Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis

Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis View ORCID Profile Mariska de Munnik , Karina Calvopiña , View ORCID Profile Patrick Rabe , View ORCID Profile Christopher J. Schofield doi: https://doi.org/10.1101/2025.01.28.635326 Mariska de Munnik 1 Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute of Antimicrobial Research, University of Oxford , 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mariska de Munnik Karina Calvopiña 1 Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute of Antimicrobial Research, University of Oxford , 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Patrick Rabe 1 Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute of Antimicrobial Research, University of Oxford , 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom 2 Diamond Light Source, Diamond House, Harwell Science and Innovation Campus , Didcot OX11 0DE, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Rabe Christopher J. Schofield 1 Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute of Antimicrobial Research, University of Oxford , 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher J. Schofield For correspondence: christopher.schofield{at}chem.ox.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract β-Lactams demonstrate promising in vitro activity against Mycobacterium species and are being explored for tuberculosis treatment; however, evidence of their in vivo efficacy versus Mycobacterium tuberculosis remains limited. To achieve broad clinically relevant potency, optimisation of the classical β-lactam scaffolds or development of new or non-β-lactam inhibitors for mycobacterial transpeptidases is likely required. In mycobacteria, potential targets of β-lactams include L,D-transpeptidases (Ldts) and penicillin-binding proteins (PBPs). Reports suggest that dual inhibition of Ldts and PBPs may be necessary to achieve effective anti-mycobacterial activity, yet the specific contributions of Ldt and PBP inhibition to the β-lactam antibacterial mechanisms are not understood. We used fluorogenic substrate mimics to investigate the effects of β-lactams and reported Ldt Mt2 inhibitors on Mycobacterium smegmatis ( Msm ), assessing their impacts on the transpeptidase activities of Ldts and PBPs in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; though under the tested conditions a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, inhibition of both PBPs and Ldts was observed in all active inhibitors, though β-lactams manifest increased potency of PBP inhibition. The combination of the β-lactams meropenem and faropenem with selected Ldt Mt2 inhibitors manifested an additive inhibitory effect against Msm . Our results highlight the importance of further optimising PBPs and Ldt transpeptidase inhibition, particularly of Ldts, to improve β-lactam efficacy versus mycobacteria. Introduction The discovery of β-lactam antibiotics represented a revolution in treatment of infections, and they remain the most important class of antibiotics in clinical use.( 1 ) β-Lactams act as mechanism based inhibitors of the penicillin-binding proteins (PBPs), an essential class of enzymes involved in the biosynthesis of the peptidoglycan layer which is present in every bacterial species, which have D,D-transpeptidase or D,D-carboxypeptidase activities ( Figure 1A ).( 2 , 3 ) The effectivity of β-lactams, including penams, cephalosporins, carbapenems, penems, and monobactams ( Figure 1B ), is increasingly compromised by resistance, in particular by β-lactamases which are enzymes that catalyse β-lactam hydrolysis ( Figure 1A ).( 1 ) The presence of a chromosomally encoded Ambler class A β-lactamase (BlaC) has been commonly thought to be a reason why Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB) that is estimated to cause 1.6 million deaths per year,( 4 ) is resistant to β-lactams.( 5 ) However, the promising in vitro activities of carbapenems in combination with the β-lactamase inhibitor clavulanic acid against Mtb have generated interest in reappraisal of β-lactams for TB treatment,( 6 – 9 ) including with respect to optimisation of cephalosporins for TB treatment. ( 10 – 12 ) Download figure Open in new tab Figure 1. Inhibition of mycobacterial cell wall biosynthesis by β-lactams. A. The mycobacterial cell wall is made up of three core components: the mycolic acid, arabinogalactan, and peptidoglycan layers. The final steps of the peptidoglycan biosynthesis include the formation of 3→3 cross-links (catalysed by Ldts) and 4→3 cross-links (catalysed by PBPs), and cleavage of the terminal D-Ala of the pentapeptide monomer to form tetrapeptide monomers (catalysed by PBPs with D,D-carboxypeptidase activity). Cross-links in the peptidoglycan layer of the mycobacterial cell wall can be inhibited by β-lactams (represented here with the core motif of carbapenems); the serine β-lactamases (BlaC in Mtb , and BlaS and BlaE in Msm ) confer resistance. Hexagons in green are N-acetylglucosamine (GlcNAc), hexagons in teal are N-acetylmuramic acid (MurNAc), purple circles are D-Ala, dark blue circles are m - DAP, light blue circles are amino acids, dark blue lines are cross-linked amino acids. The figure was created using BioRender.com. B. Core motifs of clinically relevant classes of β-lactams. The activity of β-lactams against Mtb is further complicated by the presence of L,D- transpeptidases (Ldts), an important class of enzymes involved in the bacterial cell wall biosynthesis. Ldts are nucleophilic cysteine enzymes that catalyse 3→3 cross-links in the peptidoglycan layer, which are present in exceptionally high quantities in mycobacteria during the stationary phase, compared to the 4→3 cross-links catalysed by the nucleophilic serine PBPs ( Figure 1 ).( 13 , 14 ) Several mycobacterial Ldts have been identified as being essential for cell morphology, virulence, aging, and resistance to β-lactams;( 15 , 16 ) Ldts are not generally thought to be susceptible to inhibition by most β-lactams, though carbapenems and selected cephalosporins have been observed to inhibit Ldts.( 17 – 20 ) Mtb contains at least seven PBPs, and five Ldts.( 21 ) It has been suggested that inhibition of Ldt Mt2 alone should be sufficient to treat Mtb ,( 22 ) and several PBPs been identified as being individually essential for growth (PBP3) and/or infection ( e.g. , PonA1, PonA2, and PBPA).( 23 , 24 ) However, there is evidence that inhibition of both Ldts and PBPs may be required for therapeutically relevant inhibition of Mtb .( 7 – 9 ) Dual treatment of amoxicillin/clavulanic acid and meropenem improved inhibition in both Mtb and Mycobacterium smegmatis ( Msm ).( 16 , 25 ) An anecdotal report of a patient treated with the meropenem and amoxicillin/clavulanic acid combination describes the early clearance of Mtb from the sputum.( 26 ) However, clinical trials have yielded inconclusive results.( 26 – 28 ) The available results suggest that for clinically relevant inhibition of Mtb via inhibition of PBPs and Ldts, inhibitors tailored towards the Mtb transpeptidase targets will be required. Given the intricate network of cysteine and serine transpeptidases, along with presence of a serine β-lactamase (SBL), the exact modes of action of β-lactams and β-lactam-based inhibitors during mycobacterial chemotherapy are unclear. Recent studies using fluorescent derivatives of β-lactams have been used to address this challenge by identifying and studying the targets of β-lactams in cell lysates of Mtb , revealing their binding to various PBPs, Ldts, and BlaC.( 29 ) Pidgeon et al. (2019) have also reported on the use of fluorescently labelled substrate mimics of Ldts (TetraRh) and PBPs (PentaFI) to assess Ldt and PBP activity, as well as inhibition by the β-lactams ampicillin and meropenem in whole cells of Mtb and Msm .( 30 ) We are interested in further investigating the inhibition of mycobacteria by different classes of β-lactams, as well as selected new types of Ldt Mt2 inhibitors (including cephalosporins) recently reported by us.