Ursolic Acid Conjugates: A New Frontier in Anticancer Drug Development.

New Ursolic Acid (UA) conjugates were synthesized using optimized synthetic protocols through the molecular hybridization approach at C-3 and C-28. This resulted in the targeted molecules being produced in good yields. Some of the synthesized conjugates showed significantly relevant bioactivity against mammalian cells and in animal models of cancers. Selected UA conjugates were tested against bladder and breast cancer cell lines. The conjugates showed moderate to significantly enhanced antiproliferative activities against Triple Negative Breast Cancer (TNBC; MDA-MB 231), which is an aggressive tumor making up about 10-15 % of all breast cancers and bladder (T24 and 5637) cancer cell lines. These properties were superior to the parent UA. Among all the synthesized compounds, 18 c and 18 d have exhibited promising antiproliferative and cytotoxic properties against all tested cancer cell lines. However, 18 d has proved to be exceptionally selective for cancer cell lines, showing more cytotoxicity towards them than normal epithelial cells (MCF-12A). Compound 18 d has demonstrated cytotoxicity against tumor cells, including those intrinsically resistant to chemotherapy drugs such as 2-difluoro-deoxy cytidine (Gemcitabine). The activity of the UA conjugates on tumor cells was mediated by multiple cytotoxic mechanisms, including drug-induced cytotoxic autophagy and programmed cell death, indicating a novel possibility of combination therapy.

Characterization of mRNA therapeutics.

Therapeutic messenger RNAs (mRNAs) have emerged as powerful tools in the treatment of complex diseases, especially for conditions that lack efficacious treatment. The successful application of this modality can be attributed to its ability to encode entire proteins. While the large nature of these molecules has supported their success as therapeutics, its extended size creates several analytical challenges. To further support therapeutic mRNA development and its deployment in clinical trials, appropriate methods to support their characterization must be developed. In this review, we describe current analytical methods that have been used in the characterization of RNA quality, identity, and integrity. Advantages and limitations from several analytical techniques ranging from gel electrophoresis to liquid chromatography-mass spectrometry and from shotgun sequencing to intact mass measurements are discussed. We comprehensively describe the application of analytical methods in the measurements of capping efficiency, poly A tail analysis, as well as their applicability in stability studies.

Considerations and perspectives on the practice of incurred sample reanalysis assessment for chiral drugs.

Given the inherent complexities of bioanalysis, the role of incurred sample reanalysis (ISR) is increasingly appreciated in regulatory bioanalysis. Incurred sample reanalysis has evolved as an integral part of an assay to ensure method reproducibility. The current regulatory ISR guidelines do not provide clarity regarding ISR assessment for chiral drugs comprising enantiomers. Because chiral assays evaluate two enantiomers, there are additional complexities associated with the ISR data generation and interpretation. Based on the current literature, the practices for conducting ISR in chiral methods were reviewed and assessed. While ISR was conducted in chiral methods for both enantiomers using the acceptance criteria prescribed for non-chiral methods, there may be a need to streamline the nuances of ISR data interpretation and define the ISR requirements for chiral methods. The article provides perspectives on the ISR of enantiomeric drugs, including strategy development, by providing various hypothetical scenarios and possible considerations for defining ISR evaluation for chiral assays.

Identification of lysine isobutyrylation as a new histone modification mark.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Drug-Induced Conformational Dynamics of P-Glycoprotein Underlies the Transport of Camptothecin Analogs.

