we expressed mtb proteasome from the genes encoding proteasome components a ( for  chain ) and b ( for  chain ) ( prcba ) with and without the n - terminal octapeptide deleted from the  chains . the latter  open - gate mutant ( mtb20sog ) is thought to mimic a physiologic mechanism for gate opening and has a higher specific activity than the wild type without a change in substrate preference . by following mtb20sog s hydrolysis of suc - llvy-7-amido-4-methylcoumarin ( amc ) , we screened 20,000 compounds ( supplementary information ) and identified two inhibitors , 5-(5-methyl-2-(methylthio)thiophen-3-yl)-1,3,4-oxathiazol-2-one ( gl5 ) and 5-(2-methyl-3-nitrothiophen-2-yl)-1,3,4-oxathiazol-2-one ( ht1171 ) , as well as an inactive congener , 5-(5-methyl-2-(methylsulfonyl)thiophen-3-yl)-1,3,4-oxathiazol-2-one ( gl6 ) ( fig . like bortezomib , oxathiazol-2-ones were able to cross mtb s cell wall , insofar as gl5 and ht1171 inhibited proteasome activity upon treatment of mycobacterium bovis var . exposure of bcg to 25 m gl5 for 4 hours led to > 80% reduction of proteasome activity ( fig . 1c ) , while as little as ~15 min exposure to gl5 at 50 m was sufficient to reduce activity by ~50% ( fig . moreover , gl5 killed bcg alone and in synergy with sub - bacteriostatic fluxes of nitric oxide arising from the decomposition of 2 , 2-(hydroxynitrosohydrazino)-bis - ethanamine ( deta - no ) ( fig . s1 ) . gl5 and ht1171 also dose - dependently killed 1.5 - 2.5 log10 mtb over 4 days in synergy with sufficient nitric oxide to induce a pathophysiologically relevant state of bacterial non - replication ( fig . in contrast , gl5 and ht1171 showed no apparent toxicity to mammalian cells ( fig . 1f ; fig s2 ) at concentrations up to 75 m , 3000-fold greater than those at which bortezomib destroyed the epithelial cells . the oxathiazol-2-ones exerted no antibacterial activity against mycobacterium avium - intracellulare , staphylococcus aureus , salmonella enterica var . although some oxathiazol-2-ones were reported to react with thiols , those studied here did not inhibit the thiol - dependent cathepsin b ( data not shown ) . moreover , 11 of 23 oxathiazol-2-ones tested were < 5% reactive with glutathione ; the others reacted to a limited degree ( table s1 ) . thus , at a functional level , the oxathiazol-2-ones tested here appear to be relatively selective and nontoxic , although they may have additional targets . the different impact of oxathiazol-2-ones on mtb and mammalian cells prompted us to ask if these compounds differentially inhibit isolated mycobacterial and human proteasomes . in dialysis ( fig . 2a ) , while inhibition of human proteasome 1 , 2 and 5 sites was so minimal ( fig . 2b , c ; table s1 ) as to preclude definition of a mode of inhibition . after establishing that oxathiazol-2-ones spontaneously hydrolyze in tissue culture medium to the corresponding amide with t1/2 s ranging from 7 to 180 minutes ( table s2 ) , we used partition ratios ( the ratios of rate constants for catalysis and inactivation ) to assess their relative potency . by this measure , gl5 and ht1171 inhibition was competitive with the substrate benzyloxycarbonyl - valylleucylarginyl - amc ( z - vlr - amc ) ( fig . s3c , d ) , suggesting that the inhibitor binds at or near the active site , and was time - dependent ( fig . potencies of all 21 oxathiazol-2-ones tested against wild type mtb proteasome correlated closely ( r = 0.82 ) with their potencies against the open - gate form ( fig . oxathiazol-2-one - treated mtb proteasomes lost the ability to degrade not only oligopeptides , but also a protein substrate , -casein ( fig . s6 ) . in contrast , oxathiazol-2-ones were inactive or very weak inhibitors of trypsin ( ic50 > 50 m ) , cathepsin b ( ic50 > 50 m ) , matrix metalloproteinase-2 ( ic50 > 100 m ) and mycobacterial blac - encoded-lactamase , a serine protease - like enzyme ( ic50 > 100 m ) . although gl5 reversibly inhibited -chymotrypsin with ki 64 nm , anti - chymotryptic potency was minimal in 13 other oxathiazol-2-ones tested ( table s4 ) . table s1 summarizes biochemical and mycobactericidal properties of 24 oxathiazol-2-ones , including 21 we synthesized as described in supplemental information . competitive , irreversible , mechanism - based inhibition suggested that the oxathiazol-2-ones inactivate the mtb proteasome by covalent attack on the active site thr1 . to test this , we trypsinized mtb proteasomes that had been treated or not with ht1171 . only one peptide ion was unique to the mtb proteasome  subunit treated with ht1171 ( fig . its mass was 26 da higher than that of the n - terminal heptapeptide ( ttivalk ) identified only in the untreated samples ( figs . 