GPC-100, a novel CXCR4 antagonist, improves in vivo hematopoietic cell mobilization when combined with propranolol.

Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits ß-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (ß2AR). Co-treatment with CXCL12 and the ß2AR agonist epinephrine synergistically increases ß-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.

Optimization of multi-enzyme cascade process for the biosynthesis of benzylamine.

Benzylamine is a valuable intermediate in the synthesis of organic compounds such as curing agents and antifungal drugs. To improve the efficiency of benzylamine biosynthesis, we identified the enzymes involved in the multi-enzyme cascade, regulated the expression strength by using RBS engineering in Escherichia coli, and established a regeneration-recycling system for alanine. This is a cosubstrate, coupled to cascade reactions, which resulted in E. coli RARE-TP and can synthesize benzylamine using phenylalanine as a precursor. By optimizing the supply of cosubstrates alanine and ammonia, the yield of benzylamine produced by whole-cell catalysis was increased by 1.5-fold and 2.7-fold, respectively, and the final concentration reached 6.21 mM. In conclusion, we achieved conversion from l-phenylalanine to benzylamine and increased the yield through enzyme screening, expression regulation, and whole-cell catalytic system optimization. This demonstrated a green and sustainable benzylamine synthesis method, which provides a reference and additional information for benzylamine biosynthesis research.

Economic evaluation of plerixafor addition in the mobilization and leukapheresis of hematopoietic stem cells for autologous transplantation: a systematic review.

INTRODUCTION: Although plerixafor in association with granulocyte colony-stimulating factor (G-CSF) can improve mobilization and collection of hematopoietic stem cells (HSC) by leukapheresis, cost may limit its clinical application. The present study systematically reviews economic evaluations of plerixafor plus G-CSF usage compared to G-CSF alone and compares different strategies of plerixafor utilization in multiple myeloma and lymphoma patients eligible for autologous HSC transplantation. AREAS COVERED: Relevant economic evaluations, partial or complete, were searched on PubMed, Embase, LILACS, and Cochrane Central Register of Controlled Trials for a period ending 30 June 2021. This systematic review was reported following the PRISMA Statement. Six economic evaluations were included, considering the use of upfront or just-in-time plerixafor compared to G-CSF alone or other plerixafor strategies. Most comparisons showed both increased cost and health benefits with the addition of plerixafor. Most analyses favored just-in-time plerixafor compared to upfront plerixafor, with a probable preference for broader cutoffs for just-in-time plerixafor initiation. EXPERT OPINION: Plerixafor is a potentially cost-effective technology in the mobilization of HSC in patients with multiple myeloma and lymphomas eligible for autologous HSC transplantation. There is a decreased number of leukapheresis sessions and remobilizations and a higher yield of CD34+ cells.

Effects of Chemical Structures Interacting with Amine Oxidases on Glucose, Lipid and Hydrogen Peroxide Handling by Human Adipocytes.

Benzylamine is a natural molecule present in food and edible plants, capable of activating hexose uptake and inhibiting lipolysis in human fat cells. These effects are dependent on its oxidation by amine oxidases present in adipocytes, and on the subsequent hydrogen peroxide production, known to exhibit insulin-like actions. Virtually, other substrates interacting with such hydrogen peroxide-releasing enzymes potentially can modulate lipid accumulation in adipose tissue. Inhibition of such enzymes has also been reported to influence lipid deposition. We have therefore studied in human adipocytes the lipolytic and lipogenic activities of pharmacological entities designed to interact with amine oxidases highly expressed in this cell type: the semicarbazide-sensitive amine oxidase (SSAO also known as PrAO or VAP-1) and the monoamine oxidases (MAO). The results showed that SZV-2016 and SZV-2017 behaved as better substrates than benzylamine, releasing hydrogen peroxide once oxidized, and reproduced or even exceeded its insulin-like metabolic effects in fat cells. Additionally, several novel SSAO inhibitors, such as SZV-2007 and SZV-1398, have been evidenced and shown to inhibit benzylamine metabolic actions. Taken as a whole, our findings reinforce the list of molecules that influence the regulation of triacylglycerol assembly/breakdown, at least in vitro in human adipocytes. The novel compounds deserve deeper investigation of their mechanisms of interaction with SSAO or MAO, and constitute potential candidates for therapeutic use in obesity and diabetes.

Identification and Strategies to Mitigate High Total Clearance of Benzylamine-Substituted Biphenyl Ring Systems.