P-glycoprotein (Pgp) plays a pivotal role in drug bioavailability and multi-drug resistance development. Understanding the protein's activity and designing effective drugs require insight into the mechanisms underlying Pgp-mediated transport of xenobiotics. In this study, we investigated the drug-induced conformational changes in Pgp and adopted a conformationally-gated model to elucidate the Pgp-mediated transport of camptothecin analogs (CPTs). While Pgp displays a wide range of conformations, we simplified it into three model states: 'open-inward', 'open-outward', and 'intermediate'. Utilizing acrylamide quenching of Pgp fluorescence as a tool to examine the protein's tertiary structure, we observed that topotecan (TPT), SN-38, and irinotecan (IRT) induced distinct conformational shifts in the protein. TPT caused a substantial shift akin to AMPPNP, suggesting ATP-independent 'open-outward' conformation. IRT and SN-38 had relatively moderate effects on the conformation of Pgp. Experimental atomic force microscopy (AFM) imaging supports these findings. Further, the rate of ATPase hydrolysis was correlated with ligand-induced Pgp conformational changes. We hypothesize that the separation between the nucleotide-binding domains (NBDs) creates a conformational barrier for substrate transport. Substrates that reduce the conformational barrier, like TPT, are better transported. The affinity for ATP extracted from Pgp-mediated ATP hydrolysis kinetics curves for TPT was about 2-fold and 3-fold higher than SN-38 and IRT, respectively. On the contrary, the dissociation constants (KD) determined by fluorescence quenching for these drugs were not significantly different. Saturation transfer double difference (STDD) NMR of TPT and IRT with Pgp revealed that similar functional groups of the CPTs are accountable for Pgp-CPTs interactions. Efforts aimed at modifying these functional groups, guided by available structure-activity relationship data for CPTs and DNA-Topoisomerase-I complexes, could pave the way for the development of more potent next-generation CPTs.

BIOANALYSIS AND BIOTRANSFORMATION OF OLIGONUCLEOTIDE THERAPEUTICS BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY.

Since 2016, eight new oligonucleotide therapies have been approved which has led to increased interest in oligonucleotide analysis. There is a particular need for powerful bioanalytical tools to study the metabolism and biotransformation of these molecules. This review provides the background on the biological basis of these molecules as currently used in therapies. The article also reviews the current state of analytical methodology including state of the art sample preparation techniques, liquid chromatography-mass spectrometry methods, and the current limits of detection/quantitation. Finally, the article summarizes the challenges in oligonucleotide bioanalysis and provides future perspectives for this emerging field. © 2020 John Wiley & Sons Ltd.

Evaluation of alkylamines and stationary phases to improve LC-MS of oligonucleotides.

This study evaluated four bridged-ethylene hybrid (BEH) columns containing C18 (130 Å), peptide C18 (300 Å), phenyl, or a mixed-mode charged surface hybrid (CSH C18 ) using a wide range of antisense oligonucleotide therapeutics. The BEH C18 , peptide, and phenyl columns were all capable of providing significant retention of oligonucleotide samples across multiple ion-pairing systems using alkylamines and 1,1,1,3,3,3,-hexafluoroisopropanol (HFIP). The retention of the oligonucleotides varied depending on the choice of alkylamine, with the order of retention being dimethylcyclohexylamine > diisopropylethylamine > triethylamine. The selectivity of these columns for several closely eluting impurities was similar. Although overall the C18 , peptide, and phenyl columns were all found to be capable of analyzing oligonucleotide therapeutics, the phenyl column was found to be the most retentive and the C18 column provided the best peak shape. The CSH C18 column was found to be degraded by the alkylamine-HFIP mobile phase despite the mobile phase being within the pH stability range of the column.

Assessment of brain-to-blood drug distribution using liquid chromatography.

Delivery of already existing and new drugs under development to the brain necessitates passage across the blood-brain barrier (BBB) with its tight intercellular junctions, molecular components and transporter systems. Consequently, it is critical to identify the extent of brain permeation and the partitioning across the BBB. The interpretation of brain-to-blood ratios is considered to be a significant and fundamental approach for estimating drug penetration through BBB, the brain-targeting ability and central nervous system (CNS) pharmacokinetics. Among the different bioanalytical techniques, liquid chromatography with various detectors has been widely used for determination of these ratios. This review defines the different approaches for sample preparation, extraction techniques and liquid chromatography procedures concerned with the determination of drugs in blood and brain tissues and the assessment of brain-to-blood levels. These approaches are expanded to cover the analysis of several drug classes such as CNS-acting drugs, chemotherapeutics, antidiabetics, herbal medicinal products, radiopharmaceuticals, antibiotics and antivirals. Accordingly, stability in biological matrices and matrix effects are investigated. The different administration/formulation effects and the possible deviations in these ratios are also disscussed.

