Squaramides constitute a novel class of RNA polymerase inhibitors of which genetic evidence and computational modeling previously have suggested an inhibitory mechanism mediated by binding to the RNA polymerase switch region. non-template DNA a mechanism akin to that of natural product myxopyronin. Furthermore the structures confirmed the chemical features required for biochemical potency. The terminal isoxazole and benzyl rings bind into distinct relatively narrow hydrophobic pockets and both are required for biochemical potency. In contrast the linker composed of squarate and piperidine accesses different conformations in their respective co-crystal structures with RNA polymerase reflecting its main role of proper orientation of the aforementioned terminal rings. These observations further explain the tolerance of hydrophilic substitutions in the linker region that was exploited to improve the fraction unbound to human plasma protein while retaining biochemical potency. infections responsible for 15 0 0 deaths in the United States each year 18. Unlike rifampin fidaxomicin binds to the RNAP switches thus preventing formation of the transcription ready promoter DNA open complex 19. Rifampin-resistant mutants were not cross-resistant to the switch region binding RNAP inhibitors 20-23. Furthermore the importance of mobility of the clamp during the transcription-competent open Choline Fenofibrate complex Choline Fenofibrate formation and identification Rabbit Polyclonal to EIF5B. of a number of natural and synthetic antimicrobial molecules that bind the switches have raised the importance of studying and subsequent exploitation of the molecular basis of their action and these compounds could be Choline Fenofibrate promising antibiotic candidates for treatment of multi-drug resistant tuberculosis. These include chemically diverse natural compounds such as the α-pyrones myxopyronin (Myx) (Fig. 1A) and corallopyronin and the macrocycles ripostatin and fidaxomicin 19 23 More recently synthetic scaffolds including ureidothiophene-2-carboxylic acid-based inhibitors 21 26 and squaramide-based inhibitors (Fig. 1B) 27 have been added. Structural and biochemical studies of Myx-mediated inhibition of RNAP culminated into two possible hypotheses regarding its mechanism. In the hinge-jamming model Myx prevents conformational changes of the RNAP clamp required for the promoter DNA melting 8 25 Alternatively in the steric clash model Myx alters the conformation of switch 2 not preventing the nucleation of the promoter DNA melting but interfering with its further accommodation into the active-center cleft 24. Physique 1 Chemical structures of (A) Myxopyronin and (B) Squaramide (compound 14). The chemical moieties are indicated. The stereocenter at the C7-position on Myx is usually indicated by an asterisk. In a previous study a series of antimicrobial squaramides were synthesized of which a subset acts by binding Choline Fenofibrate to the switches as concluded from mapping of resistance mutations to the β and β′ subunits 27. Using the crystal structure of RNAP – Myx complex 25 a computational model of the RNAP and squaramide complex was constructed to predict possible squaramide binding modes around the switches. Here in an extension of these studies crystal structures of RNAP holoenzyme in complex with squaramides were decided structurally confirming that this squaramides bind the RNAP switches. This revealed that Myx and squaramides although chemically distinct bind to the same region of RNAP and identified new binding crevices within the switches that could be explored for the design of a new class of antibiotics. RESULTS AND DISCUSSION Determination of structure activity relationships of squaramides A high-throughput screen of a corporate compound library using S30 extracts of in a coupled Transcription-Translation assay (TT assay) identified a series of squaramides of which its most active member compound 1 (Table 1) had a sub-micromolar half maximal inhibitory concentration (IC50)27. Since this assay was 10-100-fold more sensitive than an RNAP assay using purified components27 the TT assay was used to monitor biochemical potency. In addition compound 1 was selective as decided in an eukaryotic TT assay using rabbit reticulocytes with an IC50 of >100 μM (data not shown). Although hydrophobicity and solubility of the.