Anal. Chem. 1995, 67, 3697-3701
Capillary Electrophoresis Study of the Hydrolysis of a /?-Lactamase inhibitor George N. Okafo,t Paul Cutler,t David J. Knowles,* and Patrick Camilld*gt
SmithKline Beecham Pharmaceuticals, The Ftythe, Welwyn, Hefts AL69AR, U.K., and SmithKline Beecham Pharmaceuticals, Brockham Park, Betchworth, Surrey RH3 7AJ, U.K.
(5R)-6(Z)-[ (1-Methyl-1,2,3-triazo1-4-yl)methylenelpenem-3-carboxylic acid monohydrate (BRL 42715) is a potent /?-lactamase inhibitor. We report a study on the hydrolysis of the four-membered lactam ring of this compound mediated either by Tris buffer or by Tem-2 /?-lactamase. In both studies free-zone capillary electrophoresis proved to be ideal in the identification of the products of reaction, the interpretation of the mechanism of hydrolysis, and the determination of kinetic parameters and stoichiometry. j3-Lactam antibiotics are commonly used in the treatment of bacterial infections. However, the elaboration of hydrolytic enzymes (4-lactamases) by many bacterial pathogens renders many such antibiotics ineffective in the clinical environment. The discovery of j3-lactamase inhibitors, such as clavulanic led to the successful development of a variety of antibiotic/inhibitor combinations for therapeutic use.495 The majority of known j3-lactamase inhibitors, including all those used clinically, contain the characteristic four-membered,&lactam ring and act as suicide inhibitors; cleavage of the j3-lactam amide bond gives rise to a stably inhibited enzyme species, acylated on the active site serine. Such inhibitors frequently exhibit branched kinetic pathways with appreciable turnover (acylation/deacylation) before the stably inhibited enzyme species is More recently, a novel class of j3-lactamase inhibitors, the &(substituted methylene) penems, have been reported? One such inhibitor, BRL 42715 [ (5R)-(Z)-&(l-methyl-1,2,3triazol-4-yl)methylenelpenem-3-carboxylicacid], is highly potent with an initial stoichiometry of inhibition of Tem-2 ,!l-lactamase of 1:l. Regeneration of active enzyme has been found to be slow and incomplete, suggesting partitioning of the inhibited enzyme to give a very stable inactivated species.’O One of the simplest methods commonly used to follow a reaction in solution is to monitor directly changes in the ultraviolet SmithKline Beecham Pharmaceuticals, Surrey. SmithKline Beecham Pharmaceuticals, Herts. (1) Howarth, T. T.; Brown, A. G.; King, T. J. ]. Chem. Soc., Chem. Commun. 1976,266-269. (2) Reading, C.; Cole, M. Antimicrob. Agents Chemother. 1970,1 1 , 852-857. (3) Hunter, P. A; Coleman, IC;Fisher, J.; Taylor, D. J. Antimicrob. Chemother. 1980,6.455-470. (4) English, A. R; Retsema, J. A; Girard, A E.; Lynch, J. E.; Bath, W. E. Antimicrob. Agents Chemother. 1978,14, 414-419. (5) Aronoff, S. C.; Jacobs, M. R; Johenning, S.; Yamabe, S. Anfimicrob.Agents Chemother. 1984,26, 580-582. (6)Knowles, J. R Antibiotics. Vol. 6: Modes andMechanisms ofMicrobial Growth Inhibitors; Hahn, B., Ed.; Springer-Verlag: New York, 1992; pp 99-107. (7) Cartwright, S. J.; Waley, S. G. Med. Res. Rev. 1983,3,341-382. (8) Reading, C.; Cole, M. J. Enzyme Inhib. 1986,1. 83-104. (9) Bennet, I. S.; Brooks, G.; Broom, N. J.; Calvert,S. N.; Coleman, K; Francois, .]I Antibiot. 1991,44, 969-978.
