Design, synthesis, and crystal structures of 6-alkylidene-2'-substituted penicillanic acid sulfones as potent inhibitors of Acinetobacter baumannii OXA-24 carbapenemase.
Class D β-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial β-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel β-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2'-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important β-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 β-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC(50) values against OXA-24 and two OXA-24 β-lactamase variants ranged from 10 ± 1 (4 vs WT) to 338 ± 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K(i) (500 ± 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k(inact)/K(i) = 0.21 ± 0.02 μM(-1)  s(-1)). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 Å) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2'-substituted penicillin sulfones are effective mechanism-based inactivators of class D β-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D β-lactamases is proposed.
Bou
German
G
Instituto de Investigación Biomédica de A Coruña (INIBIC), Complejo Hospitalario Universitario A Coruña, 15006-A Coruña, Spain.
Santillana
Elena
E
Sheri
Anjaneyulu
A
Beceiro
Alejandro
A
Sampson
Jared M
JM
Kalp
Matthew
M
Bethel
Christopher R
CR
Distler
Anne M
AM
Drawz
Sarah M
SM
Pagadala
Sundar Ram Reddy
SR
van den Akker
Focco
F
Bonomo
Robert A
RA
Romero
Antonio
A
Buynak
John D
JD
D040981:Acinetobacter baumannii / Q000187:drug effects / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D010397:Penicillanic Acid / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013450:Sulfones / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D013450:Sulfones; D065093:beta-Lactamase Inhibitors; D010397:Penicillanic Acid; D001618:beta-Lactamases; C063912:carbapenemase
Adv Exp Med Biol. 1998;456:71-98
10549364
J Biol Chem. 2006 Sep 8;281(36):26734-44
16803899
Bioorg Med Chem Lett. 1999 Jul 19;9(14):1997-2002
10450969
Antimicrob Agents Chemother. 2005 Oct;49(10):4240-6
16189104
Biochem J. 1991 May 15;276 ( Pt 1):269-70
2039479
Biochem Soc Trans. 1999 Feb;27(2):58-63
10093707
Proc Natl Acad Sci U S A. 2007 Mar 27;104(13):5354-9
17374723
Antimicrob Agents Chemother. 2006 Dec;50(12):4114-23
17000742
Antimicrob Agents Chemother. 2010 Jan;54(1):24-38
19721065
Antimicrob Agents Chemother. 1997 Jun;41(6):1322-5
9174192
J Am Chem Soc. 2007 Apr 4;129(13):3834-5
17343387
Proc Natl Acad Sci U S A. 1987 Nov;84(21):7378-82
2823258
Antimicrob Agents Chemother. 2000 Jun;44(6):1556-61
10817708
J Biol Chem. 2003 Dec 26;278(52):52724-9
14534312
Biochemistry. 2003 Nov 11;42(44):12835-43
14596597
Antimicrob Agents Chemother. 2005 Jul;49(7):2941-8
15980372
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14280-5
11724923
J Biol Chem. 2009 Jan 9;284(2):945-53
18955486
J Comput Chem. 2004 Oct;25(13):1605-12
15264254
J Biol Chem. 2004 Nov 5;279(45):47278-87
15322076
Biochim Biophys Acta. 2001 Jun 11;1547(2):196-205
11410275
J Biol Chem. 2005 Oct 21;280(42):35528-36
15987690
Biochemistry. 1996 Sep 24;35(38):12421-32
8823177
Clin Microbiol Rev. 2007 Jul;20(3):440-58, table of contents
17630334
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Proteins. 1991;11(4):281-96
1758883
J Biol Chem. 2000 Sep 1;275(35):26674-82
10837472
J Antibiot (Tokyo). 1997 Feb;50(2):184-5
9099233
Clin Microbiol Rev. 2010 Jan;23(1):160-201
20065329
Antimicrob Agents Chemother. 2008 Sep;52(9):3135-43
18559643
Arch Immunol Ther Exp (Warsz). 1999;47(4):211-6
10483868
Antimicrob Agents Chemother. 2007 Oct;51(10):3471-84
17646423
Biochem Pharmacol. 2006 Mar 30;71(7):930-40
16359643
Proc Natl Acad Sci U S A. 1981 Aug;78(8):4897-901
6795623
Curr Opin Microbiol. 2009 Oct;12(5):476-81
19716760
J Am Chem Soc. 2004 Nov 3;126(43):13945-7
15506754
Clin Microbiol Rev. 2009 Jan;22(1):161-82, Table of Contents
19136439
Antimicrob Agents Chemother. 2010 Feb;54(2):890-7
20008772
Chem Biol. 2009 May 29;16(5):540-7
19477418
Acta Crystallogr D Biol Crystallogr. 2006 Jan;62(Pt 1):48-57
16369093
Bioorg Med Chem Lett. 2004 Mar 8;14(5):1299-304
14980686
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
J Am Chem Soc. 2008 Sep 24;130(38):12656-62
18761444
Antimicrob Agents Chemother. 1999 Jul;43(7):1743-6
10390233
Chem Rev. 2005 Feb;105(2):395-424
15700950
Proc Natl Acad Sci U S A. 2006 Jul 11;103(28):10630-5
16815972
J Antimicrob Chemother. 1991 Jul;28(1):35-45
1722802
Protein Sci. 2003 Jan;12(1):82-91
12493831
Antimicrob Agents Chemother. 2010 Apr;54(4):1414-24
20086146
Bioorg Med Chem Lett. 2000 May 1;10(9):853-7
10853646
Curr Protein Pept Sci. 2009 Oct;10(5):397-400
19538149
Curr Med Chem. 2004 Jul;11(14):1951-64
15279575
J Mol Biol. 2001 Jul 20;310(4):859-74
11453693
Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321-31
6109327
Biochemistry. 2001 May 29;40(21):6233-9
11371184
Mol Microbiol. 1999 Jan;31(1):89-101
9987113
Bioorg Med Chem Lett. 2000 May 1;10(9):847-51
10853645
Antimicrob Agents Chemother. 2010 Mar;54(3):969-76
19995920
Curr Opin Struct Biol. 2002 Feb;12(1):14-20
11839484
J Comput Aided Mol Des. 1996 Jun;10(3):255-62
8808741
Clin Microbiol Infect. 2008 Jan;14 Suppl 1:134-43
18154537
Curr Med Chem. 2009;16(28):3740-65
19747143
Nat Prod Rep. 1999 Feb;16(1):1-19
10101880
Biochem Pharmacol. 1987 Jul 15;36(14):2393-403
3038122
Antimicrob Agents Chemother. 2002 Sep;46(9):2901-7
12183245
J Am Chem Soc. 1975 Aug 20;97(17):5020-1
1165354
Cell Mol Life Sci. 1998 Apr;54(4):341-6
9614970