( 31 , 32 ) We envisaged that differentiating between Ldt and PBP inhibition, and correlating the extent of transpeptidase inhibition with the inhibition of growth may provide insight that will help enable focussed inhibitor optimisation efforts. Here we report studies using Msm , a well validated model organism for Mtb ,( 33 ) which contains at least five PBPs and six Ldts, as well as the Ambler class A β-lactamase BlaS,( 34 – 36 ) to assess the activities of PBP and Ldt Mt2 inhibitors, and their ability to inhibit PBPs and Ldts in live Msm cells, using PentaFI and TetraRh, respectively. The results reveal a statistically significant correlation between both Ldt and PBP inhibition with suppression of Msm growth and imply that a combination of Ldt and PBP inhibition will be beneficial for antibacterial activity, with particular potential for improving Ldt inhibition. Results Inhibition of Mycobacterium smegmatis by β-lactams We first aimed to assess the activity of different classes of β-lactams against Msm ( Figure 2 , Table S1). Selected carbapenems (meropenem, imipenem, ertapenem, and doripenem; 2 - 5 ) manifested potent inhibition of Msm (MIC 0.125-4 μg/mL) in the absence of clavulanic acid. This observation is in line with the proposal that carbapenems act as inhibitors of the Mtb SBL BlaC.( 6 , 37 , 38 ) Imipenem ( 2 ) was particularly potent (MIC 0.125 μg/mL), in accord with reported susceptibility of Msm towards imipenem as determined by disc diffusion.( 36 ) The penem faropenem ( 1 ) also manifested inhibition of Msm without the presence of clavulanic acid, though was less potent than the carbapenems (MIC of 16 µg/mL). Download figure Open in new tab Figure 2. Minimum inhibitory concentrations of 1-31 against Mycobacterium smegmatis . The MICs of 1 - 31 against Msm were determined with three independent repeats. Note that the highest tested concentration was 128 µg/mL, and inactive compounds are represented here as exceeding an MIC of 128 µg/mL. MIC values and compound structures are given in Table S1. None of the tested cephalosporins, penams, or the monobactam aztreonam inhibited Msm in the absence of clavulanic acid ( 6 - 16 ; MIC >128 μg/mL), in line with previously reported susceptibility studies as determined by disc diffusion of Msm .( 36 ) This observation is likely in part due to β-lactamase activity, because the presence of clavulanic acid (100 µg/mL),( 39 ) which by itself did not potently inhibit Msm (MIC 128 µg/mL), significantly improved the activities of ampicillin ( 11 ) and amoxicillin ( 12 ) (MICs of 2 µg/mL and 1 µg/mL, respectively). The activities of ampicillin and amoxicillin versus a ΔblaS strain has been reported (MIC of 16 µg/mL and 1 µg/mL, respectively).( 40 ) In contrast, the inhibitory potencies of oxacillin ( 13 ), penicillin G ( 14 ), and carbenicillin ( 15 ) varied from poor to moderate (MIC >128 µg/mL, 32 µg/mL, and 64 µg/mL, respectively). The presence of clavulanic acid had no impact on the cephalosporins ceftazidime ( 6 ), ceftriaxone ( 7 ), and cefepime ( 10 ) (MIC 128 µg/mL), in contrast to the reported MIC of ceftriazone of 32 μg/mL in the presence of clavulanic acid.( 36 ) However, cephalothin ( 8 , MIC 2 µg/mL) and cefmetazole ( 9 , MIC 4 µg/mL) showed potent inhibition. Addition of clavulanic acid (100 µg/mL) did not enable inhibition of Msm by the monobactam aztreonam ( 16 , MIC >128 µg/mL). To evaluate the potential of various classes of β-lactamase inhibitors in restoring cephalosporin activity, we compared the MICs of cephalothin in the presence of clavulanic acid, tazobactam,( 41 ) sulbactam,( 42 ) BLI-489,( 43 ) and the non β-lactam SBL inhibitors xeruborbactam, a bicyclic boronate and the diazabicyclooctane (DBO) avibactam (Table S2). ( 44 , 45 ) The addition of sulbactam, xeruborbactam, or avibactam did not enhance the potency of cephalothin. In contrast, tazobactam or BLI-489 (100 µg/mL) increased cephalothin activity to 32 µg/mL and 8 µg/mL, respectively. Clavulanic acid (100 µg/mL) reached the most potent recovery of cephalothin activity (MIC 2 µg/mL). None of the tested β-lactamase inhibitors exhibited inhibitory potency against Msm on their own, with MIC values exceeding 128 µg/mL. Inhibition of M ycobacterium smegmatis by inhibitors of Ldt Mt2 A high-throughput screen for Mtb Ldt Mt2 inhibitors has identified diverse potent electrophilic compounds reacting with the active site nucleophilic cysteine.( 31 , 32 ) Despite not being optimised for pharmacokinetic properties, a selection of the inhibitors were active against Mtb in macrophages.( 31 ) We set out to investigate the activity of these inhibitors against Msm , and additionally tested representative Ldt Mt2 inhibitors that did not show activity against Mtb , the results of which are summarised in Figure 2 and Table S1. We included the established anti-TB therapeutics ethambutol, isoniazid, and rifampicin as control compounds, which manifested inhibition similar to the reported values (Table S3);( 33 , 46 ) note that MIC values reported in the literature for isoniazid vary significantly, and that the Clinical & Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) do not provide breakpoints for Msm , as it is not a primary clinical pathogen.( 47 , 48 ) The most potent activity against of Msm was observed with the sulfonyl pyridine 31 , with an MIC of 8 µg/mL. Interestingly, 31 was also among the most potent compounds tested against Mtb (MIC 50 11 ± 1.2 μM), though was not active against Mtb residing inside the macrophage (MIC 50 >50 μM). Cyanamide 26 exhibited promising activity with an MIC of 16 µg/mL. Ebsulfur 21 and cyanamide 25 manifested MICs of 32 µg/mL and 48 µg/mL, respectively. Poor activity against Msm was observed with 19 , 20 , 24 and 27 (MIC of 64 µg/mL). No inhibition of Msm was detected with 18 , 22 and 23 (MIC >128 µg/mL), despite exhibiting moderate inhibition of Mtb , while 29 - 30 were inactive versus both Msm and Mtb .( 31 ) Dual treatment of Mycobacterium smegmatis with Ldt Mt2 inhibitors and β-lactams To evaluate potential synergy between Ldt Mt2 inhibitors and β-lactams, we assessed the effect of dual treatment with the two transpeptidase inhibitor classes. For these studies we selected Ldt Mt2 inhibitors with varying efficacy against Msm ( 19 , 21 , 24 - 26 , and 31 ), alongside the inactive compounds 29 and 30 (MIC >128 µg/mL). Meropenem and faropenem were selected as representative β-lactams, based on their inhibitory potency against Msm without the presence of clavulanic acid. We initially assessed effect of Ldt Mt2 inhibitors on the MIC of the selected β-lactams in three concentrations (64 µg/mL, 16 µg/mL, and 4 µg/mL; Table S4). The presence of 21 , 25 and 31 , increased the observed activities of both faropenem and meropenem. The increase in activity was apparently more pronounced with the combinations with faropenem. Specifically, the MIC of faropenem decreased from 16 μg/mL to 8 μg/mL, 4 μg/mL, and <0.25 in the presence of 4 μg/mL of 21 , 25 , and 31 , respectively. The MIC of meropenem was only altered from 2 µg/mL to <0.25 µg/mL and 1 µg/mL in the presence of 16 μg/mL 21 and 25 , respectively at a concentration of 16 µg/mL. Further, in the presence of 26 (4 µg/mL), the MIC of faropenem decreased to 8 μg/mL, though 26 (4 µg/mL) did not influence the MIC of meropenem. The presence of 19 , 24 , 29 and 30 had no effect on the MIC of faropenem or meropenem. Given that compounds 21 , 25 and 31 exhibited increased antibacterial activity in combination with the selected β-lactams, we performed checkerboard analyses of these compounds in combination with faropenem or meropenem ( Table 1 , Figure S1). We observed increased activity of the Ldt Mt2 inhibitor-β-lactam combinations against Msm in all cases. According to the recommended interpretation of the determined Fractional Inhibitory Concentration (FIC) indexes of the assessed inhibitor combinations,( 49 ) the results suggest that the combinations of Ldt Mt2 inhibitors with faropenem manifest a potentially synergistic effect, with FIC indexes ranging from 0.4 to 0.5, while the combinations with meropenem yielded to an additive effect, with FIC indexes between 0.6 and 0.7 ( Table 1 , Figure S1). View this table: View inline View popup Download powerpoint Table 1. The effects of Ldt Mt2 inhibitors 21, 25, and 31 in combination with faropenem or meropenem. FIC index represents the Fractional Inhibitory Concentration index of the indicated combinations of inhibitors, calculated using .( 76 ) FAR = faropenem (MIC alone of 16 μg/mL), MER = meropenem (MIC alone of 2 μg/mL). The results of all tested combinations are given in Figure S1. Inhibition of the incorporation of fluorescent peptides To investigate a potential correlation between Msm growth inhibition and the inhibition of PBPs and Ldts, we employed the fluorescent probes PentaFI and TetraRh, which mimic the substrates for PBPs and Ldts, respectively (Figure S2), as described by Pidgeon et al. (2019).