For most oral small-molecule projects within drug discovery, the extent and duration of the effect are influenced by the total clearance of the compound; hence, designing compounds with low clearance remains a key focus to help enable sufficient protein target engagement. Comprehensive understanding and accurate prediction of animal clearance and pharmacokinetics provides confidence that the same can be observed for human. During a MERTK inhibitor lead optimization project, a series containing a biphenyl ring system with benzylamine meta-substitution on one phenyl and nitrogen inclusion as the meta atom on the other ring demonstrated multiple routes of compound elimination in rats. Here, we describe the identification of a structural pharmacophore involving two key interactions observed for both the MERTK program and an additional internal project. Four strategies to mitigate these clearance liabilities were identified and systematically investigated. We provide evidence that disruption of at least one of the interactions led to a significant reduction in CL that was subsequently predicted from rat hepatocytes using in vitro/in vivo extrapolation and the well-stirred scaling method. These tactics will likely be of general utility to the medicinal chemistry and DMPK community during compound optimization when similar issues are encountered for biphenyl benzylamines.

The Impact of Mavacamten on the Pathophysiology of Hypertrophic Cardiomyopathy: A Narrative Review.

Hypertrophic cardiomyopathy (HCM) is a chronic, progressive disease of the cardiomyocyte with a diverse and heterogeneous clinical presentation and course. This diversity and heterogeneity have added to the complexity of modeling the pathophysiological pathways that contribute to the disease burden. The development of novel therapeutic approaches targeting precise mechanisms within the underlying biology of HCM provides a tool to model and test these pathways. Here, we integrate the results of clinical observations with mavacamten, an allosteric, selective, and reversible inhibitor of cardiac myosin, the motor unit of the sarcomere, to develop an integrated pathophysiological pathway model of HCM, confirming the key role of excess sarcomeric activity. This model may serve as a foundation to understand the role of HCM pathophysiological pathways in the clinical presentation of the disease, and how a targeted therapeutic intervention capable of normalizing sarcomeric activity and repopulating low-energy utilization states may reduce the impact of these pathways in HCM and potentially related disease states.

Prevalence of toxigenic fungi and mycotoxins in Arabic coffee (Coffea arabica): Protective role of traditional coffee roasting, brewing and bacterial volatiles.

Fungal infection and synthesis of mycotoxins in coffee leads to significant economic losses. This study aimed to investigate the prevalence of toxigenic fungi, their metabolites, and the effect of traditional roasting and brewing on ochratoxin A (OTA) and aflatoxins (AFs) contents of naturally contaminated coffee samples. In addition, in vivo biocontrol assays were performed to explore the antagonistic activities of Bacillus simplex 350-3 (BS350-3) on the growth and mycotoxins synthesis of Aspergillus ochraceus and A. flavus. The relative density of A. niger, A. flavus, Penicillium verrucosum and A. carbonarius on green coffee bean was 60.82%, 7.21%, 3.09% and 1.03%, respectively. OTA contents were lowest in green coffee beans (2.15 µg/kg), followed by roasted (2.76 µg/kg) and soluble coffee (8.95 µg/kg). Likewise, AFs levels were highest in soluble coffee (90.58 µg/kg) followed by roasted (33.61 µg/kg) and green coffee (9.07 µg/kg). Roasting naturally contaminated coffee beans at three traditional methods; low, medium and high, followed by brewing resulted in reduction of 58.74% (3.50 µg/kg), 60.88% (3.72 µg/kg) and 64.70% (4.11 µg/kg) in OTA and 40.18% (34.65 µg/kg), 47.86% (41.17 µg/kg) and 62.38% (53.73 µg/kg) AFs contents, respectively. Significant inhibitions of AFs and OTA synthesis by A. flavus and A. carbonarius, respectively, on infected coffee beans were observed in presence of Bacillus simplex BS350-3 volatiles. Gas chromatography mass spectrochemistry (GC-MS/MS) analysis of head-space BS350-3 volatiles showed quinoline, benzenemethanamine and 1-Octadecene as bioactive antifungal molecules. These findings suggest that marketed coffee samples are generally contaminated with OTA and AFs, with a significant level of roasted and soluble coffee contaminated above EU permissible limits for OTA. Further, along with coffee roasting and brewing; microbial volatiles can be optimized to minimize the dietary exposure to mycotoxins.

Four-Step Pathway from Phenylpyruvate to Benzylamine, an Intermediate to the High-Energy Propellant CL-20.

