Oral administration of protein nanoparticles: An emerging route to disease treatment.

Over the last two decades, developments in nanomedicine have resulted in technical advances with application to clinical science. Both organic and inorganic nanoparticles (NPs) have shown tolerability, pharmacologic specificity and biodegradability. A subclass of NPs, protein NPs, have garnered recent attention due to the inherent biocompatibility of protein substrates. Protein NPs are currently being employed widely in pharmaceuticals development with applications in nasal, pulmonary, intravenous, ocular and oral delivery. Despite the distinct advantages of orally administered pharmaceuticals, the development of oral delivery systems has been comparatively limited. Therefore, this review attempts to discuss the most recent experimental and pre-clinical findings in the development of protein NPs for oral delivery, while envisioning upcoming challenges.

Novel Acetamide Indirectly Targets Mycobacterial Transporter MmpL3 by Proton Motive Force Disruption.

To identify novel inhibitors of Mycobacterium tuberculosis cell envelope biosynthesis, we employed a two-step approach. First, we screened the diverse synthetic small molecule 71,544-compound Enamine library for growth inhibitors using the non-pathogenic surrogate Mycobacterium bovis BCG as screening strain and turbidity as readout. Second, 16 confirmed hits were tested for their ability to induce the cell envelope stress responsive promoter piniBAC controlling expression of red fluorescent protein in an M. bovis BCG reporter strain. Using a fluorescence readout, the acetamide E11 was identified. Resistant mutant selection and whole genome sequencing revealed the mycolic acid transporter Mmpl3 as a candidate target of E11. Biochemical analysis using mycobacterial spheroplasts and various membrane assays suggest that E11 indirectly inhibits MmpL3-facilitated translocation of trehalose monomycolates by proton motive force disruption. E11 showed potent bactericidal activity against growing and non-growing M. tuberculosis, low cytotoxic, and hemolytic activity and a dynamic structure activity relationship. In addition to activity against M. tuberculosis, E11 was active against the non-tuberculous mycobacterium M. abscessus, an emerging opportunistic pathogen. In conclusion, we identified a novel bactericidal anti-mycobacterial lead compound targeting MmpL3 providing an attractive starting point for optimization.

Mycobacterial Cell Wall Synthesis Inhibitors Cause Lethal ATP Burst.

Mycobacterial cell wall inhibitors interfere with targets involved in synthesis of mycolic acids, arabinogalactan and peptidoglycan. These antibiotics corrupt structural integrity of the cell envelope and this is believed to be the cause of drug mediated cell death. Here, we show that treatment of Mycobacterium bovis BCG with these mechanistically different classes of cell wall inhibitors at MIC caused a 4 to 5-fold increase in intrabacterial ATP concentration. This effect on ATP homeostasis was specific to inhibitors of cell wall synthesis and not observed for other anti-tuberculosis drugs. Treating M. bovis BCG with sub-MIC concentrations of the ATP synthase inhibitor bedaquiline or the uncoupler carbonyl cyanide 3-chlorophenylhydrazone suppressed drug induced ATP surge, suggesting that the increase in ATP concentration was due to increased oxidative phosphorylation. Pharmacological suppression of the ATP burst attenuated bactericidal activity of the cell wall-targeting drugs up to 100-fold, suggesting that increased ATP levels are associated with the lethal effect of these antibiotics. Interestingly, inhibition of the ATP burst also suppressed induction of the promoter of the cell envelope stress response operon iniBAC by cell wall inhibitors suggesting a link between ATP surge and iniBAC expression. In conclusion, we show that treatment of M. bovis BCG with inhibitors of cell wall synthesis causes a burst of intrabacterial ATP by increasing oxidative phosphorylation. This ATP surge appears to be required for induction of the iniBAC cell envelope stress response operon and to contribute to drug induced cell death. Hence, this work revealed links between inhibition of cell wall synthesis, oxidative phosphorylation, iniBAC induction and cell death. The identification of the molecular mechanisms linking these processes may reveal novel targets for the discovery of bactericidal antibiotics.

Verapamil Targets Membrane Energetics in Mycobacterium tuberculosis.

Mycobacterium tuberculosis kills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. Verapamil, an FDA-approved calcium channel blocker, potentiates the effect of several antituberculosis (anti-TB) drugs in vitro and in vivo This potentiation is widely attributed to inhibition of the efflux pumps of M. tuberculosis, resulting in intrabacterial drug accumulation. Here, we confirmed and quantified verapamil's synergy with several anti-TB drugs, including bedaquiline (BDQ) and clofazimine (CFZ), but found that the effect is not due to increased intrabacterial drug accumulation. We show that, consistent with its in vitro potentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. We recapitulated these activities in vitro using inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. We observed bactericidal activity against nonreplicating "persister" M. tuberculosis that was consistent with such a mechanism of action. In addition, we demonstrated a pharmacokinetic interaction whereby human-equivalent doses of verapamil caused a boost of rifampin exposure in mice, providing a potential explanation for the observed treatment-shortening effect of verapamil in mice receiving first-line drugs. Our findings thus elucidate the mechanistic basis for verapamil's potentiation of anti-TB drugs in vitro and in vivo and highlight a previously unrecognized role for the membrane of M. tuberculosis as a pharmacologic target.