( 30 ) Incubating Msm with the inhibitor of interest alongside PentaFI or TetraRh should lead to a dose-dependent reduction in fluorescent signal as a result of inhibition of PBPs or Ldts, respectively (Figure S2), as demonstrated with meropenem and ampicillin with Msm .( 30 ) Optimisation of the fluorescence assays was carried out by assessing the time-dependent incorporation of PentaFI and TetraRh into Msm . With 50 µM PentaFI, we observed a time-dependent increase in the fluorescence signal over 24 h, while the negative control, treated with meropenem-clavulanic acid (100 µg/mL), showed no such increase (Figure S3A). Incubation with 50 µM TetraRh exhibited high levels of fluorescent incorporation within the first hour, with minimal subsequent increase. The negative control (100 µg/mL meropenem-clavulanic acid) exhibited reduced fluorescence, but manifested some fluorescence (Figure S3B). When the concentration of TetraRh was reduced to 0.5 µM, a time-dependant increase in the fluorescence signal was observed over 24 h, with the negative controls lacking an apparent fluorescent signal (Figure S3D). As reported,( 30 ) simultaneous incubation of both TetraRh (50 µM) and PentaFI (50 µM) showed only detectable incorporation of TetraRh, likely due to the hight emission intensity of TetraRh. We performed dose-response studies using this assay to determine pIC 50 values for cellular inhibition of Ldts and PBPs in Msm , focussing on representative β-lactams from different classes and Ldt Mt2 inhibitors ( Figure 3 , Table S1). In most cases, the inhibition of PBPs was more pronounced than Ldt inhibition, with the exceptions being the Ldt Mt2 inhibitors 25 and 31 , where a modest dominance of Ldt inhibition was observed ( Figure 3A ). Amongst all the tested compounds, the carbapenem imipenem displayed the most potent apparent inhibition of both the Ldts and PBPs in Msm , with pIC 50 values of 7.5 ± 0.047 and 9.2 ± 0.054, respectively ( Figure 3B,C , Figure S4). Other carbapenems, including meropenem, ertapenem and doripenem, and the penem faropenem, also exhibited potent inhibition of transpeptidase activity of PBPs (pIC 50 values ranging from 5.7 to 6.6) and Ldts (pIC 50 values ranging from 5.1 to 6.1). Download figure Open in new tab Figure 3. Fluorescent peptide incorporation assay investigating the inhibition of Ldts and PBPs in live Mycobacterium smegmatis cells. A. The inhibition of the PBPs and the Ldts at the MIC of the respective inhibitors was investigated. Bars represent the average between Ldt and PBP inhibition. B . The inhibitory potencies (pIC 50 ’s) of the inhibitors for Msm Ldts were determined using the dose-response assays with TetraRh, and plotted against the Msm pMIC. C. The inhibitory potencies (pIC 50 ’s) of the inhibitors for Msm PBPs were determined using the dose-response assays with PentaFI, and plotted against the Msm pMIC. Note that pMIC values arise from logarithmic transformation of MIC (in g/mL) and that pIC 50 values arise from logarithmic transformation of IC 50 (in g/mL). Data points represent mean and error bars represent standard deviation. Values and compound structures are given in Table S1. In contrast, all the assessed cephalosporins (ceftazidime, ceftriaxone, cephalothin, cefmetazole, and cefepime) showed limited inhibition of TetraRh or PentaFI incorporation, despite the presence of clavulanic acid (100 µM). PBP inhibition ranged between pIC 50 values of 4.1 ± 0.069 for ceftazidime and 5.2 ± 0.11 for cefepime, while Ldt inhibition ranged between pIC 50 of 3.8 ± 0.69 for ceftazidime and 4.8 ± 0.13 for cefmetazole. The penams ampicillin, amoxicillin, penicillin G, and carbenicillin exhibited moderate to potent inhibition of PentaFI incorporation in the presence of clavulanic acid (100 µM), with the pIC 50 values ranging between 5.2 ± 0.042 for carbenicillin and 6.2 ± 0.056 for ampicillin. However, inhibition of TetraRh incorporation was very limited, with the most potent Ldt inhibition being a pIC 50 value of 4.7 ± 0.079 for ampicillin. Oxacillin and the monobactam aztreonam, which showed no inhibition of growth of Msm , did not apparently inhibit either Ldts or PBPs. The inhibitors identified in the HTS against Ldt Mt2 ( 21 , 25 , and 31 ) demonstrated limited inhibition of TetraRh incorporation, with pIC 50 values ranging between 3.8 and 4.8, correlating with their relatively poor MIC values. Interestingly, these compounds also acted as PBP inhibitors of similar potency (pIC 50 values ranging between 3.7 and 4.6), despite being identified as Ldt inhibitors. In contrast, 31 , which manifested a significantly lower MIC of 8 µg/mL, showed only limited Ldt inhibition (pIC 50 value 4.2 ± 0.071), and no PBP inhibition, suggesting a potentially alternative mechanism of action. When evaluating the inhibition of PentaFI and TetraRh incorporation at inhibitor concentrations corresponding to the MIC, combined PBP and Ldt transpeptidase inhibition varied in most cases from 64% with ertapenem to ∼40% with cefmetazole, amoxicillin, and doripenem ( Figure 3A ). Outliers to this observation are the ∼90% inhibition of PBP and Ldt transpeptidase activity in the cases of imipenem and faropenem, and 15% inhibition in the case of the sulfonyl pyridine derivative 31 . Apparently dual inhibition of PBPs and Ldts was observed with all the β-lactam inhibitors, except for the penams penicillin G and carbenicillin. Additionally, ampicillin and amoxicillin manifested considerably lower Ldt inhibition compared to PBP inhibition. We observed a relatively strong correlation between Ldt inhibition and Msm MIC ( Figure 3B ); Pearson correlation analysis revealed a significant positive correlation (r = 0.75, p < 0.001, n = 19), though the correlation was stronger on exclusion of the penams (r = 0.83, p < 0.001, n = 15). A weaker, but significant correlation was observed between PBP inhibition and MIC (r = 0.69, p < 0.05, n = 19; Figure 3C ). Biochemical inhibition of the Mycobacterium tuberculosis PBP3 Following the observation that Ldt Mt2 inhibitors, in particular the ebsulfur derivative 21 (pIC 50 4.6 ± 0.080), apparently inhibit PBPs in a cellular context, we investigated the inhibition of the essential isolated recombinant PBP3 of Mtb by 18 - 31 ( Figure 4A , Table S1, Figure S5).( 23 , 50 , 51 ) Interestingly, despite cellular inhibition of Msm PBPs, most of the assessed compounds manifested limited inhibitory potency (pIC 50 ≤ 4.5 for 19 - 21 and 26 ) or no significant inhibition (pIC 50 < 3.4 for 18 , 22 , 25 , and 28 - 31 ) against PBP3. The exception was cephalosporin 24 (pIC 50 of 6.1 ± 0.089). Notably, 24 has been identified as an inhibitor of both Ldt Mt2 and BlaC, although it does not display potent anti-TB activity,( 31 ) or significant anti- Msm activity (MIC 64 μg/mL). Download figure Open in new tab Figure 4. Inhibition of Mycobacterium tuberculosis PBP3 by 1-31. A. The inhibitory potencies (pIC 50 ’s) of the inhibitors for Msm Ldts were determined using the dose-response assays using the fluorometric S2d assay,( 72 – 74 ) applying the procedure optimised for P. aeruginosa PBP3.( 75 ) B. The Mtb PBP3 pIC 50 values did not show a significant correlation with the Msm PBP inhibition determined in the fluorescent peptide incorporation assays. C. The Mtb PBP3 pIC 50 values did not show a strong correlation with the MIC’s of Msm . Values and compound structures are given in Table S1, and data curves are given in Figure S5. We also evaluated inhibition of isolated Mtb PBP3 by β-lactams ( Figure 4A , Table S1, Figure S5). Most β-lactams potently inhibited PBP3, with pIC 50 values approaching, or reaching, the upper assay limit (pIC 50 of 6.8). Ceftazidime, known for its potent inhibition of PBP3 in Gram-negative bacteria,( 52 ) and carbenicillin displayed slightly lower potencies (pIC 50 values of 5.5 and 5.4, respectively). Additionally, aztreonam, which is effective against PBP3 in Gram-negative but not Gram-positive bacteria,( 53 ) manifested very low inhibition of Mtb PBP3 with a pIC 50 value of 3.8. No significant correlation was observed between the cellular inhibition of the PBPs of Msm by 1 - 31 and the inhibition of Mtb PBP3 under current assays conditions as evidenced by Pearson’s r analysis (r = 0.25, p > 0.05; Figure 4B ). Only weak correlation between the MIC against Msm and the inhibition of Mtb PBP3 was observed (r = 0.45, p = 0.05; Figure 4C ). Sulfonyl pyridines exhibit promising anti-mycobacterial activity Of the inhibitors identified in the HTS against Mtb Ldt Mt2 ,( 31 ) the sulfonyl pyridine 31 manifested the most potent inhibition of Msm . However, the fluorescent