3a and s7a , b ) , suggesting the addition of a carbonyl at the expense of two hydrogen atoms ( iii in fig . s7a ) indicated that the modification was on one of the first two residues . to determine if the n - terminal residue was modified , the tryptic peptides from untreated and treated mtb proteasome  subunits were subjected to reductive glutaraldehydation , which modifies amino groups of proteins and peptides . subjecting the trypsinized oligopeptides to reductive glutaraldehydation without prior exposure to oxathiazol-2-one increased the mass of the n - terminal heptapeptide by 136.13 da , consistent with modification of two primary amines , those of thr1 and lys7 ( fig . s7c ) . in contrast , applying the same procedure to proteasomes that had been pre - treated with ht1171 increased the mass of the same peptide by only 68.06 da ( fig . s7d ) , indicating that one primary amine was no longer available for reductive alkylation . s7d ) of this mono - alkylated peptide showed that only the lysine was modified , confirming that the n - terminal thr was modified upon ht1171 treatment . the mass spectrometric results were the same when ht1171 was replaced by gl5 or gl3 and when the proteasome was wild type or open - gate ( not shown ) . 3b : the attack of the oxathiazol-2-one by the oh of thr1 forms a carbonated or carbonothioated enzyme intermediate on thr1 that can either undergo hydrolysis to reactivate the enzyme , or donate a carbonyl to thr1 that links its -amino and -hydroxyl groups to form an oxazolidin-2-one , a chemically stable moiety , consonant with the ability of oxathiazol-2-ones to cyclocarbonylate 1,2-aminoalcohols . to determine the basis for species selectivity , we solved four crystal structures : wild type mtb proteasome following exposure to gl1 at 2.4  resolution and to ht1171 at 2.5  resolution , and the open - gate variant ( 20sog ) alone at 2.5  resolution or following exposure to ht1171 at 2.9  resolution . n - terminal octa - peptide deletion in the -subunit of 20sog did not alter the overall structure of the mtb proteasome ( fig . wild type and open - gate proteasomes underwent the same conformational changes ( described below ) upon inhibitor treatment ( fig . the three structures ( figs . 4 , s8 , s9 ; table s5 ) each confirmed that oxathiazol-2-ones cyclo - carbonylate thr1 . not only the oxazolidin-2-one ring , but also its protruding methyl group and carbonyl oxygen , were resolved in the electron density ( fig . the oxazolidin-2-one ring is stabilized by an h - bond network involving ala180 , ser141 , asn24 of the neighboring -subunit , and a water molecule in the substrate cavity ( fig . s9 ) . use of ht1171 and gl1 in the crystallographic studies and ht1171 , gl5 and gl3 in the mass spectroscopic studies brought to 4 the number of oxathiazol-2-ones for which the same suicide - substrate inhibition mechanism was confirmed . surprisingly , the substrate - binding pocket of the mtb proteasome underwent a major conformational change upon cyclo - carbonylation of thr1 by ht1171 or gl1 . such a change is unprecedented among the dozens of crystal structures of proteasomes in complex with inhibitors . an ~8 downward tilt of the h1 helix in the -subunit moved the n - terminal end of h1 ( ala49-phe55 ) downward by as much as 4.2  ( fig . the h1 shift brought with it a 3-amino - acid stretch ( ala46-thr48 ) of the s4 -strand , converting this segment into a short loop ( s4-h1 ) . as a consequence , another short loop ( met95gln96gly97 ) between h2 and s5 lost stabilizing contacts with the shifted components and became disordered , as illustrated by a black dashed curve in fig . its downward shift constricted the pocket to the point that it could not accommodate a peptide substrate , affording an additional mechanism of inhibition over and above incorporation of the active site hydroxyl into an oxazolidin-2-one . in the native mtb -subunit , the three s4-h1 loop amino acids ala46 , gly47 and thr48 are in strand configuration and form a -sheet interaction with leu101 , ala100 and leu99 , respectively , in the s5 -strand ( fig . none of these residue pairs is conserved in the human proteasome 5 subunit and only one of them ( g47-a96 ) in the human 1 and 2 subunits . one direct h - bond and three pairs of water - mediated h - bonds stabilized the new position of the s4-h1 loop ( fig . 4d ) : between glu54 and trp129 of neighboring -subunit ( 3.0  ) ; between thr48 , a water , and asp124 in the s6-s7 loop of neighboring -subunit ( 2.8  and 3.1  , respectively ) ; between ala50 , a water , and the carbonyl o of trp129 in the short s7 -strand of the neighboring -subunit ( 3.0  and 3.1  , respectively ) ; and between ala 49 , a water , and ser20 in the same -subunit ( 3.2  and 3.1  , respectively ) . this last pair of h - bonds cross - linked the upper and the lower substrate - binding surfaces , sealing the entrance of the substrate pocket . the residue pairs involved in the post - shift h bonds are not well conserved in the human proteasome ( fig . we speculate that the upper substrate - binding surfaces ( s4-h1 loops ) in the 3 catalytic -subunits of the human proteasome might have difficulty breaking off from their corresponding -sheet cores upon initial modification of thr1 by oxathiazol-2-ones . thus , selectivity of oxathiazol-2-ones appears to be imparted in 3 ways : by the presence of a 1,2-aminoalcohol at the active site of the target , which , among enzymes , is likely to be limited to the n - terminal thr hydrolase family ; by the ability of the inhibitor to bind rapidly ( before its spontaneous decay ) and precisely adjacent to the 1,2-aminoalcohol ; and the