2D-MoS2 based Î²-lactamase Inhibitor for Combination Therapy

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2D-MoS2 based #-lactamase Inhibitor for Combination Therapy against Drug Resistant Bacteria Sk Rajab Ali, Subhendu Pandit, and Mrinmoy De ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00105 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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ACS Applied Bio Materials

2D-MoS2 Based β-Lactamase Inhibitor for Combination Therapy against Drug Resistant Bacteria Sk Rajab Ali, Subhendu Pandit and Mrinmoy De* Department of Organic Chemistry, Indian Institute of Science, Bangalore, India E-mail: [email protected]

Abstract: Despite of remarkable improvement in modern medicine, the ever-increasing abundance of antibiotic-resistant microorganisms remains a catastrophic threat to global health care. β-lactamase is playing one of the major roles in antibiotic resistance by making the conventional antibacterial agents abortive by destroying their lactam ring. The combination therapy of traditional antibiotics along with β-lactamase inhibitors is a potential solution to this problem. In this work, we have screened various functionalized two-dimensional molybdenum disulfide (2D-MoS2) nanomaterials as enzyme inhibitors that effectively bind with β-lactamase enzyme and reveal competitive inhibition. Among these, carboxylate functionalized negatively charged 2D-MoS2 is the most potent inhibitor and in vitro combinatorial application of this with conventional antibiotic has been able to remarkably suppress relevant drug resistant bacterial growth rate. This study will help to further explore different surface functionalized 2D nanomaterials with improved β-lactamase inhibition to fight against multidrug-resistant bacterial infections. Keywords: β-lactamase inhibitor, functionalized 2D-MoS2, antibacterial activity, multidrug resistant bacteria, combination therapy

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Introduction: The development of inhibitor for β-lactamase enzyme, which is translated by drug-resistant bacteria, remains to be one of the most notable advances in the modern medicine.1 In 2004, the Infectious Diseases Society of America (IDSA) published a report titled “Bad Bugs, No Drugs: As Antibiotic Discovery Stagnates, A Public Health Crisis Brews,” and that had set a great concern worldwide.2 The first widespread clinical use of penicillin as an antibacterial agent paved the way to search its new analogues. They function as an antibacterial drug by attacking a specific enzyme, DD-transpeptidase, which is required for bacterial cell wall synthesis.3 Thus, the β-lactam-mediated inhibition of transpeptidation leads to cell lysis.1,

4

However, after

introduction of penicillin, within a few years various bacterial strains started showing drug resistance by naturally producing an enzyme, penicillinase. This class of enzyme known as βlactamase that can destroy β-lactam ring structure of penicillin.5 The emergence of bacterial resistance to penicillin was first observed by Abraham and Chain in 1940.6 After that there are several penicillin like antibiotics containing a β-lactam moiety in their structure was introduced but all faced the same consequences. Not only the ineffectiveness of the β-lactam based drugs but also the extensive use of those drugs is directly associated with the spread of antibacterial resistance and its effectiveness was virtually annulled by “plasmid epidemics”.5 In the appearance of multidrug-resistant (MDR) and extensive drug-resistant (XDR) tuberculosis, βlactamase played a key role.7 Hence, β-lactamase mediated resistance becomes a significant clinical threat and creating a great challenge worldwide. In this scenario inhibition of βlactamase enzyme is one of the important strategy for the development of novel therapeutics to treat antibiotic resistant infections.1 Thus, finding an appropriate inhibitor is of paramount importance.


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The possible strategy can be taken to inhibit β-lactamase is by either binding to the active site reversibly/irreversibly to make it inactive towards antibiotics or sterically blocking the active site using macromolecules to stop the enzymatic activity. So far mostly small molecule based inhibitors are reported by targeting the active site of various β-lactamases which mainly rely on specific mechanism based inhibition. Most of the β-lactamase inhibitors contain lactam ring and structurally similar to the penicillin e.g. clavulanic acid, sulbactam, tazobactam etc.8 The biggest disadvantage of those inhibitors is that they undergo different inactivation mechanism against different classes of β-lactamases. Hence the efficacy of one inhibitor will vary between two classes of enzymes even though they are very similar in structure. So, it is impossible to develop any general inhibitor using small molecules. For example, one of the well-studied and clinically approved β-lactamase inhibitor, calvulanate required only one molecule to deactivate single βlactamase from Staphylococcus aureus PC1,9 whereas more than 16000 molecules are needed for the same enzyme from Bacillus cereus I.10 Another disadvantage of the structurally similar inhibitors is the substantial loss of antibiotics during combination therapy due the initial competitive binding between the inhibitor and antibiotics. This leads to the excess use of antibiotics and as usual bacteria find a way to overcome the inhibitory effect through the active site modification. In comparison to target specific mechanism, substrate which are effective for multiple β-lactamase inhibitory mechanism by completely blocking the active site would be more attractive and can extend the “last line of defense” against MDR and XDR pathogens. Functionalized nanomaterials based enzyme inhibition is an alternative potential way to control the biological activity in parallel to small molecule based enzymatic inhibition. In contrast to the small molecules, nanomaterials with enormous surface and variable functionality can bind and alter the enzymatic activity very efficiently. Nanomaterials-based inhibition mainly happens