peptide incorporation assays revealed only limited evidence for Ldt and PBP inhibition ( Figure 3 ), suggesting a potential alternative mechanism of action. However, given its notable activity, we evaluated a series of derivatives of 31 against Msm ( Figure 5 , Table S5). Download figure Open in new tab Figure 5. Sulfonyl pyridines are inhibitors of Mycobacterium smegmatis . A. Sulfonyl pyridines 31 - 46 were tested against Msm . Full compound structures and MICs are given in Table S5. B. Mass spectrometry studies suggest selected sulfonyl pyridines to react with the nucleophilic cysteine of Ldt Mt2 via nucleophilic aromatic substitution, as exemplified with 31 . C. Mass spectrometry studies with 32 suggest that repositioning of the nitrogen atom on the pyridine ring reduces reactivity with Ldt Mt2 and alters the leaving group. D. The MIC against Msm does not correlate with the rate of inhibition against Ldt Mt2 ( k inact /K I , in red) or with the rate of intrinsic thiol reactivity ( k chem , in blue). E. Findings of the structure-activity relationship studies with 31-46 based on the MIC against Msm . For derivative 32 , altering the position of the nitrogen atom in the pyridine ring reduced the MIC to 4 µg/mL. Modifications to the phenyl ring, such as addition of a methyl group ( 33 ) or bromide ( 35 ), did not change the MIC, while substitution with a chloride ( 34 ) decreased the MIC to 4 µg/mL. Moving of the nitro group from the para to the ortho position ( 36 ) increased the MIC to 16 µg/mL. Notably, the removal of the nitro group entirely abolished activity against Msm , regardless of the position of the nitrogen atom on the pyridine ring ( 37 - 39 ), or substitution of the nitro group with a methoxy group ( 40 ) or a nitrile in para ( 41 ) or ortho ( 42 ) positions. Substituting the phenylsulfonyl group with a chloride increased the MIC to 32 µg/mL ( 43 ). Similarly to 37 - 42 , elimination of the nitro group in that moiety resulted in the loss of activity against Msm ( 44 ). Furthermore, nitrophenyl sulfonyl derivatives 45 and 46 did not exhibit activity against Msm . While Ldts may well not be the (only) cellular target of the sulfonyl pyridines, we examined their reactivity with the nucleophilic thiol of Ldt Mt2 . Protein-observed solid-phase extraction MS (SPE-MS) assays with Ldt Mt2 showed that 31 , 33 - 36 , 41 , 42 and 46 all reacted with Ldt Mt2 through nucleophilic aromatic substitution of the sulfonyl group on the pyridine ring ( Figure 5B , Table S5, Figure S9). In contrast, the mass shift observed with 32 corresponds to nucleophilic substitution at the nitro group rather than the sulfonyl group ( Figure 5C ). Derivatives 37 - 40 , 43 and 44 did not react with Ldt Mt2 ; of these only 43 exhibited moderate activity against Msm (MIC 32 µg/mL). Kinetic studies of the reaction with Ldt Mt2 (Table S5, Figure S6, S7), and with L-glutathione (GSH; Table S5, Figure S8) were performed to profile the electrophilic reactivity of this class of inhibitors. We observed no correlation between reactivity with Ldt Mt2 or GSH and activity against Msm ; for example, 32 demonstrated significantly lower reactivity with both Ldt Mt2 and GSH compared to 31 and 33 - 35 , yet showed increased activity against Msm ( Figure 5D ). Discussion Historically, β-lactams have been considered to be ineffective for treating TB. However, emerging in vitro studies demonstrating promising activity against Mtb have led to a re-evaluation of this paradigm.( 7 – 9 ) Nevertheless, clinical studies have yielded inconclusive results regarding the utility of β-lactam antibiotics for the treatment of TB,( 26 , 28 , 54 ) suggesting that optimisation of inhibitors specifically tailored for Mtb may be required. Due to the high level of 3→3 cross-links in the peptidoglycan layer catalysed by the Ldts, Ldts are considered a promising target for the development of inhibitors. Some evidence, however, indicates that effective inhibition may require targeting both Ldts and PBPs.( 16 ) A deeper understanding of the mechanism of action of β-lactams will facilitate targeted efforts these compounds and promote the development of non-β-lactam inhibitors against Mtb . Our results reveal a correlation between MIC values and the extent of inhibition of both PBP and Ldt transpeptidase activities, though a stronger correlation between Ldt inhibition and MIC was observed. Importantly, we observed dual inhibition of PBPs and Ldts for all the tested inhibitors of Msm . However, we noted multiple instances (e.g., with meropenem, imipenem, faropenem, ampicillin, penicillin G, and amoxicillin) where complete inhibition of cellular PBP transpeptidase activity did not correspond to growth inhibition. These findings support the importance of Ldts as an important target for Mycobacterium spp. inhibition, particularly in combination with PBP inhibition, at least under the tested conditions. They suggest that optimising β-lactams against Mycobacterium spp. should focus on enhancing their potency against Ldts, as well as PBPs. However, to better define the relationship between Ldt inhibition and MIC independent PBP inhibition, the development of Ldt-specific inhibitors is of considerable interest. Carbapenems and penems are known for their ability to inhibit both Ldts and PBPs.( 55 – 58 ) Correspondingly, we observed potent inhibition of both classes of transpeptidases, particularly by imipenem in the fluorescence based Msm cell assays. While faropenem has been identified as the most potent inhibitor of Ldts in recombinant enzyme assays,( 18 , 59 , 60 ) our findings indicate lower inhibitory potency of faropenem against the Ldts in live Msm cells, in comparison to the tested carbapenems. Interestingly, the penams ampicillin and amoxicillin exhibit significant inhibition of Msm in the presence of clavulanic acid, correlating with PBP transpeptidase inhibition, but showing much less inhibition of Ldts. While we observed that 21 and 25 appear to inhibit both Ldts and PBPs in Msm , 31 appears to operate via an alternative mechanism of action. Notably, all three compounds manifested similar levels of synergistic or additive inhibition when combined with faropenem or meropenem. Further research is warranted to elucidate the mechanism behind the benefit of dual treatment of β-lactams and 31 , while in the cases of 21 and 25 the increased inhibition of both Ldts and PBPs in the presence of β-lactams is likely to play a role. However, both 21 and 25 contain a reactive electrophilic group, and may act as relatively non-specific inhibitors. Though the cellular activity of sulfonyl pyridine 31 could not be correlated to the extent of inhibition of PBPs or Ldts, 31 displayed unexpectedly potent activity against Msm , likely involving another or an additional mechanism of action. Inhibitory studies with derivatives of 31 suggest that the nitropyridine sulfonyl moiety is critical for its inhibitory activity against Msm , as removal of the nitro group or substitution with other electron withdrawing groups, as well as substitution of the pyridine ring for a phenyl ring, abolished activity. Reactivity with nucleophilic cysteine residues (as evidenced by interactions with Ldt Mt2 and GSH) did not correlate with activity against Msm . Therefore, identification of the mechanism of action of the sulfonyl pyridine compounds is of interest. Following on the observation that Ldt Mt2 inhibitors were able to inhibit both Ldts and PBPs in Msm , we assessed the biochemical inhibition of the essential PBP3 of Mtb . Interestingly, the extent of inhibition of PBP3 by the Ldt Mt2 inhibitors was limited. In contrast, most tested β-lactams demonstrated potent inhibition of Mtb PBP3. An exception was aztreonam, which has been shown to interact with Mtb PBP3 in crystallographic studies.( 61 ) Aztreonam is known as a potent broad spectrum inhibitor of Gram-negative bacteria, but is less effective against Gram-positive bacteria.( 53 ) Mtb , as an acid-fast bacillus, falls in neither these categories, though they can be considered a subclass of Gram-positive bacteria.( 62 ) Overall, using the current assays we found no clear correlation between cellular inhibition of the PBPs and Ldts of Msm and the inhibition of recombinant Mtb PBP3 and Ldt Mt2 , though all cellularly active inhibitors were also inhibitors of isolated Mtb PBP3, with the exception of 25 . Our studies utilised Msm as a validated model organism for Mtb .( 33 ) LdtC is reported to be the main functional Ldt of Msm ,( 63 ) as well as the main contributor to TetraRh incorporation.