Modeling cationic adduction of oligonucleotides using electrospray desorption ionization.

RATIONALE: Cationic adduction causes poor sensitivity and increases spectral complexity during mass spectral analysis of oligonucleotides and alkylamines are used to reduce this adduction. It is unclear the effect of the physiochemical properties of the alkylamines on the reduction of the cationic adduction. METHODS: All samples were directly infused into a Synapt G2 HDMS quadrupole time-of-flight (TOF) hybrid mass spectrometer in negative ion electrospray ionization mode through the native built-in fluidics system. The infusion flow rate was set to 50 µL/min. The TOFMS tuning parameters were as follows: capillary voltage -2.0 kV, cone voltage 25 V, extraction cone voltage 2 V, source temperature 125°C, desolvation temperature 450°C, cone gas flow rate 0 L/h, and desolvation gas (nitrogen) flow rate 1000 L/h. RESULTS: A quantitative model was created to predict the optimized alkylamine for MS analysis, while a qualitative model was generated to explain the most important physiochemical properties: proton affinity (13.83%), gas-phase basicity (11.79%), pKa (11.47%), boiling point (10.73%), MW (10.3%), Henry's Law Constant (9.56%), and partition coefficient (logP) (9.44%). The quantitative model was applied to RNA (microRNA) and a phosphorothioate and predicts the trend of cationic adduction. CONCLUSIONS: Two models are described to understand the physiochemical properties that contribute to the adduction and to provide users a quick mathematical tool to predict the best choice of alkylamine to lower cationic adduction and decrease spectral complexity while enhancing sensitivity.

Recent advances in liquid chromatographic methods for the determination of selective serotonin reuptake inhibitors and serotonin norepinephrine reuptake inhibitors.

Depression is now the second largest public health burden throughout the world. Selective serotonin reuptake inhibitors (SSRIs) and serotonin norepinephrine reuptake inhibitors (SNRIs) have replaced older antidepressants to become first-line medications to treat this disease with increased remission rates and markedly decreased incidence of severe adverse events. Traditional and modern bioanalytical strategies for SSRI and SNRI determination are being continuously improved. There has also been a recent increase in the use of unconventional sample preparation methods. This review critically evaluates the development of SSRI and SNRI liquid chromatographic analytical methods published between 2014 and mid-2019, with special attention to novel sample preparation methods.

In vitro metabolism of 2'-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry.

Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC-tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3'-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5'-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.

Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first).

Short-chain acylation of lysine residues has recently emerged as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which shows that the function of MOF goes far beyond its canonical histone H4 lysine 16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl-coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together define a novel function for the MYST KATs as lysine propionyltransferases and suggest much broader physiological impacts for this family of enzymes.

Dietary palmitate cooperates with Src kinase to promote prostate tumor progression.

Numerous genetic alterations have been identified during prostate cancer progression. The influence of environmental factors, particularly the diet, on the acceleration of tumor progression is largely unknown. Expression levels and/or activity of Src kinase are highly elevated in numerous cancers including advanced stages of prostate cancer. In this study, we demonstrate that high-fat diets (HFDs) promoted pathological transformation mediated by the synergy of Src and androgen receptor in vivo. Additionally, a diet high in saturated fat significantly enhanced proliferation of Src-mediated xenograft tumors in comparison with a diet high in unsaturated fat. The saturated fatty acid palmitate, a major constituent in a HFD, significantly upregulated the biosynthesis of palmitoyl-CoA in cancer cells in vitro and in xenograft tumors in vivo. The exogenous palmitate enhanced Src-dependent mitochondrial ß-oxidation. Additionally, it elevated the amount of C16-ceramide and total saturated ceramides, increased the level of Src kinase localized in the cell membrane, and Src-mediated downstream signaling, such as the activation of mitogen-activated protein kinase and focal adhesion kinase. Our results uncover how the metabolism of dietary palmitate cooperates with elevated Src kinase in the acceleration of prostate tumor progression.

Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.

