Search

siRNAs can be harnessed as the next generation of oligonucleotide-based antiviral therapeutics.

Despite the presence of strategically positioned anatomical barriers designed to protect the central nervous system (CNS), it is not entirely isolated from the immune system1,2. In fact, it remains physically connected to, and can be influenced by, the peripheral immune system1. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding question. Here, in searching for molecular cues that derive from the CNS and enable its direct communication with the immune system, we identified an endogenous repertoire of CNS-derived regulatory self-peptides presented on major histocompatibility complex class II (MHC-II) molecules in the CNS and at its borders. During homeostasis, these regulatory self-peptides were found to be bound to MHC-II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. However, in neuroinflammatory disease, the presentation of regulatory self-peptides diminished. After boosting the presentation of these regulatory self-peptides, a population of suppressor CD4+T cells was expanded, controlling CNS autoimmunity in a CTLA-4- and TGFβ-dependent manner. CNS-derived regulatory self-peptides may be the molecular key to ensuring a continuous dialogue between the CNS and the immune system while balancing overt autoreactivity. This sheds light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.

Traumatic injuries to the central nervous system (CNS) afflict millions of individuals worldwide1, yet an effective treatment remains elusive. Following such injuries, the site is populated by a multitude of peripheral immune cells, including T cells, but a comprehensive understanding of the roles and antigen specificity of these endogenous T cells at the injury site has been lacking. This gap has impeded the development of immune-mediated cellular therapies for CNS injuries. Here, using single-cell RNA sequencing, we demonstrated the clonal expansion of mouse and human spinal cord injury-associated T cells and identified that CD4+T cell clones in mice exhibit antigen specificity towards self-peptides of myelin and neuronal proteins. Leveraging mRNA-based T cell receptor (TCR) reconstitution, a strategy aimed to minimize potential adverse effects from prolonged activation of self-reactive T cells, we generated engineered transiently autoimmune T cells. These cells demonstrated notable neuroprotective efficacy in CNS injury models, in part by modulating myeloid cells via IFNγ. Our findings elucidate mechanistic insight underlying the neuroprotective function of injury-responsive T cells and pave the way for the future development of T cell therapies for CNS injuries.

A fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-μg sample amount, while a 10-μg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

ABSTRACTN6-methyladenosine (m6A) modification acts as the most prevalent internal modification in eukaryotic mRNA. Emerging evidence shows the critical biological roles of m6A key enzymes in human cancers. However, the roles of m6A binding protein IGF2BP2 in gastric cancer (GC) progression are still unclear. In this study, we confirmed that IGF2BP2 was highly expressed in GC cell lines and tumor tissues. Knocking down of IGF2BP2 suppressed cell proliferation and migration, and repressed xenograft tumor growth in vivo, while IGF2BP2 overexpression promoted the proliferation and migration. Mechanistically, we identified that IGF2BP2 regulated GC the proliferation/migration through recognizing the m6A modification sites of SIRT1 mRNA. In general, our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner, suggesting that m6A may be a therapeutic target for GC.

Extracorporeal membrane oxygenation(ECMO) has emerged as a viable treatment in severe cases of acute respiratory distress syndrome, acute respiratory failure, and adult respiratory distress syndrome. However, thromboembolic events stemming from the use of ECMO devices results in significant morbidity and mortality rates; the inner surface of the ECMO tubing comes into contact with the blood and can readily initiate coagulation. In addition, the tubing needs to be continually replaced due to thromboses on the inner tube wall, which not only increases the risk of infection but also the economic burden. Despite considerable effort, a surface modification strategy that effectively addresses these challenges has not yet been realized. In this study, we developed an integrated hollow core-shell-shell hydrogel tube of gelatin/alginate/acrylamide-bacterial nanocellulose(GAA) that meets the anticoagulant requirements for the inner tubing layer as well as the highly elastic soft material needed for the outer layer. Using static blood from healthy volunteers, we confirmed that the platelets or coagulation is not stimulated by the GAA tubing. Importantly, experiments with dynamic blood also demonstrated that the inner layer of the tubing does not elicit blood clotting. The one-pot-synthesized process may provide guidance for the design of anticoagulation tubes used clinically.

