Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED₉₉ of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.     

Attachment and Biofilm Forming Capabilities of Staphylococcus epidermidis Strains Isolated from Preterm Infants

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.     

11 Findings and hurdles on the path leading from formamide to the spontaneous generation of RNA

Our laboratories analyze the synthetic reactions leading from formamide, NH₂COH to prebiotically relevant compounds in the presence of catalysts. We have described the formation of all the biological nucleic bases of carboxylic acids of two aminoacids, and of condensing agents in the presence of catalysts of terrestrial origin (Saladino et al., 2012) and of one meteorite. Heat-dependent synthetic reactions from NH₂COH lead to the synthesis of acyclonucleosides, not (yet?) to that of nucleosides [hurdle # 1]. Nucleosides are phosphorylated in the presence of NH₂COH and a phosphate source yielding cyclic nucleotides as well. (Costanzo et al., 2007). 3′,5′-cyclic GMP nonenzymatically polymerizes up to at least 25mers, as shown by PAGE, MALDI ToF, ³¹P-NMR, specific RNAse and inhibitors analyses (Costanzo et al., 2012).The reaction is stimulated by 1,8-diazabicycloundec-7-ene and dimethylformamide. 3′,5′-cUMP does not polymerize spontaneously [hurdle # 2], 3′,5′-cAMP polymerizes very poorly [hurdle # 3]. We will discuss data on the polymerization of 3′,5′-cCMP and on a ribozyme activity exerted by oligomers neosynthesized from cyclic nucleotides. This approach finds its larger perspective in the evolutionary scenario depicted by Trifonov (2009).     

Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F₀F₁ ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F₁ sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F₀F₁ complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F₀F₁ complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

A class of pyrrole derivatives endowed with analgesic/anti-inflammatory activity

We report the synthesis and bio-pharmacological evaluation of a class of pyrrole derivatives featuring a small appendage fragment (carbaldehyde, oxime, nitrile) on the central core. Compound 1c proved to be extremely effective in vivo, showing an interesting anti-nociceptic profile that is comparable to reference compounds already marketed, hence representing a great stimulus for a further improvement of this class of molecules.     

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4–6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H₂O₂, MPP+ and MnCl₂ were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.     

A Low Cost Metal-Free Vascular Access Mini-Port for Artifact Free Imaging and Repeated Injections in Mice

Purpose

Small injection ports for mice are increasingly used for drug testing or when administering contrast agents. Commercially available mini-ports are expensive single-use items that cause imaging-artifacts. We developed and tested an artifact-free, low-cost, vascular access mini-port (VAMP) for mice.

Procedures

Leakage testing of the VAMP was conducted with high speed bolus injections of different contrast agents. VAMP-induced artifacts were assessed using a micro-CT and a small animal MRI (9.4T) scanner ex vivo. Repeated contrast administration was performed in vivo.

Results

With the VAMP there was no evidence of leakage with repeated punctures, high speed bolus contrast injections, and drawing of blood samples. In contrast to the tested commercially available ports, the VAMP did not cause artifacts with MRI or CT imaging.

Conclusions

The VAMP is an alternative to commercially available mini-ports and has useful applications in animal research involving imaging procedures and contrast agent testing.