyclic natural products (31). However, despite many of these having good inhibitory properties, only a few thiols have a broad spectrum of inhibition for MBLs.…”

Comparative Study of the Inhibition of Metallo-.BETA.-Lactamases (IMP-1 and VIM-2) by Thiol Compounds That Contain a Hydrophobic Group

“…The X-ray crystal structures of MBLs complexed with inhibitors have been reported, including CcrA-4-morpholineethanesulfonic acid, 39) CfiA-tricyclic natural products, 36) IMP-1-mercaptocarboxylate, 12) 2,3-disubstituted succinic acid derivatives, 35) BlaB-D-captopril, 40) and FEZ-D-captopril. 41) From these reports, these enzymes have conserved the amino acid residues and the binuclear metal center at the ac- 17) The thiol group of inhibitors was assumed to coordinate with the metal ions in the active site, and this was supported by an X-ray crystallographic analysis of 2-[5-(1-tetrazolyl-methyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutyrylglycine)] and the IMP-1 complex.…”

“…A number of compounds have been examined as inhibitors of MBLs since 1996, including trifluoromethyl alcohols and ketones, 15) hydroxamates, 16) thiols, [17][18][19][20][21][22][23][24][25] thioester derivatives, 19,[26][27][28][29] cysteinyl peptides, 30) biphenyl tetrazoles, 31,32) mercaptocarboxylates, 12,21,29) 1b-methylcarbapenem derivatives, 33,34) 2,3-disubstituted succinic acid derivatives, 35) tricyclic natural products, 36) sulfonyl hydrazones, 37) and a synthetic cephamycin. 38) Most of these compounds, however, have been shown to exert their adverse effects only on a limited number of MBLs.…”

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase

“…They are generally considered to play a critical role for catalytic activity, especially in the ability of MBLs to hydrolyze ␤-lactam antibiotics of very diverse structures (11,17). In particular, the ␤3-␤4 loop (32-38(59 -66)) (also known as the "mobile loop" or "flap"), which is conserved in most class B1 MBLs, has been shown to move toward bound inhibitors and substrates and thus to contribute to the formation of a hydrophobic pocket, which is important for ligand binding (31,40,(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90). Together with the flap, the ␤11-␣4 loop (170 -188(223-241)) on the other side of the active site also contributes to the capacity of MBLs to bind a structurally broad range of substrates and inhibitors (77,79,80,90).…”