The cell wall of Mycobacterium spp. is required for growth and survival of the organism in the host (10). The infrastructure, or core, of the cell wall is composed of a covalently linked complex of mycolic acids, the heteropolysaccharide arabinogalactan (AG), and peptidoglycan (15). Much of the primary structure is known from recent (2) and earlier work (16). However, new evidence and concepts have emerged, supportive of the theme of an asymmetric, staggered lipid bilayer within the cell wall, in which the mycolic acids anchored to the arabinan of AG form an inner leaflet and the outer monolayer contains a mixture of phospholipids and members of several classes of glycolipids, depending on the species and/or serotype (4).One of the most prominent substances intercalating the core cell wall or perhaps anchored in the outer lipid barrier is lipoarabinomannan (LAM) and its simpler version, lipomannan (LM) (6). LAM may have important immunoregulatory functions in tuberculosis and leprosy (1). Disruption of the biosynthesis of any of these components, especially the mycolic acids, AG, or peptidoglycan, should destroy the integrity of the macromolecular assembly. In fact, some of the most effective antituberculosis drugs, isoniazid and ethambutol (EMB), affect mycolic acid and arabinan biosynthesis, respectively (23). The effects of EMB are extremely pleiotropic, and proposals for the primary site of action of EMB have ranged from trehalose dimycolate (TDM) (14) and mycolate (20) metabolism, AG synthesis (21), and glucose metabolism (18) to spermidine biosynthesis (17). However, emphasis is now on inhibition of arabinan biosynthesis, in that Takayama and Kilburn (21) ]glucose into sugar components of AG. Delipidated cell pellets from the control (Con), EMB-treated, and EMB-R cultures were extracted with 50% ethanol and 2% SDS in phosphate-buffered saline. The insoluble cell walls were hydrolyzed with 2 M trifluoroacetic acid at 120ЊC for 3 h, and ca. 50,000 cpm of each extract was applied to cellulose TLC plates. The plates were developed three times in formic acid-water-ethylmethylketonetert-butyl alcohol (15:15:30:40) and exposed to film for 3 days.

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

No abstract

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure–property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

Despite significant research efforts, treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain limited. This is due in part to a lack of therapeutics that increase host defense to the virus. Replication of SARS-CoV-2 in lung tissue is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses that amplify tissue injury. Antagonists of the androgen (AR) and glucocorticoid (GR) receptors have shown efficacy in models of COVID-19 and in clinical studies because the cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2), are transcriptionally regulated by these receptors. We postulated that the GR and AR modulator, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID-19 by down-regulating expression of critical genes regulated through these receptors. Animals were infected intranasally with 2.5 × 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were examined at 3, 5 and 7 days post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the progression of SARS-CoV-2 infection. Results indicated that oral administration of PT150 caused a dose-dependent decrease in replication of SARS-CoV-2 in lung, as well as in expression of ACE2 and TMPRSS2. Lung hypercellularity and infiltration of macrophages and CD4+ T-cells were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular docking studies suggest that PT150 binds to the co-activator interface of the ligand-binding domain of both AR and GR, thereby acting as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR revealed a high degree of sequence identity maintained across multiple species, including humans, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID-19 across variants of SARS-CoV-2.