Ipomoeassin F (Ipom-F) is a plant-derived macrocyclic resin glycoside that potently inhibits cancer cell growth through blockage of Sec61-mediated protein translocation at the endoplasmic reticulum. Recently, detailed structural information on how Ipom-F binds to Sec61α was obtained using Cryo-EM, which discovered that polar interactions between asparagine-300 (N300) in Sec61α and four oxygens in Ipom-F are crucial. One of the four oxygens is from the carbonyl group at C-4 of the fatty acid chain. In contrast, our previous structure–activity relationship (SAR) studies suggest that the carbonyl group is not essential. To resolve this discrepancy, we designed and synthesized two new open-chain analogues (10 and 11); 10 without the C-4 carbonyl had a dramatic activity loss, whereas 11 with an amide functional group was even more potent than Ipom-F. These new SAR data, in conjunction with some previous SAR information, imply two functional roles of the C-4 carbonyl: (1) to form H-bonds with N300; and (2) to regulate interactions of the fatty acid chain with membrane lipids. Impacts of these dual functions on antiproliferation depend on the overall structure of an Ipom-F derivative. Moreover, 11 can serve as a lead compound for developing future amino acid/peptide-modified analogues of Ipom-F with improved therapeutic properties.

Ipomoeassin F (Ipom-F) is a plant-derived macrocyclic resin glycoside that potently inhibits cancer cell growth through blockage of Sec61-mediated protein translocation at the endoplasmic reticulum. Recently, detailed structural information on how Ipom-F binds to Sec61α was obtained using Cryo-EM, which discovered that polar interactions between asparagine-300 (N300) in Sec61α and four oxygens in Ipom-F are crucial. One of the four oxygens is from the carbonyl group at C-4 of the fatty caid chain. In contrast, our previous structure–activity relationship (SAR) studies suggest that the carbonyl group is not essential. To resolve this discrepancy, we designed and synthesized two new open-chain analogues (10 and 11). 10 without the C-4 carbonyl had a dramatic activity loss, whereas 11 with an amide functional group was even more potent than Ipom-F. These new SAR data, in conjunction with some previous SAR information, imply two functional roles of the C-4 carbonyl: 1) to form H-bonds with N300; and 2) to regulate interactions of the fatty acid chain with membrane lipids. Impacts of these dual functions on antiproliferation depend on the overall structure of an Ipom-F derivative. Moreover, 11 can serve as a lead compound for developing future amino acid/peptide-modified analogues of Ipom-F with improved therapeutic properties.

BACKGROUND:Chronic infection by Staphylococcus aureus drives pathogenesis in important clinical settings, such as recurrent pulmonary infection in cystic fibrosis and relapsing infection in osteomyelitis. Treatment options for intracellular S. aureus infection are limited. Rifampin, a lipophilic antibiotic, readily penetrates host cell membranes, yet monotherapy is associated with rapid antibiotic resistance and development of severe adverse events. Antibiotic cotreatment can reduce this progression, yet efficacy diminishes as antibiotic resistance develops. ML141 and simvastatin inhibit S. aureus invasion through host-directed rather than bactericidal mechanisms.OBJECTIVE:To determine whether cotreatment of ML141 or of simvastatin with rifampin would enhance rifampin efficacy.METHODS:Assays to assess host cell invasion, host cell viability, host cell membrane permeability, and bactericidal activity were performed using the human embryonic kidney (HEK) 293-A cell line infected with S. aureus (29213) and treated with vehicle control, simvastatin, ML141, rifampin, or cotreatment of simvastatin or ML141 with rifampin.RESULTS:We found cotreatment of ML141 with rifampin reduced intracellular infection nearly 85% when compared to the no treatment control. This decrease more than doubled the average 40% reduction in response to rifampin monotherapy. In contrast, cotreatment of simvastatin with rifampin failed to improve rifampin efficacy. Also, in contrast to ML141, simvastatin increased propidium iodide (PI) positive cells, from an average of 10% in control HEK 293-A cells to nearly 20% in simvastatin-treated cells, indicating an increase in host cell membrane permeability. The simvastatin-induced increase was reversed to control levels by cotreatment of simvastatin with rifampin.CONCLUSION:Taken together, rifampin efficacy is increased through host-directed inhibition of S. aureus invasion by ML141, while efficacy is not increased by simvastatin. Considerations regarding novel therapeutic approaches may be dependent on underlying differences in pharmacology.