Receptor interacting protein kinase 3 (RIP3 or RIPK3), the critical executor of cell programmed necrosis, plays essential roles in maintaining immune responses and appropriate tissue homeostasis. Although the E3 ligases CHIP and PELI1 are reported to promote RIP3 degradation, however, how post-translational modification regulates RIP3 activity and stability is poorly understood. Here, we identify the tripartite motif protein TRIM25 as a negative regulator of RIP3-dependent necrosis. TRIM25 directly interacts with RIP3 through its SPRY domain and mediates the K48-linked polyubiquitination of RIP3 on residue K501. The RING domain of TRIM25 facilitates the polyubiquitination chain on RIP3, thereby promoting proteasomal degradation of RIP3. Also, TRIM25 deficiency inhibited the ubiquitination of RIP3, thus promoting TNF-induced cell necrosis. Our current finding reveals the regulating mechanism of polyubiquitination on RIP3, which might be a potential therapeutic target for the intervention of RIP3-dependent necrosis-related diseases.

Gasdermin-D (GSDMD), the executioner of pyroptotic cell death when it is cleaved by inflammatory caspases, plays a crucial role in host defense and the response to danger signals. So far, there are no known mechanisms, other than cleavage, for regulating GSDMD. Here, we show that tripartite motif protein TRIM21 acts as a positive regulator of GSDMD-dependent pyroptosis. TRIM21 interacted with GSDMD via its PRY-SPRY domain, maintaining GSDMD stable expression in resting cells yet inducing the N-terminus of GSDMD (GSDMD-N) aggregation during pyroptosis. TRIM21-deficient cells displayed a reduced cell death in response to NLRP3 or NLRC4 inflammasome activation. Genetic ablation of TRIM21 in mice conferred protection from LPS-induced inflammation and dextran sulfate sodium-induced colitis. Therefore, TRIM21 plays an essential role in GSDMD-mediated pyroptosis and may be a viable target for controlling and treating inflammation-associated diseases.

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1–5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and ‘cleaves’ a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody–drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3could release a client protein—including an active gasdermin—from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.

Cancer cells are highly dependent on NAD+/NADH produced via the nicotinamide salvage pathway. The rate-limiting enzyme in this pathway is the nicotinamide phosphoribosyltransferase (NAMPT), which we have targeted with novel NAMPT inhibitors. NAMPT inhibition elicits depletion of total cellular NAD+levels and ultimately cytotoxicity via depletion of cellular ATP levels. 18F-fluorodeoxyglucose– positron emission tomography (FDG-PET) is a translational imaging tool to assess glucose utilization in tumors and normal tissue. We used FDG-PET to understand the timing of ATP depletion in vivo and better understand the pharmacology of NAMPT inhibitors. Because of the intimate relationship between cellular ATP levels and cell viability, we developed an in-depth understanding of our NAMPT inhibitor pharmacology and the relationship with changes in tumor FDG uptake. Taken together, we show that FDG-PET could be used as a biomarker in clinical studies to understand dose and provide proof of mechanism for NAMPT inhibitors.

Crystallization of drug from an amorphous formulation is expected to negatively impact its bioperformance following oral delivery. In evaluating this in vivo, neat crystalline drug is typically mixed with the amorphous formulation. However, this approach may not adequately mimic the effect of drug crystals that form within the amorphous matrix, because crystal properties are highly dependent on the crystallization environment. The aim of this study was to evaluate the in vivo impact of crystals formed in a generic tacrolimus amorphous formulation, relative to noncrystallized formulations and a reference suspension containing neat crystalline drug. Crystallization of tacrolimus was induced in the generic product by exposing it to moderate temperatures and high relative humidity. Controlled levels of crystallinity in the formulations were achieved by mixing maximally crystallized and fresh formulations at the desired ratios. These formulations were then characterized in vitro and used for oral dosing to beagle dogs. Analysis of blood concentrations versus time revealed that formulations containing 50 and 100% crystalline tacrolimus resulted in lower area under the curve (AUC) and maximum concentration (Cmax) values as compared to the fresh amorphous formulation. However, the AUC and the Cmaxvalues for these formulations were significantly higher than those observed after dosing the pure crystalline tacrolimus suspension. The innovator formulation, Prograf, showed comparable pharmacokinetics before and after exposure to accelerated stability conditions, confirming the robustness of the innovator product to drug crystallization. This study provides insight into the impact of endogenously crystallized material on the oral absorption of a poorly water-soluble compound and highlights the importance of using representative crystalline material when undertaking risk assessment of amorphous formulations.

