Compounds with longchain aliphatic acidscould potentially disrupt the Mtb membrane, allowing for entry of protons into the intracellular space, even though they had no such effect in the counter-screens. Compounds 12F10 and 12H5 contain reactive aldehyde moieties that are likely to allow them to bind to diverse proteins as well as DNA; additionally, 12H5 caused more than a 1.5-fold increase in fluorescence quench rate. Supplies of compound 17D7 were insufficient to support more detailed studies. Efforts to synthesize 17D7 in-house were Y-27632 dihydrochloride unsuccessful at producing the final product, but generated the closely related compound 1048, in which a phenolic hydroxyl replaces the methoxy in 17D7. Compound 1048 was 2- to 4-fold more potent than 17D7 in the pHIB assayand was used in subsequent studies. A summary of the selection process is depicted in figure 2D. This work describes a novel whole-cell assay suitable for a HTS format that allows identification of small molecules that perturb pHIB homeostasis. Compared to other whole-cell screening assays for Mtb, which have incubation periods of several days or weeks, the pHIB homeostasis assay is rapid, with results in as little as 4 hours, although we conducted our initial screen at two days. We have adapted this assay to a 384-well format and helped distribute it to another screening center in order to identify additional tool compounds and, potentially, precursors of lead compounds for the treatment of tuberculosis. Secondary screens eliminated compounds with protonophoric and membrane-perturbing properties. Although we developed the assay to identify inhibitors of pHIB homeostasis, the assay may also identify compounds with other activities against Mtb. For example, agrimophol disrupted Mtb��s pH homeostasis and killed Mtb in CHIR-99021 acidic conditions, but it also killed Mtb near neutrality in replicating conditions. Moreover, the assay may identify compounds that kill Mtb whose replication is halted not only by physiologic levels of acid but by other hostimposed stresses as well. Non-replicating subpopulations of Mtb are phenotypically relatively resistant to most standard chemotherapeutics used to treat tuberculosis. To our knowledge, this is the first report of an assay for compounds that disrupt intrabacterial pH homeostasis. This may also be the first report of PZA��s effects on pHIB in Mtb. PZA is a clinically important but paradoxical and unconventional drug. Despite its remarkable sterilizing activity in vivo, it is inactive against Mtb under standard culture conditionsbut weakly active against Mtb exposed to an acidic pH, conditions under which Mtb replicates little. Fatty acid synthase-I has been proposed as a target for PZA, but while 5-Clpyrazinamide targets this protein, PZA does not. Recent studies point to RpsA and trans-translation as a target of pyrazinoic acid. It has also been proposed that POA does not have a specific cellular target but simply functions to shuttle protons from the extracellular space into the intrabacterial space, resulting in decreased pHIB, collapse of membrane potential, and bacterial death. Our results provide direct evidence that PZA lowers Mtb��s pHIB in an acidic environment. This assay may select for compounds with similar sterilizing abilities as PZA, an important goal, as resistance to PZA is increasing. We chose to screen a natural product library because of natural products�� structural diversity and greater propensity for antiinfective activity than seen with compounds produced by conventional combinatorial chemistry. A particular challenge in the chemical biology of Mtb is its thick cell wall comprised largely of mycolic acids and their esters.