through either multivalent interactions or steric blockage which is generally difficult to overcome through biological modification.11 Earlier in many reports 3D and 2D materials are reported as potent enzyme inhibitor through steric blockage of active site and multivalent interactions. For example, inhibition of chymotrypsin by graphene oxide12 and gold nanoparticle,13 suppression of activity of β-galactosidase by shape based zinc oxide nanoparticle14 and many more.15 Furthermore, there is no any antibacterial resistance reported for nanomaterial based antibacterial agents whereas the latest marketed antibiotic Ceftobiprole, reported to have some resistance to βlactamase producing enterobacteria.16 Most of the reported inhibitor of β-lactamase enzymes are based on small molecules. In this regard we want to develop nanomaterial based inhibitor for β-lactamase and we have considered 2D layered material as it can give the maximum surface to volume ratio compared to any other nanomaterials.12 After the reinvention of 2D Materials, specifically graphite and several other 2D materials were developed and standardized such as silicate clays, transition metal dichalcogenides (TMDs), transition metal oxides (TMOs) etc. and they have their own unique properties.17The recent development on 2D nanomaterials helped many cutting-edge biomedical applications, where graphene has been widely used to construct nano-devices for disease diagnosis and therapy.18-19 Like graphene based materials, layered TMDs such as MoS2 is also used for various biomedical applications.20 It has been shown that functionalized 2D TMD nanosheets are nontoxic and biocompatible.21 It has also been used in photothermal therapeutics considering its intrinsic material property.22 Regarding surface modification it has been reported that, 2D-MoS2 is more convenient and it can be functionalized by using simple thiol coordination, like noble metal nanoparticles.23 So far only native graphene oxide and carbon nanotube are reported as nanomaterial based enzymatic inhibitor for β-lactamase but the lack of


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functionalization restrict the further development of those materials. So we have considered functionalized 2D-MoS2 in our study for further surface modification and better efficacy.24 In this study we have used chemically exfoliated MoS2 as 2D nanomaterial and functionalized with different thiolated ligands to develop the β-lactamase inhibitor. Herein, we are reporting that the functionalized layer MoS2 material can effectively inhibit βlactamase activity to combat with multidrug resistant bacteria. Where, among various ligands such as neutral, positive and negatively charged ligand molecule, only negative ligand functionalized 2DMoS2 can effectively bind with β-lactamase enzyme and prevent hydrolysis of β-lactam antibacterial drugs. We have also performed the in vitro antibacterial assay using ampicillin as an antibiotic for Methicillin-resistant Staphylococcus Aureus (MRSA) bacteria, where the growth of bacteria in presence of β-lactamase inhibitor was significantly suppressed for longer period of time than the antibiotic alone.

Results and Discussion: Exfoliation and ligand conjugation of MoS2. For our experiment Bulk MoS2 was exfoliated by the lithium intercalation method.25Intercalated MoS2 was suspended and sonicated in water to achieve high quality water dispersible chemically exfoliated single layered 2D MoS2. Exfoliated material was then functionalized with different thiol ligands with variable charges using our earlier reported protocol.23,