absorbance of either a reactant or a product with time.11J2 This methodology is especially useful when the reaction followed is first order or pseudo first order, and there is no interference due to absorbance by other species. In cases where it is not possible to choose an appropriate wavelength to follow a reaction due either to more than one absorbing species in a reaction mixture or to consecutive reactions, the change in concentration-time profile of reactants and products is commonly determined using highperformance liquid chromatography (HPLC)13J4with W detection. This separation technique is reliable and is ideal when relatively hydrophobic and neutral molecules are involved. The more recently introduced analytical technique of capillary electrophoresis (CE)15J6is complementary to HPW. CE can be most effective in providing high resolution of both neutral and ionic species when used in the micellar electrokinetic capillary electrophoresis (MECC) or freezone electrophoresis modes, respectively. This technique can also be fast, allowing more frequent sampling during the time course of a reaction. It is also allows the decay or formation of both simple and much more complex molecules to be monitored simultaneously. We now report the use of freezone CE to study the mechanism of hydrolysis of the /?-lactam ring of BRG42715 catalyzed either by tris(hydroxymethy1)aminomethane (Tris) or by Tem-2 ,&lactamase. Unlike conventional direct W spectral analysis, our investigations have allowed the simultaneous analysis of BRL 42715, enzyme, intermediates, and products with high resolution and low sample consumption.
BRL 42715 (1)
+
$
0003-2700/95/0367-3697$9.00/0 0 1995 American Chemical Society
EXPERIMENTAL SECTION Materials. Buffer constituents used in capillary and gel electrophoresis were of Analar grade and were purchased from BDH (Lutterworth, U.K.) and from Sigma (Poole, U.K). BRL 42715 and the dihydrothiazepine derivative BRL 44516 were supplied by the Medicinal Chemistry Department at SmithKline (10) Farmer, T. H.; Page, J. W. J. Payne, D. J.; Knowles, D. J. C. Biochem. ]., in press. (11) Jencks, W. P. Cufalysis in Chemistly and Enzymology;McGraw-Hik New York, 1969 Chapter 11. (12) Kandanarachchi, P.; Sinnott, M. L. ]. Am. Chem. Soc. 1994,116, 55925600. (13) Breslow, R; Xu, R]. Am. Chem. SOC.1993,115, 10705-10713.
Analytical Chemistty, Vol. 67, No. 20, October 15, 1995 3697
Beecham. Tem-2 p-lactamase (75% protein by weight) from Escherichia coli was obtained from Porton as a white lyophilized powder containing potassium phosphate. Equipment. The CE system consisted of a Beckman P/ACE Model 5000 equipped with an autosampler, diode array detector, and temperaturecontrolled sampler carousel equilibrated at 20 "C. The conditions used in the study of the reaction of BRL 42715 with Tris were as follows: capillary, 27 cm x 50 pm i.d. untreated fused-silica capillary (effective length, 20 cm); buffer, 20 mM sodium phosphate, pH 7.30; separation voltage, 10 kV; detection, W absorbance at a wavelength of 200 nm; temperature, 37 "C; sample introduction, 1s hydrodynamic pressure (0.5 psi) injection of the reaction solution. The hydrolysis of BRL 42715 by Tem-2 was studied under the following CE conditions: capillary, 24 cm x 50 pm i.d. (effective length, 17 cm) untreated fused-silica capillary; buffer, 20 mM sodium dihydrogen phosphate, pH 7.3; detection, W absorbance, 200 nm; applied voltage, gradient voltage separation of 1 kV min-' over 15 min; temperature, 22 "C; sample, the Tem-2/BRL 42715 1:l complex was injected hydrodynamically as before. Isoelectric Focusing (IEF). Samples were focused on precast agarose gels (FMC Bioproducts) against an ampholyte pH gradient covering the pZ range 3.0-10.0 at 1.5 kV, 20 mA, 25 W for 30 min at 9 "C. Protein bands were visualized by staining by either Coomassie Blue or silver stain. The gels were loaded with 10 pg of protein per track (unless otherwise stated). A reference sample of proteins of defined pZs (Pharmacia LKB) was run on each gel, and the banding patterns were compared by eye. Hydrolysis of BRL 42715 Mediated by Tris. BRL 42715 (1.28 mg, 4.21 pmol) was dissolved in an aliquot of 80, 300, 450, or 600 mM Tris (PH 9.54, equilibrated at 20 "C) to give a final concentration of 4.96 mM. The solution was then immediately transfered to a plastic vial and analyzed by CE at 20 min intervals for a total time of 300 min. The reaction product was isolated as follows: solid Tris (9.6 mg, 79.3 pmol) was dissolved in a glass vial containing distilled water (1 mL). The solution was adjusted to pH 9.54 using dilute HCl before transfer to another vial containing BRL 42715 (10 mg, 32.9 pmol). An aliquot (200 pL) of the solution was immediately placed in a CE vial and the reaction monitored by CE until the inhibitor had been consumed. After 2 h, the resulting solution was acidified by the addition of 1.0 M HCl (200 pL) and then evaporated to dryness (Genevac centrifugal evaporator). The residue was extracted twice with methanol (2 x 500 mL) and dried in a stream of NZ gas. The structure of the resulting solid was then determined by proton and 13CNMR and mass spectrometric analysis: H (400 MHz; DzO) 6 7.82 (lH, C=CH), 7.58 (lH, NCH=C), 6.38 (lH, SCH=C), 5.84 (lH, SCH), 4-4.1 (2H, OCHzC), -4.00 (3H, CH3), 3.4 (3 x 2H, CH20); C (100 MHz; DzO) 6 171.25 (C=O), 171.05 (C=O), 153.06 @C=), 144.98 (NHC=), 141.55 (=C(NH)carboxylic), 126.78 @IC=), 112.46 (SC=), 109.67 @=Carboxylic), 68.16 (C-0 Vris moiety), 65.78 (CHzOH), 58.63 (C(NHz)), 44.42 (OCO), 39.38 (NCH3). FAB (nitrobenzyl alcohol matrix), m/z 386 (M H)+.