( 30 ) LdtC is a closer homologue of Mtb Ldt Mt5 than Ldt Mt2 , the latter of which is the main Ldt of Mtb , which is a close homologue of Msm LdtB.( 60 ) Superimposition of a Ldt Mt2 structure with a structural prediction model of LdtC created with AlphaFold,( 64 ) manifests high structural similarity of the two active site regions (Cα RMSD 0.75 Å) and of key residues (Figure S10A). PBP3 from Msm and Mtb share ∼79% sequence similarity, and manifest structural similarity of their active site domains (RMSD 0.48 Å; Figure S10B).( 61 ) It has yet to be determined which PBPs in Msm contribute to the incorporation of PentaFI into the cell wall. However, considering the nature of the assay, it can be inferred likely that only PBPs with D,D- transpeptidase activity are involved, and not those with D,D-carboxypeptidase activity. The primary β-lactamase of Msm , BlaS, has ∼40% sequence similarity and high structural homology with BlaC, the major β-lactamase of Mtb , and exhibits particularly efficient penicillinase and cephalosporinase activity.( 36 ) Similarly to BlaC, BlaS can be inhibited by clavulanic acid.( 36 ) However, Msm also expresses an additional cephalosporinase, BlaE, which is less sensitive to clavulanic acid inhibition,( 36 ) knowledge consistent with the resistance to several cephalosporins (e.g. ceftazidime and ceftriaxone) observed in our experiments, despite the presence of clavulanic acid. Conclusion Certain β-lactams exhibit promising activity against Mycobacterium spp. though optimisation will likely be required to obtain clinically relevant potency. Relevant targets for β-lactams in Mycobacterium spp . include the PBPs and the Ldts, with emerging evidence suggesting that dual inhibition of these targets may be essential for optimised antibacterial efficacy, via transpeptidase inhibition. In living Msm cells, β-lactams (particularly the carbapenems, penems, and penams) are potent inhibitors of PBPs with transpeptidase activity, reinforcing their potential as therapeutic options. Notably, our results reveal a strong correlation between Ldt inhibition and MIC values, implying the critical role of Ldt inhibition in the inhibition of Msm . In addition, we identified the sulfonyl pyridines as promising inhibitors of Msm , though they could not be related to inhibition of transpeptidase activity of Ldts or PBPs. Overall, our results suggest that future efforts to optimise β-lactams, alongside non-β-lactam inhibitors of transpeptidases, that focus on enhancing potency against Ldts, may lead to effective inhibition of Mycobacterium spp. Methods Materials S2d and 2-(6-(((2,4-dinitrophenyl)sulfonyl)oxy)-3-oxo-3H-xanthen-9-yl)benzoic acid (Probe 1) were synthesised as described.( 65 – 67 ) 18 - 31 were obtained from the GSK HTS compound library. 32 , 36 , and 38 - 42 were from Cortex Organics, 37 and Fmoc-D-iGln-OH were from AmBeed, 33 - 35 and 46 were from Key Organics, 44 and 45 were purchased from Enamine, and 5( 6 )-TAMRA was from MedChemExpress. Faropenem was purchased from Fluorochem Ltd, meropenem was from Glentham Life Sciences, ceftazidime was purchased from TOKU- E, ceftriaxone and aztreonam were from Molekula Ltd, ampicillin was from Apollo Scientific, and ampicillin was from Alfa Aesar. Isoniazid, ethambutol, and rifampicin were from Cambridge Bioscience Ltd; all other compounds were purchased from Merck. Synthesis of fluorescent peptides PentaFI (D-Ala-D-Ala-L-Lys-D-iGln-L-Ala-Fluorescein) and TetraRh (D-Ala-L-Lys-D-iGln-L-Ala-Rhodamine) were synthesised by solid phase peptide synthesis using a Liberty Blue Automated Microwave Peptide Synthesiser, following stepwise coupling reactions to D-Alanine-Wang resin (715 mg, 0.5 mmol). The respective amino acids D-Ala (1.5 mmol, 3 eq.), L-Lys (2.5 mmol, 5 eq.), D-iGln (2.5 mmol, 5 eq.), and L-Ala (2.5 mmol, 5 eq.), were coupled using the standard procedure with N,N-diisopropylethylamine (DIPEA) in ethyl cyanohydroxyiminoacetate (Oxyma, 1 M; DIPEA/Oxyma 1.5 % ( v / v )) and 7.8% ( v / v ) N,N′-diisopropylcarbodiimide (DIC) in DMF. After each coupling step, the resin was washed with DMF and the Fmoc group was deprotected with 20% ( v / v ) piperidine in DMF using the standard deprotection procedure. The fluorescent tags 5( 6 )-carboxyfluorescein (2 eq) and 5( 6 )-TAMRA (2 eq) were coupled manually, using hexafluorophosphate benzotriazole tetramethyl uronium (HBTU; 2 eq) and DIPEA (6 eq). The resin bound TetraRh and PentaFI were washed with dichloromethane (DCM), methanol and DCM, and treated with a 5 mL solution of 2:1 TFA/DCM (2 h, rt). Solvents were removed by evaporation in vacuo and purified by HPLC on a C 18 column (5 µm, 10 x 150 mm; SunFire, Waters) at a flowrate of 3 mL/min with a gradient of 98% ( v / v ) buffer A (0.1% ( v / v ) formic acid in H 2 O) and 2% buffer B (0.1% ( v / v ) formic acid in acetonitrile) to 50% buffer A and 50% buffer B over 22 min. TetraRh and PentaFI eluted after 18.8 min and 22.1 min, respectively. TetraRh and PentaFI were characterised by 1 H as well as two-dimensional NMR experiments including 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13 C-HMBC (Figure S12-13, Table S6-7). Recombinant protein production and purification Ldt Mt2 was produced in Escherichia coli and purified (>95% purity by SDS-PAGE analysis) as previously described.( 68 ) A codon-optimised synthetic gene (GeneArt, Thermo Fisher Scientific) encoding for Mtb PBP3 Δ1-122 was amplified and cloned into the expression vector pCold using Sal1-HF (New England BioLabs) and Not1-HF (New England BioLabs) digestion and ligation using T4 DNA ligase (New England BioLabs) according to the manufacturer’s protocol. The ampicillin resistance gene of the vector was exchanged for the kanamycin resistance gene using Gibson Assembly.( 69 ) A culture of E. coli BL21(DE3) pCold-PBP3 Δ1-122 was grown at 37 °C at 180 rpm in 2xYT media (with 50 µg/mL kanamycin) to OD 600 of 0.6. Isopropyl β-D-thiogalactopyranoside (IPTG) (0.5 mM) was then added and the culture was incubated at 15 °C at 180 rpm for an additional 16 h. Cells were collected by centrifugation (11,000 x g, 8 min), and stored at −80 °C. The cell pellet was resuspended in HisTrap Buffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole) in the presence of DNase I, and lysed using a Continuous Flow Cell Disruptor (Constant Systems, 25 kpsi). The lysates were centrifuged (32,000 x g, 20 min), passed through a 0.45 µm filter, and loaded onto a 5 mL HisTrap column (GE Life Sciences) that had been pre-equilibrated in HisTrap Buffer A. The column was washed with HisTrap Buffer A, followed by a gradient running from 0 % to 100 % ( v/v ) HisTrap Buffer B (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole). Fractions containing PBP3 (as observed by SDS- PAGE) were combined, the buffer was exchanged to HisTrap Buffer A, and the HisTag was cleaved using recombinant 3C protease at 4 °C, over 12 h. The HisTag cleaved PBP3 was passed through a 5 mL HisTrap column (GE Life Sciences) and subsequently loaded onto a 300 mL Superdex 75 column (GE Life Sciences) pre-equilibrated in gel filtration buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl), and eluted over 1 column volume in gel filtration buffer. The identity and purity of PBP3 was confirmed by mass spectrometry (calculated mass 60148 Da, observed deconvoluted mass 60152 Da) and SDS-PAGE (>95% purity). Bacterial strain and growth conditions All experiments using Msm refer to strain ATCC 607. Msm was grown on Columbia blood agar supplemented with 5% sheep blood at 37 °C. Liquid cultures of Msm were grown in Middlebrook 7H9 broth supplemented with 0.1% ( v / v ) Tween-80, 0.5 % ( w / v ) bovine albumin fraction V, 0.2% ( w / v ) dextrose, and 0.3 % ( v / v ) catalase (beef). Susceptibility testing Antibiotic susceptibility was assessed using the broth microdilution method for MIC determination. The effect of two combined inhibitors was determined using a checkerboard assay.