As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from targetbased antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.acetylcoenzyme A carboxylase ͉ biotin carboxylase ͉ crystal structure ͉ high-throughput screening ͉ fatty acid biosynthesis

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Several tablets of a formulation containing 1% w/w of the desired active pharmaceutical ingredient (API) form are spiked with minimal amounts of two different anhydrous polymorphs and an amorphous form. The amount of contaminant form was 2.5 to 10% of the total API concentration (0.025 to 0.1% w/w in the tablet), with five spiked tablets prepared. The presence of these contaminant particles are then identified using Raman microscopy/mapping. The entire surface of each of these tablets is Raman-probed through a grid based on our previous proposal (Šašić, S.; Whitlock, M. Appl. Spectrosc.2008, 62, 916) about the minimal number of spectra to acquire that would guarantee identification of the targeted component (taking into account the limit of detection). All three forms have been clearly identified in the Raman mapping spectra of prepared "calibration" tablets; particularly of note is the 2.5% spike (0.025% w/w in the tablet) of the relatively weakly scattering amorphous form. The same method is then applied to packaged tablets on stability and demonstrates that none of the previously analyzed contaminant forms is detected, hence building confidence that the desired API form does not change during stability testing.

The present study investigated the pharmacokinetic/pharmacodynamic (PK/PD) relationships of a prototype biotin carboxylase (BC) inhibitor, PD-0162819, against Haemophilus influenzae 3113 in static concentration time-kill (SCTK) and onecompartment chemostat in vitro infection models. H. influenzae 3113 was exposed to PD-0162819 concentrations of 0.5 to 16؋ the MIC (MIC ‫؍‬ 0.125 g/ml) and area-under-the-curve (AUC)/MIC ratios of 1 to 1,100 in SCTK and chemostat experiments, respectively. Serial samples were collected over 24 h. For efficacy driver analysis, a sigmoid maximum-effect (E max ) model was fitted to the relationship between bacterial density changes over 24 h and corresponding PK/PD indices. A semimechanistic PK/PD model describing the time course of bacterial growth and death was developed. The AUC/MIC ratio best explained efficacy (r 2 ‫؍‬ 0.95) compared to the peak drug concentration (C max )/MIC ratio (r 2 ‫؍‬ 0.76) and time above the MIC (T>MIC) (r 2 ‫؍‬ 0.88). Static effects and 99.9% killing were achieved at AUC/MIC values of 500 and 600, respectively. For time course analysis, the net bacterial growth rate constant, maximum bacterial density, and maximum kill rate constant were similar in SCTK and chemostat studies, but PD-0162819 was more potent in SCTK than in the chemostat (50% effective concentration [EC 50 ] ‫؍‬ 0.046 versus 0.34 g/ml). In conclusion, basic PK/PD relationships for PD-0162819 were established using in vitro dynamic systems. Although the bacterial growth parameters and maximum drug effects were similar in SCTK and the chemostat system, PD-0162819 appeared to be more potent in SCTK, illustrating the importance of understanding the differences in preclinical models. Additional studies are needed to determine the in vivo relevance of these results. Steadily increasing bacterial resistance to existing antibiotics continues to be a major public health concern (3, 8). Because most new antibacterial agents represent chemical modifications of existing chemical classes of antibacterial agents (5), it is suspected that the limited options of chemically distinct antibiotics have led to extensive drug resistance among bacterial pathogens. Therefore, it is of the utmost importance to identify novel, safe, and effective antibacterial agents that work through unique antibacterial biological mechanisms. The discovery of a new chemical class of antibacterial compounds, the pyridopyrimidines, targeting bacterial biotin carboxylase (BC), was recently reported (14, 15) and offers the potential that this novel chemical class, targeting a unique antibacterial mechanism, can be developed into drugs effective against multidrug-resistant bacteria.Compared to the development of drugs from an existing chemical class, the discovery of a novel class of compounds presents extra challenges (1, 5). The translation of pharmacokinetic/ pharmacodynamic (PK/PD) relationships between animal infection models and human patients has been well established for several existing chemical classes across a variety of in...