+
(14) Martin, M. T.; Angeles, T. S.; Sugasawara, R.; Aman, N.1.; Napper, A. D.; Booth, P.; Titmas, R C.J. Am Chem. SOC.1994, Darsley, M. J.; Sanchez, R1.; 116,6508-6512. (15) Jorgenson. J. W.; Lukacs, K D. Anal. Chem. 1991,63, 802-807. (16) Camillen, P., Ed. Capillaly Electrophoresis: T h e o y and Practice; CRC Press: Boca Raton, FL, 1993.
3698 Analytical Chemistry, Vol. 67,No. 20,October 15, 1995
Tem2 @-Lactamase-MediatedHydrolysisof BRL 427 15. BRL 42715 (0.86 mg, 2.83 mmol) was dissolved in a volume (27.2 mL) of 5 mM Tris buffer adjusted to pH 7 using dilute HC1 to give a final concentration of 104 pM. An aliquot (104 pL, representing 10.8 nmol of inhibitor) of solution was then transferred to a CE vial and allowed to equilibrated to 30 "C within the Beckman P/ACE sample tray. A separate Tris solution containing 1 molar equiv (0.40 mg, 10.8 nmol dissolved in 104 mL of Tris buffer) of Tem-2 P-lactamase, preincubated at 30 "C, was also prepared. Both the enzyme and the inhibitor solutions were then mixed thoroughly on a vortex mixer before incubation at 30 "C and CE analysis. The reaction was sampled every 60 min for 24 h and then finally after 48 h. RESULTS AND DISCUSSION
The proposed mechanism of action of BRL 42715 with Tem P-lactamase is the rapid formation of an acyl enzyme species. At low inhibitor/enzyme ratios, free enzyme is slowly regenerated.1° Spectroscopic data suggest that the reaction proceeds via the formation of the seven-membered dihydrothiazepine, BRL 44156,17 as was shown unequivocally for the interaction of BRL 42715 with K1 P-lactamase.18 The interaction of a 2C-fold molar excess of BRL 42715 to Tem P-lactamase led to irreversible inhibition.I6The amino acid residue parti~ipating'~ in the acylation of the main P-lactamases is serine-70. The possible involvement of the stronger alkoxide nucleophile rather than the hydroxy group of this amino acid may be related to the fact that a common feature present around the serine-70 active site is a lysine residue (Lys 73 or Lys 83 in class A and class C enzymes, respectively). The facile protonation of the amino group of lysine can stabilize a zwitterionic form where the negative charge lies on the alkoxide form of serine-70. As a simple model for the interaction of BRL 42715 with p-lactamase, we studied the reaction of this compound with Tris. The nature of the product of reaction provides information on whether one of the hydroxyl groups (in the neutral or anionic form) or the amino group is involved in nucleophilic attack. The alternative products are the dihydrothiazepine derivatives 2 and 4, respectively. We have used free-zone capillary electrophoresis to distinguish between these two possible products and the free acid BRL 44516, using the expected charge density differences of these species at alkaline pH.