Mass Spectrometric Quantitation of Tubulin Acetylation from Pepsin-Digested Rat Brain Tissue Using a Novel Stable-Isotope Standard and Capture by Anti-Peptide Antibody (SISCAPA) Method.

Acetylation of α-tubulin at Lys-40 is a potential biomarker for cognitive deficits in various neurological disorders. However, this key post-translational modification (PTM) has not been previously studied with mass spectrometry, due to the inadequate distribution of tryptic cleavage sites. Following peptic digestion, a surrogate sequence containing this key PTM site was identified and was found to be stable and quantitatively reproducible. A highly sensitive and specific SISCAPA-LC-MS method for quantitating rat brain tubulin acetylation was developed, validated, and applied, and only required a small amount of tissue (2.2 mg). This workflow includes peptic digestion, stable-isotope dilution, capture with antiacetylated peptide antibody bound on protein G beads, and quantitation using LC-MS. The method allowed a lower limit of quantitation at 2.50 pmol/mg and provided a linear range of 2.50-62.50 pmol/mg. Selectivity, intra and interday precision and accuracy were also validated. This method has been successfully applied in a preclinical study of organophosphate neurotoxicity, and we found that chronic exposure to chlorpyrifos led to a significant and persistent inhibition of brain tubulin acetylation.

Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities.

Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and multistep synthesis and purification processes, generation of low-level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency. In this review, the different types of oligonucleotide impurities and degradation products, with an emphasis on their origin, mechanism of formation and methods to reduce, prevent or even eliminate their production, will be extensively discussed. This review will focus mainly on the application of chromatographic techniques to determine these impurities but will also discuss other approaches such as mass spectrometry, capillary electrophoresis and nuclear magnetic resonance spectroscopy. Finally, the chirality and formation of diastereomer mixtures of PS oligonucleotides will be covered as well as approaches used for their characterization and the application for the development of stereochemically-controlled PS oligonucleotides.

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of two novel highly lipophilic anticancer drug candidates in rat plasma and tissues.

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for determination of two highly lipophilic anticancer drug candidates, LG1980 and GH501, in rat plasma and tissues (liver, kidney and femur bones). LG1980 and GH501 were extracted from rat plasma and tissue homogenates using liquid-liquid extraction. The method provided a linear range of 1.0-200.0 ng/mL for GH501 in plasma and LG1980 in plasma and liver. For both analytes in other tissue homogenates the linear range was 2.0-400.0 ng/mL. The method was validated with precision within 15% relative standard deviation, accuracy within 15% relative error and a consistent recovery. This method has been successfully applied in two preclinical studies for LG1980 and GH501 to determine their concentrations in rat plasma, liver, kidney and bone over 24 h after intravenous injection of compounds.

Development of a Method for the Determination of Acyl-CoA Compounds by Liquid Chromatography Mass Spectrometry to Probe the Metabolism of Fatty Acids.

Acyl-Coenzyme As (acyl-CoAs) are a group of activated fatty acid molecules participating in multiple cellular processes including lipid synthesis, oxidative metabolism of fatty acids to produce ATP, transcriptional regulation, and protein post-translational modification. Quantification of cellular acyl-CoAs is challenging due to their instability in aqueous solutions and lack of blank matrices. Here we demonstrate an LC-MS/MS analytical method which allows for absolute quantitation with broad coverage of cellular acyl-CoAs. This assay was applied to profile endogenous acyl-CoAs under the challenge of a variety of dietary fatty acids in prostate and hepatic cells. Additionally, this approach allowed for detection of multiple fatty acid metabolic processes including the biogenesis of acyl-CoAs, and their elongation, degradation, and desaturation. Hierarchical clustering in the remodeling of acyl-CoA profiles revealed a fatty-acid-specific pattern across all tested cell lines, which provides a valuable reference for making predictions in other cell models. Individual acyl-CoAs were identified which were altered differentially by exogenous fatty acids in divergent tumorigenicity states of cells. These findings demonstrate the power of acyl-CoA profiling toward understanding the mechanisms for the progression of tumors or other diseases in response to fatty acids.