Benzylamine is a commodity chemical used in the synthesis of motion-sickness treatments and anticonvulsants, in dyeing textiles, and as a precursor to the high-energy propellant CL-20. Because chemical production generates toxic waste streams, biosynthetic alternatives have been explored, recently resulting in a functional nine-step pathway from central metabolism (phenylalanine) in E. coli. We report a novel four-step pathway for benzylamine production, which generates the product from cellular phenylpyruvate using enzymes from different sources: a mandelate synthase (Amycolatopsis orientalis), a mandelate oxidase (Streptomyces coelicolor), a benzoylformate decarboxylase (Pseudomonas putida), and an aminotransferase (Salicibacter pomeroyi). This pathway produces benzylamine at 24 mg/L in 15 h (4.5% yield) in cultures of unoptimized cells supplemented with phenylpyruvate. Because the yield is low, supplementation with pathway intermediates is used to troubleshoot the design. This identifies conversion inefficiencies in the mandelate synthase-mediated synthesis of (S)-mandelic acid, and subsequent genome mining identifies a new mandelate synthase (Streptomyces sp. 1114.5) with improved yield. Supplementation experiments also reveal native redirection of ambient phenylpyruvate away from the pathway to phenylalanine. Overall, this work illustrates how retrosynthetic design can dramatically reduce the number of enzymes in a pathway, potentially reducing its draw on cellular resources. However, it also shows that such benefits can be abrogated by inefficiencies of individual conversions. Addressing these barriers can provide an alternative approach to green production of benzylamine, eliminating upstream dependence on chlorination chemistry.

Oral Supplementation with Benzylamine Delays the Onset of Diabetes in Obese and Diabetic db-/- Mice.

Substrates of semicarbazide-sensitive amine oxidase (SSAO) exert insulin-like actions in adipocytes. One of them, benzylamine (Bza) exhibits antihyperglycemic properties in several rodent models of diabetes. To further study the antidiabetic potential of this naturally occurring amine, a model of severe type 2 diabetes, the obese db-/- mouse, was subjected to oral Bza administration. To this end, db-/- mice and their lean littermates were treated at 4 weeks of age by adding 0.5% Bza in drinking water for seven weeks. Body mass, fat content, blood glucose and urinary glucose output were followed while adipocyte insulin responsiveness and gene expression were checked at the end of supplementation, together with aorta nitrites. Bza supplementation delayed the appearance of hyperglycemia, abolished polydypsia and glycosuria in obese/diabetic mice without any detectable effect in lean control, except for a reduction in food intake observed in both genotypes. The improvement of glucose homeostasis was observed in db-/- mice at the expense of increased fat deposition, especially in the subcutaneous white adipose tissue (SCWAT), without sign of worsened inflammation or insulin responsiveness and with lowered circulating triglycerides and uric acid, while NO bioavailability was increased in aorta. The higher capacity of SSAO in oxidizing Bza in SCWAT, found in the obese mice, was unaltered by Bza supplementation and likely involved in the activation of glucose utilization by adipocytes. We propose that Bza oxidation in tissues, which produces hydrogen peroxide mainly in SCWAT, facilitates insulin-independent glucose utilization. Bza could be considered as a potential agent for dietary supplementation aiming at preventing diabetic complications.

Novel Facet of an Old Dietary Molecule? Direct Influence of Caffeine on Glucose and Biogenic Amine Handling by Human Adipocytes.

Caffeine is a plant alkaloid present in food and beverages consumed worldwide. It has high lipid solubility with recognized actions in the central nervous system and in peripheral tissues, notably the adipose depots. However, the literature is scant regarding caffeine's influence on adipocyte functions other than lipolysis, such as glucose incorporation into lipids (lipogenesis) and amine oxidation. The objective of this study was to explore the direct effects of caffeine and of isobutylmethylxanthine (IBMX) on these adipocyte functions. Glucose transport into fat cells freshly isolated from mice, rats, or humans was monitored by determining [3H]-2-deoxyglucose (2-DG) uptake, while the incorporation of radiolabeled glucose into cell lipids was used as an index of lipogenic activity. Oxidation of benzylamine by primary amine oxidase (PrAO) was inhibited by increasing doses of caffeine in human adipose tissue preparations with an inhibition constant (Ki) in the millimolar range. Caffeine inhibited basal and insulin-stimulated glucose transport as well as lipogenesis in rodent adipose cells. The antilipogenic action of caffeine was also observed in adipocytes from mice genetically invalidated for PrAO activity, indicating that PrAO activity was not required for lipogenesis inhibition. These caffeine inhibitory properties were extended to human adipocytes: relative to basal 2-DG uptake, set at 1.0 ± 0.2 for 6 individuals, 0.1 mM caffeine tended to reduce uptake to 0.83 ± 0.08. Insulin increased uptake by 3.86 ± 1.11 fold when tested alone at 100 nM, and by 3.21 ± 0.80 when combined with caffeine. Our results reinforce the recommendation of caffeine's potential in the treatment or prevention of obesity complications.

