Structure-Based Drug Design of ADRA2A Antagonists Derived from Yohimbine.

Yohimbine, a natural indole alkaloid and a nonselective adrenoceptor antagonist, possesses potential benefits in treating inflammatory disorders and sepsis. Nevertheless, its broader clinical use faces challenges due to its low receptor selectivity. A structure-activity relationship study of novel yohimbine analogues identified amino esters of yohimbic acid as potent and selective ADRA2A antagonists. Specifically, amino ester 4n, in comparison to yohimbine, showed a 6-fold higher ADRA1A/ADRA2A selectivity index (SI > 556 for 4n) and a 25-fold higher ADRA2B/ADRA2A selectivity index. Compound 4n also demonstrated high plasma and microsomal stability, moderate-to-low membrane permeability determining its limited ability to cross the blood-brain barrier, and negligible toxicity on nontumor normal human dermal fibroblasts. Compound 4n represents an important complementary pharmacological tool to study the involvement of adrenoceptor subtypes in pathophysiologic conditions such as inflammation and sepsis and a novel candidate for further preclinical development to treat ADRA2A-mediated pathologies.

Inhibition of FLT3-ITD Kinase in Acute Myeloid Leukemia by New Imidazo[1,2-b]pyridazine Derivatives Identified by Scaffold Hopping.

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

Gas Dynamic Virtual Nozzle Sprayer for an Introduction of Liquid Samples in Atmospheric Pressure Ionization Mass Spectrometry.

Electrospray may exhibit inadequate ionization efficiency in some applications. In such cases, atmospheric-pressure chemical ionization (APCI) and photoionization (APPI) can be used. Despite a wide application potential, no APCI and APPI sources dedicated to very low sample flow rates exist on the market. Since the ion source performance depends on the transfer of analytes from the liquid to the gas phase, a nebulizer is a critical component of an ion source. Here, we report on the nebulizer with a gas dynamic virtual nozzle (GDVN) and its applicability in APCI at microliter-per-minute flow rates. Nebulizers differing by geometrical parameters were fabricated and characterized regarding the jet breakup regime, droplet size, droplet velocity, and spray angle for liquid flow rates of 0.75-15.0 µL/min. A micro-APCI source with the GDVN nebulizer behaved as a mass-flow-sensitive detector and provided stable and intense analyte signals. Compared to a classical APCI source, an order of magnitude lower detection limit for verapamil was achieved. Mass spectra recorded with the nebulizer in dripping and jetting modes were almost identical and did not differ from normal APCI spectra. Clogging never occurred during the experiments, indicating the high robustness of the nebulizer. Low-flow-rate APCI and APPI sources with a GDVN sprayer promise new applications for low- and medium-polar analytes.

Thermodynamic and structural characterization of an optimized peptide-based inhibitor of the influenza polymerase PA-PB1 subunit interaction.

Influenza virus causes severe respiratory infection in humans. Current antivirotics target three key proteins in the viral life cycle: neuraminidase, the M2 channel and the endonuclease domain of RNA-dependent-RNA polymerase. Due to the development of novel pandemic strains, additional antiviral drugs targetting different viral proteins are still needed. The protein-protein interaction between polymerase subunits PA and PB1 is one such possible target. We recently identified a modified decapeptide derived from the N-terminus of the PB1 subunit with high affinity for the C-terminal part of the PA subunit. Here, we optimized its amino acid hotspots to maintain the inhibitory potency and greatly increase peptide solubility. This allowed thermodynamic characterization of peptide binding to PA. Solving the X-ray structure of the peptide-PA complex provided structural insights into the interaction. Additionally, we optimized intracellular delivery of the peptide using a bicyclic strategy that led to improved inhibition in cell-based assays.

Discovery of Modified Amidate (ProTide) Prodrugs of Tenofovir with Enhanced Antiviral Properties.

This study describes the discovery of novel prodrugs bearing tyrosine derivatives instead of the phenol moiety present in FDA-approved tenofovir alafenamide fumarate (TAF). The synthesis was optimized to afford diastereomeric mixtures of novel prodrugs in one pot (yields up to 86%), and the epimers were resolved using a chiral HPLC column into fast-eluting and slow-eluting epimers. In human lymphocytes, the most efficient tyrosine-based prodrug reached a single-digit picomolar EC50 value against HIV-1 and nearly 300-fold higher selectivity index (SI) compared to TAF. In human hepatocytes, the most efficient prodrugs exhibited subnanomolar EC50 values for HBV and up to 26-fold higher SI compared to TAF. Metabolic studies demonstrated markedly higher cellular uptake of the prodrugs and substantially higher levels of released tenofovir inside the cells compared to TAF. These promising results provide a strong foundation for further evaluation of the reported prodrugs and their potential utility in the development of highly potent antivirals.