26

The morphology of exfoliated MoS2 sheets was

characterized from atomic force microscopy (AFM) by spin coating of the sample solution on freshly cleaved mica foil. The AFM images revealed the single layer MoS2 nanosheets with the average thickness of ~1.5 nm (Figure 1a) without any aggregation. We have also characterize the



exfoliated MoS2 by using transmission electron microscopy (TEM), and x-ray diffraction, which also support the formation of single layers from bulk MoS2 (Figure S4). We have used three different thiolated ligands with alkane chain (for stability) and polyethylene glycol (PEG) (for biocompatibility) as spacer with different charge functionality. Negative ligand has carboxylate group while positive ligand has quaternary ammonium as head group and hydroxyl group of PEG act as a neutral ligand (Figure 1b). After functionalization all the resultant materials show good stability in 50 mM HEPES buffer for several months but the native exfoliated MoS2 tends to get aggregated. The extent of ligand conjugation was examined by zeta potential measurement of each sample, which provide the nature of charge associated with the functionalized 2D-MoS2 nanosheets. As can be seen from figure 1c, positively charged ligand functionalized MoS2 have zeta potential about +24.6 mV, while neutral ligand functionalized MoS2 exhibits zeta potential around -2.94 mV. For negatively charged ligand functionalized MoS2 have higher negative charge (-46.7 mV) compare to the native exfoliated MoS2 (-32.4 mV) which indicates the presence of negative ligands. Overall these results indicate that the exfoliated MoS2 is functionalized with different thiolated ligands and possesses different charged surfaces in respect to the corresponding functionalized ligands.


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Figure 1. (a) AFM imageof exfoliated 2D MoS2 nanosheets with average thickness of ~1.5 nm, indicates the formation of single layer. (b) The structure of thiolated ligands used for 2D MoS2 functionalizationwith different charge based head-group. (c) The zeta potential distribution plots for different functionalized 2D MoS2 materials indicates the functionalization with respective ligands.

β-lactamase Activity Assay. The crystal structure analysis suggests that the active site of β-lactamase enzyme is surrounded by cationic amino acid (lysine and arginine) residues.27 Hence negatively charged MoS2should block the active site and subsequently hinder the accessibility of substrate molecule to the catalytic center, causing in the inhibition of the β-lactamase activity (Figure 2a).28To validate the hypothesis and to find the possible interactions with β-lactamase the activity assay was performed in presence of various functionalized MoS2as well as native exfoliated MoS2, which is also negatively charged. We have performed the activity assay using nitrocefin which is a chromogenic cephalosporin-based substrate. In presence of β-lactamase, the lactam ring of nitrocefin get hydrolyzed and exhibits a UV-Vis absorption shift from yellow (390 nm) to red (486 nm) which can be used to monitor the activity of the enzyme (Figure 2b). The



optimized concentration for enzyme and that of nitrocefin substrate, 2.5 nM and 20 µM respectively, were used throughout our experiments in presence of various MoS2samples at variable concentration ranging from 0 to 3.34 µg/ml.

Figure 2. (a) The structure of β-lactamase. Active site (red) of β-lactamase is surrounded by lysine and arginine (blue) residues. (b) Hydrolysis product of the chromogenic substrate, nitrocefin in presence of β-lactamase.

Based on selective host-guest interaction, the negatively charged functionalized MoS2 was found to be the most effective towards binding to the enzyme and consequently exhibits inhibition of the catalytic activity (Figure 3a). The extent of inhibition becomes higher with increased concentration of MoS2 and a significant inhibition (~90%) was achieved with only 3.34µg/ml of negatively charged MoS2. Expectedly, positively charged MoS2 does not show any inhibitory activity whereas neutral MoS2 exhibits very low inhibitory effect. More interestingly, the native exfoliated MoS2, which is also possessing negatively charged surface according to the zeta potential measurement does not show any significant loss of enzymatic activity. Thus, among fourdifferentMoS2 layered materials, only negative ligand functionalized MoS2 showed significant inhibitory effect, which indicates that, not only the electrostatic interaction is playing


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the important role in enzymatic inhibition but also the negative functionality (carboxylate group) is dictating the binding between the enzyme surface and receptor. The similar effect also has been observed in case of ketophosphonate based small molecular receptors where phosphate group binds to the positive residues near active sites.29 The association strength between β-lactamase and negatively charged MoS2 can be assessed by using nonlinear least-squares curve-fitting analysis from the activity assay data.13To establish that correlation, we have considered n number of MoS2 units (also can be considered as active site on the receptor molecule) from the layered materials are binding to the single enzyme. By assuming the diminishing of activity is proportional to the formation of complex between βlactamase and functionalized MoS2, we have considered the following relation (Eq. 1) to estimate the binding constant and number of MoS2 units responsible for inactivating of single enzyme molecule (see supporting information). Where, ∆A is the activity difference, [En]0 and [MoS2]0 denote the initial molar concentration of β-lactamase and MoS2 respectively and Ks is the binding constant.

