As an essential and uniquely prokaryotic enzyme, alanine racemase has long been pursued as a target for antimicrobial drug discovery. Numerous enzyme inhibitors have been identified. The vast majority, however, are suicide substrates that react with the enzyme cofactor and tend to indiscriminately inhibit other PLP-containing enzymes. In an effort to obtain more target-specific inhibitors, we performed HTS and identified 16 novel alanine racemase inhibitors that are not substrate analogs. A subset of these inhibitors blocked M. tuberculosis growth and showed low cytotoxicity toward mammalian cells, making them promising leads and warranting further studies into their mechanism of action.
Mass spectromentry analysis revealed that two of the inhibitors likely bind the enzyme in a reversible manner while four inhibitors are irreversible binders. It is highly unlikely that the inhibitors bind the enzyme through interaction with PLP, primarily due to a missing free amino group, requisite for external aldimine formation with the co-factor 
. Based on the crystal structure of M. tuberculosis
alanine racemase, there are at least three possible ways by which an inhibitor that does not engage the PLP could block enzyme function. An inhibitor could directly bind to the active site, or alternatively, it could block the formation of the active sites by blocking enzyme dimerization. All of these possibilities are currently being investigated with efforts to obtain enzyme-inhibitor co-crystals.
Some additional comments can be made concerning structure-activity relationships (SAR) among the inhibitors in ; however, given the relatively small data set such SAR proposals should be considered preliminary at this time. In any event, among the three thioamides (L2-7, L2-9, and L2-12), whereas they all had similar alanine racemase enzyme IC50s, only compound L2-12 had significant activity against M. tuberculosis in cell culture. This would suggest that either a hydrogen on the para position of the phenyl ring is favored over a methyl or methoxy group, or a piperidine amide rather than a morpholine amide is necessary for MICs, or both. Note that another thioketo derivative, namely the carbamothioyl analog L2-11, had activity against alanine racemase; however, it did not show significant M. tuberculosis activity. With regards to the two pyrimidine carboxamide derivatives L2-05 and L2-06, while both had similar activity against the enzyme, it would appear that more potent MIC activity is obtained with L2-06 suggesting that a phenyl amide is preferred over a benzyl amide for best cell culture activity. Inhibitors L2-01 and L2-02 are both sulfonylhydrazides, with L2-02 showing significantly improved alanine racemase enzyme inhibition over L2-01 suggesting that either the larger size of L2-02 is allowing for additional hydrophobic (possible π-stacking) interactions relative to L2-01, or else the additional sulfonylhydrazide in L2-02 is responsible for another favorable interaction with alanine racemase. The phenols L2-03 and L2-15 may share a similar binding site in the enzyme, and, if so, this would suggest that the methyl amide can be replaced with a hydroxamide without significant loss of enzyme activity. As more data is obtained on newer analogs of compounds in , these preliminary SAR suggestions will likely be both refined and expanded.
The antimycobacterial activities noted for seven of the inhibitors are particularly encouraging, and highlight the feasibility of obtaining, by HTS, not only structurally new alanine racemase inhibitors, but also compounds that are able to cross the mycobacterial cell wall. The seven inhibitors show good MICs, which range from <4.5 µM to 59 µM. However, preliminary experiments with exogenously added D-alanine suggest that the observed MICs may not be due solely to inhibition of alanine racemase, and that additional cellular targets for these inhibitors likely exist.
Cytotoxicity, which is often used as a criterion to select compounds for lead development revealed that six inhibitors, with the exception of L2-16, were not cytotoxic at their MIC concentrations. The thioamide L2-12, though not cytotoxic, is generally not considered a good therapeutic lead due to its potential oxidation into toxic reactive species in vivo. This leaves five new inhibitors as possible leads for further studies.
Alanine racemase has often been proposed as a target for drug design in antimicrobial development. Though several attempts have been made to rationally design enzyme inhibitors, none has produced candidates superior to cycloserine, the toxic TB drug that interacts with the PLP cofactor and blocks enzyme activity in a non-specific manner. To identify inhibitors that do not share features of cycloserine or any other previously known alanine racemase inhibitors, we performed HTS of chemical compound libraries. We identified several novel non-substrate alanine racemase inhibitors. These belong to different chemical classes, including hydrazide, hydroxamic acid, thiopyridine, pyrimidine carboxamide, thioamide, pyridine ester, oxadiazole N-oxide, carbamodithioate, isothiazolopyridione, benzopyranone oxime, alkoxy phenol, and quinolinedione. Seven of these compounds inhibited M. tuberculosis growth, several of which were minimally cytotoxic. Collectively, these results demonstrate the feasibility of using HTS to obtain novel alanine racemase inhibitors that are potentially useful for development as anti-TB agents.