BACKGROUND:Recurrent pharyngotonsillitis due to Streptococcus pyogenes develops regardless of whether infecting strains are resistant or susceptible to first-line antimicrobials. Causation for recurrent infection is associated with the use of first-line antimicrobials that fail to penetrate deep tissue and host cell membranes, enabling intracellular S. pyogenes to survive throughout repeated rounds of antimicrobial therapy.OBJECTIVE:To determine whether simvastatin, a therapeutic approved for use in the treatment of hypercholesterolemia, and ML141, a first-in-class small molecule inhibitor with specificity for human CDC42, limit host cell invasion by S. pyogenes.METHODS:Assays to assess host cell invasion, bactericidal activity, host cell viability, actin depolymerization, and fibronectin binding were performed using the RAW 267.4 macrophage cell line and Human Umbilical Vein Endothelial Cells (HUVEC) infected with S. pyogenes (90-226) and treated with simvastatin, ML141, structural analogs of ML141, or vehicle control.RESULTS:Simvastatin and ML141 decreased intracellular infection by S. pyogenes in a dose-dependent manner. Inhibition by simvastatin persisted following 1 h washout whereas inhibition by ML141 was reversed. During S. pyogenes infection, actin stress fibers depolymerized in vehicle control treated cells, yet remained intact in simvastatin and in ML141 treated cells. Consistent with the previous characterization of ML141, simvastatin decreased host cell binding to fibronectin. Structural analogs of ML141, designated as the RSM series, decreased intracellular infection through non-cytotoxic, nonbactericidal mechanisms.CONCLUSION:Our findings demonstrate the potential of repurposing simvastatin and of developing CDC42-targeted therapeutics for eradicating intracellular S. pyogenes infection to break the cycle of recurrent infection through a host-directed approach.

Phenylpyrazoles (or arylpyrazoles) are known to be extremely potent as noncompetitive inhibitors of the GABA-gated chloride channel on the GABA receptor. This project involves the synthesis of novel isoxazole and isoxazoline heterocycles that have similar structural features to the commercially used phenylpyrazole, fipronil. Synthesis of the appropriately substituted styrenes and phenylacetylenes is followed by 1,3-dipolar cycloaddition reaction with several aliphatic nitrile oxides, which are prepared in situ from aldoximes with bleach. Relative reaction rates were determined for these specialized alkene and alkyne dipolarophiles.

Staphylococcus aureus is a leading causative agent in sepsis, endocarditis, and pneumonia. An emerging concept is that prognosis worsens when the infecting S. aureus strain has the capacity to not only colonize tissue as an extracellular pathogen, but to invade host cells and establish intracellular bacterial populations. In previous work, we identified host CDC42 as a central regulator of endothelial cell invasion by S. aureus. In the current work, we report that ML 141, a first-in-class CDC42 inhibitor, decreases invasion and resultant pathogenesis in a dose-dependent and reversible manner. Inhibition was found to be due in part to decreased remodeling of actin that potentially drives endocytic uptake of bacteria/fibronectin/integrin complexes. ML 141 decreased binding to fibronectin at these complexes, thereby limiting a key pathogenic mechanism used by S. aureus to invade. Structural analogs of ML 141 were synthesized (designated as the RSM series) and a subset identified that inhibit invasion through non-cytotoxic and non-bactericidal mechanisms. Our results support the development of adjunctive therapeutics targeting host CDC42 for mitigating invasive infection at the level of the host.

A complete conformational analysis of the keto and enol forms of ethyl benzoylacetate (EBA), a β-ketoester, was carried out by ab initio calculations, at the density functional theory (DFT) level. The relative stabilities of cis-enol and keto forms were calculated in the gas phase and in solution. The intramolecular hydrogen bond characters of the most stable enol forms of EBA are discussed and compared with those of benzoylacetone (BA).Harmonic vibrational frequencies and 1H and 13C NMR chemical shifts of the most stable enol and keto forms were also calculated at the B3LYP/6-311++G** level and compared with the experimental data.

A complete conformational analysis of the keto and enol forms of benzyl acetoacetate (BAA), a β-dicarbonyl compound, was carried out by ab initio calculations, at the density functional theory (DFT) level. By inspection of all possible conformers and tautomers, 22 stable cis-enol, 28 stable trans-enol, and five keto conformers were obtained.Among all stable cis-enol forms only six of them are engaged in intramolecular hydrogen bond. The hydrogen bond strength of the most stable conformer of BAA is compared with that of acetylacetone (AA) and dimethyl oxaloacetate (DMOA). Harmonic vibrational frequencies of the most stable enol and keto forms and their deuterated analogues were also calculated and compared with the experimental data.According to the theoretical calculations, the hydrogen bond strength of the most stable enol conformer of BAA is 56.7kJ/mol (calculated at the B3LYP/6-311++G∗∗ level), about 10kJ/mol less than that of AA. This weakening of hydrogen bond is consistent with the spectroscopic results. NMR studies indicate that BAA exists mainly as a keto tautomer in all considered solutions.The Gibbs energies for keto/enol tautomerization were calculated at the B3LYP level, with several basis sets, in both gas phase and CH3CN solution (using PCM model), for the most stable enol and keto conformers.