( 70 ) A liquid culture of Msm was incubated at 37 °C with aeration for 48 h, after which the culture had reached the stationary phase (Figure S11), and subsequently diluted in media containing diverse concentrations of the compound of interest (final concentrations of 0.25 - 128 µg/mL, total volume 200 µL) to OD 600 0.05 in a sterile 96-well plate (Corning Costar, flat-bottom, cell culture-treated). Positive controls consisted of samples containing only Msm culture in media and negative controls omitted Msm inoculation and contained only media. In line with reported procedures for Msm MIC assays,( 33 , 46 , 71 ) we applied an incubation period of 4 days to all MIC experiments. Then, resazurin (20 µL of a 150 µg/mL stock solution) was added to all wells, and the samples were incubated at 37 °C for 12 h. The MIC was defined as the lowest concentration at which no resazurin colour change was observed. All assays were performed in triplicate. Fluorescent peptide incorporation assays A 5 mL culture of Msm was grown at 37 °C to an OD 600 of 0.8. This culture (196 µL) was added to a sterile 96-well plate (Corning Costar, flat-bottom, cell culture-treated). To this was added either TetraRh (2 µL, to a final concentration of 0.5 µM) or PentaFI (2 µL, to a final concentration of 5 µM), and varying concentrations of the compound of interest (2 µL, final concentrations ranging from 0.5 µg/mL to 256 µg/mL). A positive control (no inhibitor) and a negative control (100 µg/mL meropenem and 100 µg/mL clavulanic acid) were included. This was incubated at 37 °C for 24 h. Samples were then washed in PBS (200 µL, 3x), and fixed with 2% ( v/v ) in PBS for 30 min. The Msm cells were washed with PBS (200 µL, 3x), then resuspended in 200 µL PBS. The fluorescence of the Msm cells was analysed using an LSRFortessa™ X-20 (BD Biosciences). Cells treated with TetraRh were assessed using a 561 nm laser and a 586/15 bandpass filter. Cells treated with PentaFI were assessed using a 488 nm laser and a 530/30 bandpass filter. For each dataset 10,000 events were counted. Data were analysed using FlowJo™ Software. All assays were performed in duplicate. Biochemical inhibition assays Ldt Mt2 dose-response assays were performed as described.( 65 ) In brief, Ldt Mt2 (100 nM) was incubated with varying concentrations of the compound of interest (final concentrations ranging between 400 µM and 20.3 nM) for 10 min in the assay buffer (50 mM HEPES, pH 7.2, 0.01% ( v / v ) Triton X-100) and then assayed using Probe 1 (25 µM). The ‘intrinsic’ thiol reactivity ( k chem ) was determined as described.( 31 ) In brief, L-glutathione (500 nM) was incubated with varying concentrations of the compound of interest (final concentrations ranging between 400 µM and 20.3 nM) and Probe 1 (10 µM) for 15 h in assay buffer (50 mM HEPES, pH 7.2, 0.01% ( v / v ) Triton X-100). The second-order rate constant of covalent target inactivation ( k inact /K I ) of Ldt Mt2 was determined as described.( 31 ) In brief, Ldt Mt2 (100 nM) was incubated with varying concentrations of the compound of interest (final concentrations ranging between 400 µM and 20.3 nM) and Probe 1 (10 µM) for 3.5 h in assay buffer (50 mM HEPES, pH 7.2, 0.01% ( v / v ) Triton X-100). Dose-response assays of PBP3 were assessed using the fluorometric S2d assay,( 72 – 74 ) applying the procedure optimised for P. aeruginosa PBP3.( 75 ) PBP3 (300 nM) in assay buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 0.01% ( v / v ) Triton X-100) was incubated with varying concentrations of the compound of interest (final concentrations ranging between 400 µM and 20.3 nM) for 10 minutes in a black polystyrene, flat-bottomed 384-well μ-clear plate (clear bottomed, Greiner Bio-One, part number 781096). Then, a mixture containing S2d (1.5 mM), monobromobimane (mBBr; 0.05 mM), and D-Ala (1 mM) in assay buffer was added (final volume 25 µL). The fluorescence signal was measured using a BMG Labtech CLARIOstar instrument with λ ex =394 nm and λ em =490 nm. Data were analysed using Prism (GraphPad). Mass spectrometry assays Protein-observed SPE-MS experiments with Ldt Mt2 were performed as described.( 31 ) In brief, Ldt Mt2 (1 μM) in 50 mM tris, pH 7.5 was incubated with an inhibitor (100 µM) at room temperature. Mass spectrometry was performed using a RapidFire200 integrated autosampler/solid phase extraction (SPE) system (Agilent Technologies) employing a C4 cartridge (Agilent Technologies), coupled to an API40000 triple quadrupole mass spectrometer (Applied Biosystems) operating in the positive ionisation mode. The mass spectrometer parameters were: capillary voltage (2000 V), nozzle voltage (1500 V), fragmentor voltage (150 V), gas temperature (225 °C), gas flow (13 L/min), sheath gas temperature (300 °C), sheath gas flow (12 L/min). Author contributions M.d.M., K.C.T., and C.J.S conceived the experiments; M.d.M. carried out experiments, with assistance from K.C.T., and P.R.; M.d.M. analysed the data; M.d.M. drafted the manuscript with C.J.S, with input from all authors. Competing interests The authors have no competing interests to declare. Acknowledgements The project was co-funded by the Tres Cantos Open Lab Foundation (Project TC297). It was also supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) [BB/M011224/1] and the Wellcome Trust (106244/Z/14/Z). P.R. thanks the Wellcome Trust (227298/Z/23/Z). We thank Robert Hedley and Vasiliki Tsioligka for providing technical assistance in the analysis of the fluorescent peptide incorporation assays at The Don Mason Facility of Flow Cytometry, Sir William Dunn School of Pathology, University of Oxford. We thank Alistair Farley, University of Oxford, for helpful discussions on the selection of sulfonyl pyridines. References 1. ↵ Bush K , Bradford PA . 2016 . β-Lactams and β-Lactamase Inhibitors: An Overview . Cold Spring Harb Perspect Med 6 . 2. ↵ Georgopapadakou NH , Liu FY . 1980 . Penicillin-binding proteins in bacteria . Antimicrob Agents Chemother 18 : 148 – 57 . OpenUrl Abstract / FREE Full Text 3. ↵ Sauvage E , Kerff F , Terrak M , Ayala JA , Charlier P . 2008 . The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis . FEMS Microbiol Rev 32 : 234 – 58 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Anonymous . 2022 . Global tuberculosis report 2022 .. Geneva : World Health Organization , 5. ↵ Wivagg CN , Bhattacharyya RP , Hung DT . 2014 . Mechanisms of β-lactam killing and resistance in the context of Mycobacterium tuberculosis . J Antibiot 67 : 645 – 654 . OpenUrl CrossRef PubMed 6. ↵ Hugonnet J-E , Tremblay LW , Boshoff HI , Barry CE , Blanchard JS . 2009 . Meropenem-Clavulanate Is Effective Against Extensively Drug-Resistant Mycobacterium tuberculosis . Science 323 : 1215 – 1218 . OpenUrl Abstract / FREE Full Text 7. ↵ Gun MA , Bozdogan B , Coban AY . 2020 . Tuberculosis and beta-lactam antibiotics . Future Microbiol 15 : 937 – 944 . OpenUrl CrossRef PubMed 8. Story-Roller E , Lamichhane G . 2018 . Have we realized the full potential of β-lactams for treating drug-resistant TB? IUBMB Life 70 : 881 – 888 . OpenUrl CrossRef PubMed 9. ↵ Jaganath D , Lamichhane G , Shah M . 2016 . Carbapenems against Mycobacterium tuberculosis : a review of the evidence . Int J Tuberc Lung Dis 20 : 1436 – 1447 . OpenUrl CrossRef PubMed 10. ↵ Gold B , Smith R , Nguyen Q , Roberts J , Ling Y , Lopez Quezada L , Somersan S , Warrier T , Little D , Pingle M . 2016 . Novel cephalosporins selectively active on nonreplicating Mycobacterium tuberculosis . J Med Chem 59 : 6027 – 6044 . OpenUrl CrossRef PubMed 11. Lopez Quezada L , Li K , McDonald SL , Nguyen Q , Perkowski AJ , Pharr CW , Gold B , Roberts J , McAulay K , Saito K , Somersan Karakaya S , Javidnia PE , Porras De Francisco E , Amieva MM , DíAz SP , Mendoza Losana A , Zimmerman M , Liang H-PH , Zhang J , Dartois V , Sans S , Lagrange S , Goullieux L , Roubert C , Nathan C , Aubé J. 2019 . Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis . ACS Infectious Diseases 5 : 1433 – 1445 . OpenUrl CrossRef PubMed 12. ↵ Gold B , Zhang J , Quezada LL , Roberts J , Ling Y , Wood M , Shinwari W , Goullieux L , Roubert C , Fraisse L , Bacqué E , Lagrange S , Filoche-Rommé B , Vieth M , Hipskind PA , Jungheim LN , Aubé J , Scarry SM , McDonald SL , Li K , Perkowski A , Nguyen Q , Dartois V , Zimmerman M , Olsen DB , Young K , Bonnett S , Joerss D , Parish T , Boshoff HI , Arora K , Barry CE , III , Guijarro L , Anca S , Rullas J , Rodríguez-Salguero B , Martínez-Martínez MS , Porras-De Francisco E , Cacho M , Barros-Aguirre D , Smith P , Berthel SJ , Nathan C , Bates RH . 2022 . Identification of β-Lactams Active against Mycobacterium tuberculosis by a Consortium of Pharmaceutical Companies and Academic Institutions . ACS Infect Dis 8 : 557 – 573 . OpenUrl CrossRef PubMed 13. ↵ Lavollay M , Arthur M , Fourgeaud M , Dubost L , Marie A , Veziris N , Blanot D , Gutmann L , Mainardi JL . 2008 . The Peptidoglycan of Stationary-Phase Mycobacterium tuberculosis Predominantly Contains Cross-Links Generated by L,D-Transpeptidation . J Bacteriol 190 : 4360 – 4366 . OpenUrl Abstract / FREE Full Text 14. ↵ Wietzerbin J , Das BC , Petit JF , Lederer E , Leyh-Bouille M , Ghuysen JM . 