The oxygenation membrane, a core material of extracorporeal membrane oxygenation (ECMO), is facing challenges in balancing anti‐plasma leakage, gas exchange efficiency, and hemocompatibility. Here, inspired by the asymmetric structural features of alveolus pulmonalis, a novel triple‐functional membrane for blood oxygenation with a Janus architecture is proposed, which is composed of a hydrophobic polydimethylsiloxane (PDMS) layer to prevent plasma leakage, an ultrathin polyamide layer to enhance gas exchange efficiency with a CO2:O2permeance ratio of ≈10.7, and a hydrophilic polyzwitterionic layer to improve the hemocompatibility. During the simulated ECMO process, the Janus oxygenation membrane exhibits excellent performance in terms of thrombus formation and plasma leakage prevention, as well as adequate O2transfer rate (17.8 mL min−1m−2) and CO2transfer rate (70.1 mL min−1m−2), in comparison to the reported oxygenation membranes. This work presents novel concepts for the advancement of oxygenation membranes and demonstrates the application potential of the asymmetric triple‐functional Janus oxygenation membrane in ECMO. A novel triple‐functional membrane with Janus architecture is proposed for blood oxygenation, which is composed of hydrophobic polydimethylsiloxane (PDMS), ultrathin polyamide (PAD), and hydrophilic poly(sulfobetaine methacrylate) (PSBMA) layers. The membrane exhibits excellent oxygenation performance in terms of adequate gas transfer rate and resistances to thrombus formation and plasma leakage, thereby demonstrating novel concepts for the advancement of oxygenation membranes.

Extracorporeal Membrane Oxygenation In article 2302708, Tong Li, Song Zhao, and co‐workers develop an asymmetric triple‐functional Janus membrane for extracorporeal membrane oxygenation (ECMO) system. The PSBMA‐PAD@PDMS membrane exhibits sufficient gas exchange capacity for blood oxygenation under the premise of preventing thrombus formation and plasma leakage, and thus provides novel insights for the advancement of oxygenation membranes.

Cystic fibrosis (CF) is a multiorgan disease of the lungs, sinuses, pancreas, and gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an epithelial anion channel that regulates salt and water balance in the tissues in which it is expressed. To effectively treat the most prevalent patient population (F508del mutation), two biomolecular modulators are required: correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Despite approved potentiator and potentiator/corrector combination therapies, there remains a high need to develop more potent and efficacious correctors. Herein, we disclose the discovery of a highly potent series of CFTR correctors and the structure–activity relationship (SAR) studies that guided the discovery of ABBV/GLPG-2222 (22), which is currently in clinical trials in patients harboring the F508del CFTR mutation on at least one allele.

The nickel/photoredox dual catalysis system is an efficient conversion platform for the difunctionalization of unsaturated hydrocarbons. Herein, we disclose the first dual nickel/photoredox-catalyzed intramolecular 1,2-arylsulfonylation of allenes, which can accurately construct a C(sp2)–C(sp2) bond and a C(sp3)–S bond. The reaction exhibits excellent chemoselectivity and regioselectivity, allowing modular conformations of a diverse series of 3-sulfonylmethylbenzofuran derivatives. Control experiments showed that the bipyridine ligand is crucial for the formation of a stable σ-alkyl nickel intermediate, providing the possibility for sulfonyl radical insertion. Meanwhile, the electrophilic sulfonyl radical facilitates further oxidative addition of the σ-alkyl nickel intermediate and inhibits addition with allenes. In addition, control experiments, cyclic voltammetry tests, Stern–Volmer experiments, and density functional theory calculations afford evidence for the Ni(0)/Ni(I)/Ni(II)/Ni(III) pathway in this 1,2-arylsulfonylation.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite d-glycero-β-d-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2–4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosiswith biochemical analyses and identified ADP-β-d-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR–Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)–TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand–receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively. The bacterial metabolite ADP-heptose activates NF-κB in host cells via alpha-kinase 1 and the TIFA–TRAF signalling pathway.