∆A =

α 2

⋅ {([En ]0 +

1 [MoS2 ]0 + 1 / KS ) − ([En ]0 + 1 [MoS2 ]0 + 1 / K S ) 2 − 4 [En ]0 [MoS2 ]0 } (1) n n n

The excellent curve fit (Figure 3b) indicates the reliability of the assessment. Based on the curve fitting analysis we have observed the binding constant to be around 4.02 x 106 M-1 which is comparable to the other nanomaterial based host-guest complexation.13, 30 From the binding ratio, we have also determined that approximately 51 MoS2 unites are responsible for inactivation of single β-lactamase unit. But overall each layer of functionalized MoS2 can bind many βlactamase molecules in both sides which lead to highly efficient inhibition of enzymatic activity.



Figure 3. (a) Activity of β-lactamase in presence of increasing concentration of various 2D MoS2in 50 mM HEPES buffer (pH 7.3) using nitrocefin as a substrate. (b) The normalized activity of β-lactamase plotted as a function of MoS2concentration and fitted with nonlinear least-square curve fitting analysis. The β-lactamase concentration is 2.5 nM.

Gel electrophoresis assay was carried out to check the binding ability of MoS2 towards enzyme molecule. Where, we found that negatively charged functionalized MoS2 nanosheets were effectively interacting with the enzyme molecule, resulting in formation of a MoS2-enzyme complex with more negative charge than the native enzyme. The complex moves at a faster rate upon electrophoresis than the native enzyme. This clearly indicates the significant binding ability of MoS2 and beta lactamase. The higher protein concentration (20 µM, 8000 times) was used for gel electrophoresis to get the clear spot after coomassie blue staining. Whereas the inhibitor


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concentration was used only 12.5 times higher (42 µg/ml ) due to the stability issue, as a result excess amount of unbound protein remains, which gave the corresponding spot that of native enzyme (SI, figure S6). Inhibition mechanism. To determine the nature of inhibition the initial velocity curve of substrate hydrolysis was measured at different substrate concentrations (0-20 µM of nitrocefin) in presence of different concentrations of inhibitor (0-2 µg/ml) (Figure 4a). The initial velocity of substrate hydrolysis reaction decreases with increasing concentration of the MoS2 based inhibitor. The general expression for the determination of the velocity (V) of an enzymatic reaction in presence of inhibitor (I) is (Eq 2): =

( )

(2)

According to this equation the value of α will define the mode of inhibition. Specifically, α = 1 implies that the nature of inhibition is noncompetitive, when α > 1, that indicates binding of the inhibitor competes with binding of the substrate molecule, which is competitive inhibition. The nonlinear fitting of the activity assay data using GraphPad Prism 5, corresponds to the value, α= 7.863, is indicating that negative charged 2D MoS2 inhibits β-lactamase through competitive inhibition. The mechanism of inhibition also can easily be inferred from the Lineweaver-Burk plot (Figure 4b). The regression lines on the Lineweaver-Burk plot coincide on the y-axis at the same point, which specify a competitive type of inhibition. From the above curve fitting analysis, the observed binding constant (ki) is 3.793 µg/ml. Here we found the mechanistic behavior and the binding constant to be very similar to the interaction of other layer materials (e.g. graphene oxide) and enzymes (e.g. α-chymotrypsin).12



Figure 4. (a) Initial velocity as a function of substrate concentration at different concentrations of inhibitor (negative charged MoS2). β-lactamase concentration of 2.5 nM was used throughout the activity assay. The data fitting indicates a competitive inhibition with Ki= 3.793 µg/ml. (b) Lineweaver-Burk plot is showing a common y-intercept, which indicates the competitive inhibition.

Even though there functionalized MoS2 is very stable and ligand generally not desorb from the surface based on our earlier studies26 and many other reports.31 But to remove any possibility of effect of free ligands in enzymatic activity inhibition we have performed a controlled study only with free ligand molecule. As expected, we have observed that there is no inhibitory effect of free ligand even at very high concentration (Figure 5a). As we assume the interaction to the active site is mainly based on electrostatic interaction we have also studied the effect of ionic strength in enzyme –nanomaterial complexation. In higher salt concentration the electrostatic


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interaction will be attenuated and the enzymatic activity should be regenerated. This reversibility of MoS2 enzyme interaction was examined by performing enzymatic kinetic assay using reaction medium of low to very high ionic strength. We observed that the activity of enzyme is recovered at higher salt concentration in presence of the inhibitor. This indicates the interaction between MoS2 and enzyme is electrostatic in nature which could be reversed (Figure 5b).