1974 . Occurrence of D-alanyl-(D)-meso-diaminopimelic acid and meso-diaminopimelyl-meso-diaminopimelic acid interpeptide linkages in the peptidoglycan of Mycobacteria . Biochemistry 13 : 3471 – 6 . OpenUrl CrossRef PubMed Web of Science 15. ↵ Gupta R , Lavollay M , Mainardi J-L , Arthur M , Bishai WR , Lamichhane G . 2010 . The Mycobacterium tuberculosis protein Ldt Mt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin . Nat Med 16 : 466 – 469 . OpenUrl CrossRef PubMed 16. ↵ Baranowski C , Welsh MA , Sham LT , Eskandarian HA , Lim HC , Kieser KJ , Wagner JC , McKinney JD , Fantner GE , Ioerger TR , Walker S , Bernhardt TG , Rubin EJ , Rego EH . 2018 . Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape . Elife 7 . 17. ↵ Kumar P , Kaushik A , Lloyd EP , Li S-G , Mattoo R , Ammerman NC , Bell DT , Perryman AL , Zandi TA , Ekins S , Ginell SL , Townsend CA , Freundlich JS , Lamichhane G . 2017 . Non-classical transpeptidases yield insight into new antibacterials . Nat Chem Biol 13 : 54 – 61 . OpenUrl CrossRef PubMed 18. ↵ Steiner EM , Schneider G , Schnell R . 2017 . Binding and processing of β-lactam antibiotics by the transpeptidase Ldt Mt2 from Mycobacterium tuberculosis . FEBS J 284 : 725 – 741 . OpenUrl CrossRef PubMed 19. Cordillot M , Dubée V , Triboulet S , Dubost L , Marie A , Hugonnet J-E , Arthur M , Mainardi J-L . 2013 . In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D- transpeptidases and inactivation of these enzymes by carbapenems . Antimicrob Agents Chemother 57 : 5940 – 5945 . OpenUrl Abstract / FREE Full Text 20. ↵ Triboulet S , Arthur M , Mainardi JL , Veckerlé C , Dubée V , Nguekam Moumi A , Gutmann L , Rice LB , Hugonnet JE . 2011 . Inactivation Kinetics of a New Target of β-Lactam Antibiotics . J Biol Chem 286 : 22777 – 22784 . OpenUrl Abstract / FREE Full Text 21. ↵ Cole ST , Brosch R , Parkhill J , Garnier T , Churcher C , Harris D , Gordon SV , Eiglmeier K , Gas S , Barry CE , Tekaia F , Badcock K , Basham D , Brown D , Chillingworth T , Connor R , Davies R , Devlin K , Feltwell T , Gentles S , Hamlin N , Holroyd S , Hornsby T , Jagels K , Krogh A , McLean J , Moule S , Murphy L , Oliver K , Osborne J , Quail MA , Rajandream MA , Rogers J , Rutter S , Seeger K , Skelton J , Squares R , Squares S , Sulston JE , Taylor K , Whitehead S , Barrell BG . 1998 . Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence . Nature 393 : 537 – 544 . OpenUrl CrossRef PubMed Web of Science 22. ↵ Gupta R , Lavollay M , Mainardi JL , Arthur M , Bishai WR , Lamichhane G . 2010 . The Mycobacterium tuberculosis protein Ldt Mt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin . Nat Med 16 : 466 – 9 . OpenUrl CrossRef PubMed 23. ↵ Botta GA , Park JT . 1981 . Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation . J bacteriol 145 : 333 – 340 . OpenUrl Abstract / FREE Full Text 24. ↵ DeJesus MA , Gerrick ER , Xu W , Park SW , Long JE , Boutte CC , Rubin EJ , Schnappinger D , Ehrt S , Fortune SM , Sassetti CM , Ioerger TR . 2017 . Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis . mBio 8 : doi: 10.1128/mbio.02133-16 . OpenUrl CrossRef 25. ↵ Gonzalo X , Drobniewski F . 2013 . Is there a place for β-lactams in the treatment of multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between meropenem and amoxicillin/clavulanate . J Antimicrob Chemother 68 : 366 – 9 . OpenUrl CrossRef PubMed 26. ↵ Diacon AH , van der Merwe L , Barnard M , von Groote-Bidlingmaier F , Lange C , García- Basteiro AL , Sevene E , Ballell L , Barros-Aguirre D. 2016 . β-Lactams against Tuberculosis — New Trick for an Old Dog? N Engl J Med 375 : 393 – 394 . OpenUrl CrossRef PubMed 27. Sotgiu G , D’Ambrosio L , Centis R , Tiberi S , Esposito S , Dore S , Spanevello A , Migliori GB . 2016 . Carbapenems to Treat Multidrug and Extensively Drug-Resistant Tuberculosis: A Systematic Review . Int J Mol Sci 17 : 373 . OpenUrl CrossRef PubMed 28. ↵ Tiberi S , Sotgiu G , D’Ambrosio L , Centis R , Abdo Arbex M , Alarcon Arrascue E , Alffenaar JW , Caminero JA , Gaga M , Gualano G , Skrahina A , Solovic I , Sulis G , Tadolini M , Alarcon Guizado V , De Lorenzo S , Roby Arias AJ , Scardigli A , Akkerman OW , Aleksa A , Artsukevich J , Auchynka V , Bonini EH , Chong Marín FA , Collahuazo López L , de Vries G , Dore S , Kunst H , Matteelli A , Moschos C , Palmieri F , Papavasileiou A , Payen MC , Piana A , Spanevello A , Vargas Vasquez D , Viggiani P , White V , Zumla A , Migliori GB. 2016 . Comparison of effectiveness and safety of imipenem/clavulanate-versus meropenem/clavulanate-containing regimens in the treatment of MDR- and XDR-TB . Eur Respir J 47 : 1758 – 66 . OpenUrl Abstract / FREE Full Text 29. ↵ Levine SR , Beatty KE . 2021 . Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes . ACS Infect Dis 7 : 461 – 470 . OpenUrl CrossRef PubMed 30. ↵ Pidgeon SE , Apostolos AJ , Nelson JM , Shaku M , Rimal B , Islam MN , Crick DC , Kim SJ , Pavelka MS , Kana BD , Pires MM . 2019 . L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking . ACS Chem Biol 14 : 2185 – 2196 . OpenUrl CrossRef PubMed 31. ↵ de Munnik M , Lang PA , De Dios Antos F , Cacho M , Bates RH , Brem J , Rodríguez-Miquel B , Schofield CJ. 2023 . High-Throughput Screen with the L,D-transpeptidase Ldt Mt2 of Mycobacterium tuberculosis Reveals Novel Classes of Covalently Reacting Inhibitors Chem Sci 14 : 7262 – 7278 . OpenUrl CrossRef PubMed 32. ↵ de Munnik M , Lithgow J , Brewitz L , Christensen KE , Bates RH , Rodriguez-Miquel B , Schofield CJ. 2023 . αβ,α’β’-Diepoxyketones are mechanism-based inhibitors of nucleophilic cysteine enzymes . Chem Commun (Camb ) 59 : 12859 – 12862 . OpenUrl CrossRef PubMed 33. ↵ Lelovic N , Mitachi K , Yang J , Lemieux MR , Ji Y , Kurosu M . 2020 . Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis . J Antibiot 73 : 780 – 789 . OpenUrl CrossRef PubMed 34. ↵ Yabu K , Ochiai T , Kaneda S . 1984 . Penicillin-binding proteins in Mycobacterium smegmatis . FEMS Microbiol Lett 25 : 307 – 310 . OpenUrl CrossRef 35. Sanders AN , Wright LF , Pavelka MS . 2014 . Genetic characterization of mycobacterial L , D-transpeptidases. Microbiology 160 : 1795 – 1806 . OpenUrl PubMed 36. ↵ Flores AR , Parsons LM , Pavelka MS . 2005 . Genetic analysis of the beta-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics. Microbiology (Reading , Engl ) 151 : 521 – 532 . OpenUrl 37. ↵ Tremblay LW , Fan F , Blanchard JS . 2010 . Biochemical and structural characterization of Mycobacterium tuberculosis beta-lactamase with the carbapenems ertapenem and doripenem . Biochemistry 49 : 3766 – 73 . OpenUrl CrossRef PubMed Web of Science 38. ↵ Hazra S , Xu H , Blanchard JS . 2014 . Tebipenem, a New Carbapenem Antibiotic, Is a Slow Substrate That Inhibits the β-Lactamase from Mycobacterium tuberculosis . Biochemistry 53 : 3671 – 3678 . OpenUrl CrossRef PubMed 39. ↵ Reading C , Cole M . 1977 . Clavulanic acid: a beta-lactamase-inhiting beta-lactam from Streptomyces clavuligerus . Antimicrob Agents Chemother 11 : 852 – 7 . OpenUrl Abstract / FREE Full Text 40. ↵ Flores AR , Parsons LM , Pavelka MS , Jr . . 2005 . Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics . J Bacteriol 187 : 1892 – 900 . OpenUrl Abstract / FREE Full Text 41. ↵ Aronoff SC , Jacobs MR , Johenning S , Yamabe S . 1984 . Comparative activities of the beta-lactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin . Antimicrob Agents Chemother 26 : 580 – 2 . OpenUrl Abstract / FREE Full Text 42. ↵ English AR , Retsema JA , Girard AE , Lynch JE , Barth WE . 1978 . CP-45,899, a beta-lactamase inhibitor that extends the antibacterial spectrum of beta-lactams: initial bacteriological characterization . Antimicrob Agents Chemother 14 : 414 – 9 . OpenUrl Abstract / FREE Full Text 43. ↵ Weiss William J , Petersen Peter J , Murphy Timothy M , Tardio L , Yang Y , Bradford Patricia A , Venkatesan Aranapakam M , Abe T , Isoda T , Mihira A , Ushirogochi H , Takasake T , Projan S , O’Connell J , Mansour Tarek S . 2004 . In Vitro and In Vivo Activities of Novel 6-Methylidene Penems as β-Lactamase Inhibitors . Antimicrobial Agents and Chemotherapy 48 : 4589 – 4596 . OpenUrl Abstract / FREE Full Text 44. ↵ Hecker SJ , Reddy KR , Lomovskaya O , Griffith DC , Rubio-Aparicio D , Nelson K , Tsivkovski R , Sun D , Sabet M , Tarazi Z , Parkinson J , Totrov M , Boyer SH , Glinka TW , Pemberton OA , Chen Y , Dudley MN . 2020 . Discovery of Cyclic Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of Serine and Metallo-β-lactamases . J Med Chem 63 : 7491 – 7507 . OpenUrl CrossRef PubMed 45. ↵ Ehmann DE , Jahić H , Ross PL , Gu RF , Hu J , Kern G , Walkup GK , Fisher SL . 