A0
HO
R = OCH~C(CHZOH)~N (2)H ~ BRL 441 56, R = OH (3) R = NHCH~CH(CHZOH)~ (4)
Figure 1 shows typical electropherograms of samples from a reaction of BRL 42715 (4.96 mM) dissolved in Tris buffer (600 (17) Farmer, T. H.; Knowles, D. J. C.. unpublished observation. (18) Broom, N. J. P.; Farmer, T. H.; Osborne, N. F.; Tyler, J. W. J. Chem. Soc., Chem. Commun. 1992,1663-1664. (19) Joris, B.; Ghuysen. J.; Dive, G.; Renard, A.; Dideberg, 0.;Charlier, P.;Frere, J.; Kelly, J. A,; Boyington, J. C.; Moews, P. C.; Knox, J. R Biochem. J. 1988, 250, 313-324.
0.000 I / 0 100 200 300 400 500 600 'Tris' Concenlration (mM)
Figure 3. Linear plot of the dependence of the lint-order rate Constant, k, for the hydrolysis of BRL 42715 with Tris concentration.
0
1i ;
PI
60 min
10
c,
0
5 Time (minutes)
Figure 1. Electmphemgrams showlng lhe hydmlyss of BRL 42715 (0to form the Tns adduct (2) as the major product and BRL 44156 (31 after (a) I. (b) 22,and (c) 60 mln Tne identity of BRL 44156 was confirmed from migration t me and diode array charactenstics 01 an
4-
6.55
4-
4.55
authent c sample 38 ?I)
h t
+ 1
0
50
100
150
200
Time (mtns)
Figun 2 Kinetic profiles for the hydrolysis of BRL 42715 at dflerent Tris concentrations.
mM) at pH 9.54, after incubating at 20 "C. Under these conditions
Tris (PK, 8.1) is largely neuhal, comigrating with mesityl oxide (data not shown). BRL 42715 migrates after the elettroosmotic front due to its negative charge density. At longer times of reaction, BRL 44156 is seen as a minor product This molecule canies two negative charges so that it migrates well after BRL. 42715. m e formation of BRL 44156 is due to attack by hydroxide anions. The major product of this reaction was found to be the Tris adduct 2 and not 4. m e structure of 2 was confirmed by conducting the reaction on a larger scale (see Experimental Seaion) and NMR and MS analysis. The migration time of 2 is also consistent with a molecule that is zwitterionic in character. As expected, the rate of reaction of BRL 42715 with excess Tris follows first-xder kinetics F i r e
2
3
4
5
1. BRL 42715 2. TEM2 P-Lactamase + BRL 42715 (1:l) 3. TEM2 P-Lactamase + BRL 42715 (1:20) 4. TEM2 P-Lactamase 5. PI markers Figure 4. lsoelectic focusing gel showing the appearance of a second more acidic band due to the presence of the fl-lactamaseBRL 42715 adduct.
2). Rate constants, determined from the plots in Figure 2. were plotted against the Tris concentration to give the linear relationship (r, 0.998)shown in Figure 3. The line passes through the origin: the bimolecular reaction of BRL 42715 with Tris is much faster than with speciJicbase at the much higher concentrations of buffer used in these experiments. The formation of 2 indicates that one ofthe oxygen atoms of Tris is a stronger nucleophile toward the carbonyl group on the p-lactam ring of BRL 42715 than the more basic amine nitrogen. ?his in turn almost certainly signifies that the nucleophile is not the hydroxyl moiety but the oxygen anion in the zwitterionic form ofTris, that is (+)NH~C(CHZOH)ZCHZ@ Analytical Chemistry, Vol. 67, No.20, October 15, 1995 3699
0.06
0.05
3 0.04
0 TEM.2 p-lactamase
P
P
TEM.2Iinhib~torcomplex
m 5
2m
Hydrolysis producl
0.03
15
10
m
:
s
0.02
a"
0.01
0.0 y 0
I
I
I
I
I
150
300
450
600
750
,
I
0.00
900
Time (mins)
Figure 6. Area-time profiles showing the decay of the enzymedrug complex and the formation of free enzyme and BRL 44156. 1 I I
I I
1; 6 40
!cT,
660
6 80
I\
3
i
5
15
10 Time (minutes)
Figure 5. Capillary electrophoresis analysis of the decomposition
of the /3-lactamase-BRL 42715 complex to give free enzyme and BRL 44156 after (a) 30, (b) 240, and (c) 660 min of reaction. Insets show the relative proportions of the free enzyme and the enzymedrug complex on an expanded time scale. The labels 1, 2, and 3 refer to free enzyme, enzyme-BRL 42715 complex, and BRL 44156, respectively. Scheme 1
f
-',
\
n'
ti
0
.OH Set
.OH S er
Enz
Enz
0-
(-). Such differences have been reportedz0previously for hydroxylamine where the overall reactivity of this molecule has been found to be more than 2 orders of magnitude larger than that of an amine of comparable basicity. The mechanism of hydrolysis of BRL42715 catalyzed by Tris is in agreement with recent studies on the inactivation of B-lactamaseZ1by BRL42715. Both these studies and the X-ray structurezZ of a closely related B-lactamase (20) Jencks, W. P.: Camuolo. J. J. Am. Chem. SOC.1960,82, 178-184. (21) Bulychev, A: Massova, I.; Lerner, S. A: Mobashery, S. J. Am. Chem. SOC. 1995,117, 4797-4801.