degree to which the amino group of the 1,2-aminoalcohol has better access to the inhibitor s carbonyl ( now attached to the alcohol ) than water has . the protein landscape near the 1,2-aminoalcohol can thus determine species selectivity , both in how it binds the r group on the oxathiazol-2-one and in the conformations it adopts , which may either permit or limit access of water to the intermediate formed during reaction of the oxathiazol-2-one with the active site . detailed understanding the sequence of steps by which oxathiazol-2-ones cause a major conformational shift in the substrate - binding domain could guide design of the next generation of inhibitors selective for the mtb proteasome over the human proteasome . non - replicating mtb displays  phenotypic tolerance ,  that is , relative resistance to conventional anti - infectives , imposing the need to treat tuberculosis longer than almost any other infectious disease . hence agents are needed that can kill mtb when its replication is halted by conditions encountered in the host , such as those imposed by inducible nitric oxide synthase . oxathiazol-2-ones identified here phenocopy genetic deletion of the mtb proteasome by killing mtb rendered non - replicative by exposure to sub - lethal nitric oxide . along with thioxothiazolidines that inhibit mtb s dihydrolipoamide acyltransferase , oxathiazol-2-ones are only the second class of compounds , to our knowledge , that are selectively bactericidal for a non - replicating pathogen . they inhibit the mtb proteasome but not the human proteasome n - terminal threonine 1 , 2 or 5 proteases , cysteine proteases , serine proteases , or metalloproteases . they do not kill monkey epithelial cells , human macrophages or any bacteria we tested other than mycobacteria . however , residues distant from the active site , with which oxathiazol-2-ones do not interact , appear to impart species selectivity . inhibition of protein synthesis at the stages of transcription ( e.g. , by rifamycins ) or translation ( e.g. , by aminoglycosides and capreomycin ) are among the best validated antibiotic strategies . the ability of a brief exposure to oxathiazol-2-ones to inhibit mtb proteasomes permanently is a potential advantage . mtb may have difficulty replacing irreversibly inactivated proteasomes , not only when mtb s protein synthesis is markedly diminished in the non - replicative state , but also when its protein synthesis is impaired by antibiotics . much of biology can be viewed as the interplay of two principles , conservation and diversity . the present work demonstrates that functionally exploitable diversity exists between the proteasomes of mtb and its obligate human host . high throughput screening was performed in 384-well plates with 33 m test compounds . to test inhibition of proteasomes in intact mycobacteria , treated cells were centrifuged , washed twice and lysed mechanically , and aliquots ( 10 g protein ) of the supernates assayed with suc - llvy - amc ( 50 m ) . killing of detano - treated , non - replicating mtb erdman was tested in sauton s medium , ph 7.4 . monkey kidney cells were cultured in dulbecco s eagle s medium with 10% fetal bovine serum for cytotoxicity assessment after 48 hours by tetrazolium reduction and microscopy . kinetic studies were conducted in a molecular devices fluorescent plate reader with amc - derivatized peptide substrates according to which proteasomal protease was being studied ( mtb or human 1 , 2 or 5 ) . values of kobs were derived from the fit of data to equation ( 1 ) in prism ( graphpad software , inc . lc - ms / ms analysis used thermo ltq orbitrap and applied biosystems qstar mass spectrometers . crystallization of the mtb proteasome , diffraction data collection and structure solution and refinement are included along with other methodologic details in the methods section that appears online in the full - text html and pdf versions of this article .
summarymany anti - infectives inhibit the synthesis of bacterial proteins , but none selectively inhibits their degradation . 
 most anti - infectives kill replicating pathogens , but few preferentially kill pathogens that have been forced into a non - replicating state by conditions in the host . to explore these alternative approaches we sought selective inhibitors of the proteasome of mycobacterium tuberculosis ( mtb ) . given that proteasome structure is extensively conserved , it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes , and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts . 
 here we show that certain oxathiazol-2-ones kill non - replicating mtb and act as selective suicide - substrate inhibitors of the mtb proteasome by cyclo - carbonylating its active site threonine . 
 major conformational changes protect the inhibitor - enzyme intermediate from hydrolysis , allowing formation of an oxazolidin-2-one and preventing regeneration of active protease . 
 residues outside the active site whose h - bonds stabilize the critical loop before and after it moves are extensively non - conserved . 
 this may account for the ability of oxathiazol-2-ones to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homolog .