2012 . Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor . Proc Natl Acad Sci USA 109 : 11663 – 8 . OpenUrl Abstract / FREE Full Text 46. ↵ Agrawal P , Miryala S , Varshney U . 2015 . Use of Mycobacterium smegmatis Deficient in ADP-Ribosyltransferase as Surrogate for Mycobacterium tuberculosis in Drug Testing and Mutation Analysis . PloS one 10 : e0122076 . OpenUrl CrossRef PubMed 47. ↵ CLSI . 2023 . Performance Standards for Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes . CLSI supplement M24S ., 2nd ed . Clinical and Laboratory Standards Institute . 48. ↵ EUCAST . The European Committee on Antimicrobial Susceptibility Testing . Breakpoint tables for interpretation of MICs and zone diameters . Version 15.0, 2025 . https://www.eucast.org . Accessed January 2025 . 49. ↵ van Vuuren S , Viljoen A. 2011 . Plant-based antimicrobial studies--methods and approaches to study the interaction between natural products . Planta Med 77 : 1168 – 82 . OpenUrl CrossRef PubMed 50. ↵ Plocinska R , Martinez L , Gorla P , Pandeeti E , Sarva K , Blaszczyk E , Dziadek J , Madiraju MV , Rajagopalan M . 2014 . Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities . J Bacteriol 196 : 4120 – 4129 . OpenUrl Abstract / FREE Full Text 51. ↵ Datta P , Dasgupta A , Singh AK , Mukherjee P , Kundu M , Basu J . 2006 . Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls cell septation and mediates the formation of a trimeric complex involving FtsZ, FtsW and PBP3 in mycobacteria . Mol Microbiol 62 : 1655 – 73 . OpenUrl CrossRef PubMed 52. ↵ Hayes MV , Orr DC . 1983 . Mode of action of ceftazidime: affinity for the penicillin-binding proteins of Escherichia coli K12, Pseudomonas aeruginosa and Staphylococcus aureus . J Antimicrob Chemother 12 : 119 – 26 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Georgopapadakou NH , Smith SA , Sykes RB . 1982 . Mode of action of azthreonam . Antimicrob Agents Chemother 21 : 950 – 6 . OpenUrl Abstract / FREE Full Text 54. ↵ Sotgiu G , Centis R , D’Ambrosio L , Migliori GB . 2015 . Tuberculosis Treatment and Drug Regimens . Cold Spring Harb Perspect Med 5 : a017822 . OpenUrl Abstract / FREE Full Text 55. ↵ Chambers HF , Moreau D , Yajko D , Miick C , Wagner C , Hackbarth C , Kocagöz S , Rosenberg E , Hadley WK , Nikaido H . 1995 . Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis? Antimicrob Agents Chemother 39 : 2620 – 4 . OpenUrl Abstract / FREE Full Text 56. Kim HS , Kim J , Im HN , Yoon JY , An DR , Yoon HJ , Kim JY , Min HK , Kim S-J , Lee JY . 2013 . Structural basis for the inhibition of Mycobacterium tuberculosis L,D- transpeptidase by meropenem, a drug effective against extensively drug-resistant strains . Acta Crystallogr D 69 : 420 – 431 . OpenUrl CrossRef PubMed 57. Dubée V , Triboulet S , Mainardi JL , Ethève-Quelquejeu M , Gutmann L , Marie A , Dubost L , Hugonnet JE , Arthur M . 2012 . Inactivation of Mycobacterium tuberculosis L,D- transpeptidase LdtMt₁ by carbapenems and cephalosporins . Antimicrob Agents Chemother 56 : 4189 – 95 . OpenUrl Abstract / FREE Full Text 58. ↵ Kumar G , Galanis C , Batchelder HR , Townsend CA , Lamichhane G. 2022 . Penicillin Binding Proteins and β-Lactamases of Mycobacterium tuberculosis : Reexamination of the Historical Paradigm . mSphere 7 : e0003922 . OpenUrl CrossRef PubMed 59. ↵ Dhar N , Dubée V , Ballell L , Cuinet G , Hugonnet JE , Signorino-Gelo F , Barros D , Arthur M , McKinney JD . 2015 . Rapid cytolysis of Mycobacterium tuberculosis by faropenem, an orally bioavailable β-lactam antibiotic . Antimicrob Agents Chemother 59 : 1308 – 19 . OpenUrl Abstract / FREE Full Text 60. ↵ Zandi TA , Marshburn RL , Stateler PK , Brammer Basta LA . 2019 . Phylogenetic and Biochemical Analyses of Mycobacterial L,D-Transpeptidases Reveal a Distinct Enzyme Class That Is Preferentially Acylated by Meropenem . ACS Infect Dis 5 : 2047 – 2054 . OpenUrl CrossRef PubMed 61. ↵ Lu Z , Wang H , Zhang A , Liu X , Zhou W , Yang C , Guddat L , Yang H , Schofield CJ , Rao Z . 2020 . Structures of Mycobacterium tuberculosis Penicillin-Binding Protein 3 in Complex with Five β-Lactam Antibiotics Reveal Mechanism of Inactivation . Mol Pharmacol 97 : 287 – 294 . OpenUrl Abstract / FREE Full Text 62. ↵ Bishop PJ , Neumann G . 1970 . The history of the Ziehl-Neelsen stain . Tubercle 51 : 196 – 206 . OpenUrl CrossRef PubMed 63. ↵ Francis ZK , Sanders AN , Crick DC , Mahapatra S , Islam MN , Pavelka MS . 2023 . LdtC Is a Key L,D-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis . J Bacteriol 205 : e00424 – 22 . OpenUrl PubMed 64. ↵ Jumper J , Evans R , Pritzel A , Green T , Figurnov M , Ronneberger O , Tunyasuvunakool K , Bates R , Žídek A , Potapenko A , Bridgland A , Meyer C , Kohl SAA , Ballard AJ , Cowie A , Romera-Paredes B , Nikolov S , Jain R , Adler J , Back T , Petersen S , Reiman D , Clancy E , Zielinski M , Steinegger M , Pacholska M , Berghammer T , Bodenstein S , Silver D , Vinyals O , Senior AW , Kavukcuoglu K , Kohli P , Hassabis D. 2021 . Highly accurate protein structure prediction with AlphaFold . Nature 596 : 583 – 589 . OpenUrl CrossRef PubMed 65. ↵ de Munnik M , Lohans CT , Langley GW , Bon C , Brem J , Schofield CJ. 2020 . A Fluorescence-Based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase Ldt Mt2 . ChemBioChem 21 : 368 – 372 . OpenUrl CrossRef PubMed 66. Adam M , Damblon C , Plaitin B , Christiaens L , Frere JM . 1990 . Chromogenic depsipeptide substrates for beta-lactamases and penicillin-sensitive DD-peptidases . Biochem J 270 : 525 . OpenUrl Abstract / FREE Full Text 67. ↵ Schwyzer R . 1954 . Preparation of thio esters of amino acids and of peptides Helvetica Chimica Acta 37 : 647 – 649 . 68. ↵ Lohans CT , Chan HTH , Malla TR , Kumar K , Kamps JJAG , McArdle DJB , Van Groesen E , de Munnik M , Tooke CL , Spencer J , Paton RS , Brem J , Schofield CJ. 2019 . Non-Hydrolytic β-Lactam Antibiotic Fragmentation by L,D-Transpeptidases and Serine β- Lactamase Cysteine Variants . Angew Chem Int Ed 58 : 1990 – 1994 . OpenUrl CrossRef PubMed 69. ↵ Gibson DG , Young L , Chuang R-Y , Venter JC , Hutchison CA , Smith HO . 2009 . Enzymatic assembly of DNA molecules up to several hundred kilobases . Nat Methods 6 : 343 – 345 . OpenUrl CrossRef PubMed Web of Science 70. ↵ Bellio P , Fagnani L , Nazzicone L , Celenza G . 2021 . New and simplified method for drug combination studies by checkerboard assay . MethodsX 8 : 101543 . OpenUrl CrossRef PubMed 71. ↵ Piddock LJ , Williams KJ , Ricci V . 2000 . Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis . J Antimicrob Chemother 45 : 159 – 165 . OpenUrl CrossRef PubMed Web of Science 72. ↵ Adam M , Fraipont C , Rhazi N , Nguyen-Distèche M , Lakaye B , Frère JM , Devreese B , Van Beeumen J , van Heijenoort Y , van Heijenoort J , Ghuysen JM. 1997 . The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate . J Bacteriol 179 : 6005 – 9 . OpenUrl Abstract / FREE Full Text 73. Inglis SR , Zervosen A , Woon ECY , Gerards T , Teller N , Fischer DS , Luxen A , Schofield CJ . 2009 . Synthesis and Evaluation of 3-(Dihydroxyboryl)benzoic Acids as d,d- Carboxypeptidase R39 Inhibitors . J Med Chem 52 : 6097 – 6106 . OpenUrl CrossRef PubMed 74. ↵ Jamin M , Damblon C , Millier S , Hakenbeck R , Frère JM . 1993 . Penicillin-binding protein 2x of Streptococcus pneumoniae : enzymic activities and interactions with β-lactams . Biochem J 292 : 735 – 741 . OpenUrl Abstract / FREE Full Text 75. ↵ Smith HG , Basak S , Aniebok V , Beech MJ , Alshref FM , Allen MD , Farley AJM , Schofield CJ . 2024 . Structural basis of Pseudomonas aeruginosa penicillin binding protein 3 inhibition by the siderophore-antibiotic cefiderocol . Chem Sci 15 : 16928 – 16937 . OpenUrl CrossRef PubMed 76. ↵ Elion GB , Singer S , Hitchings GH . 1954 . Antagonists of Nucleic Acid Derivatives: VIII. Synergism in Combinations of Biochemically Related Antimetabolites . J Biol Chem 208 : 477 – 488 . OpenUrl FREE Full Text View the discussion thread. Back to top Previous Next Posted February 03, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis Mariska de Munnik , Karina Calvopiña , Patrick Rabe , Christopher J. Schofield bioRxiv 2025.01.28.635326; doi: https://doi.org/10.1101/2025.01.28.635326 Share This Article: Copy Citation Tools Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis Mariska de Munnik , Karina Calvopiña , Patrick Rabe , Christopher J. Schofield bioRxiv 2025.01.28.635326; doi: https://doi.org/10.1101/2025.01.28.635326 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)