3700
Analytical Chemistry, Vol. 67,No. 20,October 15, 1995
clearly show that, besides the lysine residues mentioned earlier, Arg 244 is close in space to Ser 70, the amino acid involved in the acylation by BRL42715. Because of the encouraging results obtained using capillary electrophoresis to follow the reaction of BRL 42715 with Tris, we used this technique to study the hydrolysis of the complex of this drug with Tem-2 p-lactamase. The formation of this complex can be seen from isoelectric focusing as shown in Figure 4. The IEF pattern for TEM-2 B-lactamase indicates a major band with an apparent prof approximately 5.8. On addition of BRL 42715 at a ratio of either 1:l or 1:20, an extra, more acidic band is generated. This results from interaction of the drug with the enzyme leading to the generation of a more acidic species (Scheme 1). Although the IEF results are good evidence for the formation of an enzymeinhibitor complex, analysis by this technique is slow and not quantitative. Moreover, this technique does not allow monitoring of BRL 44516 (3),the seven-membered ring product of hydrolysis. Free-zone CE was found to offer very clear advantages over IEF and has allowed the simultaneous monitoring with time of the disappearance of the BRL 42715-inhibitor complex and the appearance both of free enzyme and of BIU 44516 (3).As the three analytes are negatively charged at the pH of this study, they all migrate after the electroosmotic front. The order of migration can also be interpreted from expected charge densities. Thus, assuming that the volumes of enzyme and enzyme-inhibitor complex are of similar magnitude, the charge density of the latter is larger than that of the enzyme alone. The considerably longer migration time of BIU 44516 is due to its much smaller volume and the two negative charges carried by this molecule. Electropherograms obtained at three time intervals are shown in Figure 5. For an equimolar molar ratio of Tem-2 and B E 42715, no free enzyme or drug was observed at the start of the reaction. This observation is in line with 1:l stoichiometry. The kinetic profile for the interaction of BRL 42715 with Tem-2 is given in Figure 6. From the time-response curves it was found that the half-lives (22) Jelsch, C.; Mourney, L.; Masson. J. M.: Samama, J. P. Proteins: Stmcf.Funct. Genet. 1993,16, 364-371.
for the disappearance of the "EM-2 B-lactamase inhibitor complex and the appearance of free E M - 2 B-lactamase and the hydrolysis product BRL 44516 are both of the order of about 170 min, again confirming a 1:l stoichiometry. CONCLUSION
We have successfully used free-zone CE to determine some of the kinetic parameters for the reaction of BRL42715 (a B-lactamase inhibitor) with either Tris or Tem-2 /?-lactamase. The acquisition of data using this analytical technique was found to be fast, compared to other more common electrophoretic techniques such as IEF. Moreover, the methodology developed allowed the facile and simultaneous determination of changes in
the concentration of both simple and more complex analytes with time. ACKNOWLEWYENT We thank Dr. Anthony J. Kirby (University of Cambridge, Cambridge, U.K.) for helpful discussion and advice regarding the preparation of the manuscript. We also thank Dr. Colin Frydrych (SmithKline Beecham, U.K.) for supplying BRL 42715 and BRL 44156. Received for review May 31, 1995. Accepted July 31, 1995.a AC950531J @
Abstract published in Advance ACS Abstracts, September 1, 1995.
Analytical Chemistry, Vol. 67, No. 20, October 15, 1995
3701