Temperature-programmed capillary high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry for analysis of fatty acid methyl esters.

A new capillary high-performance liquid chromatography method with atmospheric pressure chemical ionization mass spectrometry was developed for the analysis of fatty acid methyl esters and long-chain alcohols. The chromatographic separation was achieved using a Zorbax SB-C18 HPLC column (0.3 × 150 mm, 3.5 µm) with a mobile phase composed of acetonitrile and formic acid and delivered isocratically at a flow rate of 10 µL/min. The column temperature was programmed simply, using a common column oven. Good reproducibility of the temperature profile and retention times were achieved. The temperature programming during the isocratic high-performance liquid chromatography run had a similar effect as a solvent gradient; it reduced retention times of later eluting analytes and improved their detection limits. Two atmospheric pressure chemical ionization sources of the mass spectrometry detector were compared: an enclosed conventional ion source and an in-house made ion source with a glass microchip nebulizer. The enclosed source provided better detectability of saturated fatty acid methyl esters and made it possible to determine the double bond positions using acetonitrile-related adducts, while the open chip-based source provided better analytical figures of merit for unsaturated fatty acid methyl esters. Temperature-programmed capillary high-performance liquid chromatography is a promising method for analyzing neutral lipids in lipidomics and other applications.

Atmospheric pressure chemical ionization mass spectrometry at low flow rates: Importance of ion source housing.

RATIONALE: Hyphenation of atmospheric pressure chemical ionization (APCI) mass spectrometry with capillary and micro high-performance liquid chromatography (HPLC) is attractive for many applications, but reliable ion sources dedicated to these conditions are still missing. There are a number of aspects to consider when designing such an ion source, including the susceptibility of the ionization processes to ambient conditions. Here we discuss the importance of ion source housing for APCI at low flow rates. METHODS: Selected compounds dissolved in various solvents were used to study ionization reactions at 10 µL/min flow rate. APCI spectra were generated using the Ion Max-S source (Thermo Fisher Scientific) operated with or without the ion source housing. RESULTS: The APCI spectra of most compounds measured in the open and enclosed ion sources were markedly different. The differences were explained by water and oxygen molecules that entered the plasma region of the open ion source. Water tended to suppress charge transfer processes while oxygen diminished electron capture reactions and prevented the formation of acetonitrile-related radical cations useful for localizing double bonds in lipids. The effects associated with the ion source housing were significantly less important for compounds that are easy to protonate or deprotonate. CONCLUSIONS: The use of ion source housing prevented alternative ionization channels leading to unwanted or unexpected ions. Compared with the conventional flow rate mode (1 mL/min), the effects of ambient air components were significantly higher at 10 µL/min, emphasizing the need for ion source housing in APCI sources dedicated to low flow rates.

Applicability of low-flow atmospheric pressure chemical ionization and photoionization mass spectrometry with a microfabricated nebulizer for neutral lipids.

RATIONALE: Mass spectrometry with atmospheric pressure chemical ionization (APCI) or photoionization (APPI) is widely used for neutral lipids involved in many fundamental processes in living organisms. Commercial APCI and APPI sources operate at high flow rates compatible with conventional high-performance liquid chromatography (HPLC). However, lipid analysis is often limited by a small amount of sample, which requires low flow rate separations like capillary or micro-HPLC. Therefore, APCI and APPI suitable for microliter-per-minute flow rates need to be developed and applied for neutral lipids. METHODS: A micro-APCI/APPI source with a heated chip nebulizer was assembled and mounted on a Thermo ion trap instrument. The ion source operated in APCI, APPI or dual mode was optimized for low microliter-per-minute sample flow rates. The source performance was investigated for squalene, wax esters, fatty acid methyl esters, triacylglycerols, and cholesterol. RESULTS: The ion source behaved as a mass-flow-sensitive detector. Direct infusion of methyl oleate showed superior analytical figures of merit when compared with high-flow ion sources. A detection limit of 200 pmol/mL and a linear dynamic range spanning three orders of magnitude were measured for micro-APCI. The mass spectra of most lipids differed from high flow rate spectra. Unlike micro-APCI, micro-APPI spectra were complicated by odd-electron species. Dual APCI/APPI mode did not show any benefits for neutral lipids. Applications for lipid samples were demonstrated. CONCLUSIONS: Micro-APCI-MS is a useful detection technique for neutral lipids at microliter-per-minute flow rates. It offers high sensitivity and high quality of spectra in direct infusion mode and promises successful utilization in capillary and micro-HPLC applications.