Biased action of the CXCR4-targeting drug plerixafor is essential for its superior hematopoietic stem cell mobilization.

Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems.

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten's ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.

Computational Fragment-Based Design Facilitates Discovery of Potent and Selective Monoamine Oxidase-B (MAO-B) Inhibitor.

Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases. Inhibition of monoamine oxidase-B (MAO-B), which is mainly found in the glial cells of the brain, may lead to an elevated level of dopamine (DA) in patients. MAO-B inhibitors have been used extensively for patients with PD. However, the discovery of the selective MAO-B inhibitor is still a challenge. In this study, a computational strategy was designed for the rapid discovery of selective MAO-B inhibitors. A series of (S)-2-(benzylamino)propanamide derivatives were designed. In vitro biological evaluations revealed that (S)-1-(4-((3-fluorobenzyl)oxy)benzyl)azetidine-2-carboxamide (C3) was more potent and selective than safinamide, a promising drug for regulating MAO-B. Further studies revealed that the selectivity mechanism of C3 was due to the steric clash caused by the residue difference of Phe208 (MAO-A) and Ile199 (MAO-B). Animal studies showed that compound C3 could inhibit cerebral MAO-B activity and alleviate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal loss.

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr-/- mice.

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr-/- mice, a model of FH. Compared to hypercholesterolemic Ldlr-/- mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr-/- mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.

Characterization of Aminobenzylphenols as Protein Disulfide Isomerase Inhibitors in Glioblastoma Cell Lines.

Disulfide bond formation is a critical post-translational modification of newly synthesized polypeptides in the oxidizing environment of the endoplasmic reticulum and is mediated by protein disulfide isomerase (PDIA1). In this study, we report a series of α-aminobenzylphenol analogues as potent PDI inhibitors. The lead compound, AS15, is a covalent nanomolar inhibitor of PDI, and the combination of AS15 analogues with glutathione synthesis inhibitor buthionine sulfoximine (BSO) leads to synergistic cell growth inhibition. Using nascent RNA sequencing, we show that an AS15 analogue triggers the unfolded protein response in glioblastoma cells. A BODIPY-labeled analogue binds proteins including PDIA1, suggesting that the compounds are cell-permeable and reach the intended target. Taken together, these findings demonstrate an extensive biochemical characterization of a novel series of highly potent reactive small molecules that covalently bind to PDI.

Glutamine as an Ammonia Donor in Catabolism of the Glucosinolate, Sinalbin, in Biosynthesis of 4-Hydroxybenzylamine.

Amines synthesized by plants may be considered a dietary source of bioactive compounds, which are of interest due to possible health promoting effects. Developing Sinapis alba sprouts are known to produce 4-hydroxybenzylamine, but the reaction mechanism has not yet been established. We propose here a suggested metabolic pathway for the formation of 4-hydroxybenzylamine in S. alba plants. The catabolic sequence starts with a reaction between l-glutamine (Gln) as ammonia donor and 4-hydroxybenzyl carbocation, the enzymatic catalyzed hydrolysis product from sinalbin (4-hydroxybenzylglucosinolate). The suggested reactions are compared with alternative plant metabolic reactions used in the biosynthesis of biogenic amines.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Preparation of cytochrome P450 enzyme-cobalt phosphate hybrid nano-flowers for oxidative coupling of benzylamine.

A novel hybrid material with flower-mimicking morphology was fabricated with a facile coprecipitation method, and cytochrome P450 enzyme and cobalt phosphate were employed as organic and inorganic components, respectively. The hybrid nano-flowers showed excellent catalytic performance in the oxidative coupling of benzylamine, including the high conversion (99.9%) and selectivity (97.9%) in mild reaction conditions, as well as the satisfactory stability in the recycling experiments. Compared to free enzyme, the as-obtained materials exhibited enhanced activity. Such results indicate that the hybrid materials are potentially good candidates in the industrial enzyme catalysis.