Figure 5. (a) β-lactamase inhibition assay in presence of only negative ligand. The plot indicates no inhibition of enzyme activity. (b) Activity of β-lactamase after pre-incubation and postincubation with 3.34 µg/ml functionalized 2D-MoS2 in various concentrations of HEPES buffer medium. The β-lactamase concentration is 2.5 nM.



Circular-dichroism (CD). After the determination of inhibition mechanism, we determined the effect on secondary structure of β-lactamase enzyme upon binding with negatively charged MoS2 by using CD spectroscopy. First, we have measured the CD spectra of native and thermally denatured enzyme as two extreme references. No CD signal was detected from functionalized MoS2. The native enzyme solution shows two characteristic minima at 208 and 220 nm which indicates the presence of large extent of α-helix.32Also we can see from the above crystal structure, β-lactamase has large fraction of α-helical structure which upon thermal denaturation completely converted to a random coil. Hence, the extent of denaturation can be monitored by measuring the diminishing of two minima (Figure 5a). To estimate the structural alteration, we have incubated the 7.14 µM of β-lactamase with 16.75 µg/ml of negatively charged MoS2 and measured the CD spectra at different time interval for a period of 24 hrs. From figure 5b, it can be seen that, even after 24 hr there is a very little change in two characteristics minima. This clearly indicates that the interaction between β-lactamase and MoS2 sheets does not disrupt the secondary structure of interacting enzyme.


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Figure 6. (a) CD spectra of β-lactamase (7.14 µM) complexed with MoS2 (16.75 µg/ml) and (b) change of CD signal at 208, 220nm at different time interval.

The antibacterial activity. To evaluate the effect of this newly developed β-lactamase inhibitor for combination antibacterial therapy, we have considered ampicillin as a β-lactam antibiotics and MRSA as the drug resistant bacterial strain. MRSA is a gram positive bacteria and highly resistant against β-lactam antibiotics such as methicilline, oxacillin, ampicillin etc. by releasing β-lactamase as an antibiotic scavenger.33-34With continued growth of resistance, currently ampicillin is an practically ineffective antibiotic against MRSA.35This combination therapy is expected to work by inhibiting β-lactamase in presence of negatively functionalized MoS2 and



thereby enhancing the antibacterial activity of conventional antibiotics such as ampicillin. The assay was carried out through microbroth dilution method by measuring absorbance at 600nm on a microplate reader equipped with a shaker and thermostat set to 370 C. The bacterial growth curves were monitored over a period of 16 hr, in a real time kinetic cycle with absorbance at 600nm (A600) nm taken at 10 min intervals followed by orbital shaking. The result showed that in the presence of either only negatively functionalized MoS2 (13.34 µg/ml) or only ampicillin (8 µg/ml) the bacterial growth is not significantly interrupted (Figure 6a). That indicates that negatively functionalized MoS2 has no microbial toxicity and only ampicillin is ineffective against MRSA. To evaluate the synergistic effect of both ampicillin and negative functionalized MoS2 we have measured the bacterial growth in presence of 8 µg/ml ampicillin and increasing concentration of MoS2 from 3.34 µg/ml to 13.34 µg/ml. Expectedly, we have observed that in presence of 6.67 µg/ml MoS2, bacterial growth was remarkably suppressed for a longer period of time (up to 12 hr), while in presence of only ampicillin the bacterial growth started just after 4 hour of incubation (Figure 6a). This indicates that the destruction of the antibiotic agent, ampicillin by the MRSA bacterial strain was prevented in the presence of negative charge MoS2 as β-lactamase inhibitor, resulting delay in further growth of the bacteria. Once the amount of the β-lactamase enzyme translated by the bacteria overcome the effect of inhibitor, subsequently destroy the antibiotic agent present in the medium and bacterial growth started. Quantitative Analysis of the combination inhibitory effect was done by using lag phase and growth rate analysis from the growth curve (Figure 6a). Lag phase of the bacterial growth curve is the time when the bacteria is adjusting to the new environment before going into rapidly growing phase. Lengthening of lag phase often correlates to the antibacterial efficiency of an antibiotic or the mixture. We have considered the stationary phase absorbance of the control


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(average absorbance of control during 12-16.5hr) to be 100% and calculated lag phase to be the time until which the bacterial growth was

2D-MoS2 based Î²-lactamase Inhibitor for Combination Therapy

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