Pyroptosis is a form of cell death that is critical for immunity. It can be induced by the canonical caspase-1 inflammasomes or by activation of caspase-4, -5 and -11 by cytosolic lipopolysaccharide. The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity. GSDMD belongs to a gasdermin family that shares the pore-forming domain. The functions and mechanisms of activation of other gasdermins are unknown. Here we show that GSDME, which was originally identified as DFNA5 (deafness, autosomal dominant 5), can switch caspase-3-mediated apoptosis induced by TNF or chemotherapy drugs to pyroptosis. GSDME was specifically cleaved by caspase-3 in its linker, generating a GSDME-N fragment that perforates membranes and thereby induces pyroptosis. After chemotherapy, cleavage of GSDME by caspase-3 induced pyroptosis in certain GSDME-expressing cancer cells. GSDME was silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibited GSDME-dependent pyroptosis upon activation of caspase-3 by chemotherapy drugs. Gsdme−/−(also known as Dfna5−/−) mice were protected from chemotherapy-induced tissue damage and weight loss. These findings suggest that caspase-3 activation can trigger necrosis by cleaving GSDME and offer new insights into cancer chemotherapy.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein–protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Fifteen berberine–bile acid analogues were synthesized. Anticancer activities of these analogues compared with berberine (BBR) were evaluated in vitro; among the analogues, A4, B4, and B5 had higher cytotoxicity than that of BBR. Most of the analogues showed higher antimicrobial activity against Staphylococcus aureus ATCC 25923 and Staphylococcus albus ATCC 8799 than that of BBR, but Bacillus subtilis AS 1.398 and Escherichia coli ATCC 31343 were not sensitive to all of the analogues. A4 and B4 were stable in the serum stability assay. B4 showed promising oral bioavailability in mice.

Androgen receptor (AR) is a steroid hormone receptor that is activated by endogenous androgens, mainly testosterone and 5- dihydrotestosterone (5-DHT). AR is also an important drug target, and AR antagonists (antiandrogens) have been widely used for prostate cancer therapy. Antiandrogens currently available on the market are all small molecules that antagonize AR function via binding to the ligand binding domain (LBD). AR peptide antagonist has been proposed as a ‘mechanism-based’ approach to directly block AR function by interrupting AR-protein interactions from the surface of the receptor. Without targeting the rigid ligand binding pocket within LBD, peptide antagonists allow more flexibility in structure design, and are likely to provide more efficient and complete blockade of AR function as compared to small molecule antagonists. AR interacts with a variety of proteins, and the interaction may be mediated by different functional domains of the receptor. Although varieties of AR-protein interfaces might serve as the target for peptide antagonist, majority of ongoing research is still focusing on peptides that target the LBD, which is mainly due to the abundance of structural information revealed by crystal structures. This review gives an overview of the current research attempts to develop AR peptide antagonists, particularly peptide antagonists that target the LBD and N-terminal domain (NTD). The challenges and opportunities for future discovery and development of peptide antagonists are discussed as well.

CYP3A4 metabolizes many drugs on the market. Although transcriptional regulation of CYP3A4 is known to be tightly controlled by some nuclear receptors (NR) including vitamin D receptor (VDR/NR1I1), posttranscriptional regulation of CYP3A4 remains elusive. In this study, we show that noncoding microRNAs (miRNAs) may control posttranscriptional and transcriptional regulation of CYP3A4 by directly targeting the 3'-untranslated region (3'UTR) of CYP3A4 and indirectly targeting the 3'UTR of VDR, respectively. Luciferase reporter assays showed that CYP3A4 3'UTR-luciferase activity was significantly decreased in human embryonic kidney 293 cells transfected with plasmid that expressed microRNA-27b (miR-27b) or mouse microRNA-298 (mmu-miR-298), whereas the activity was unchanged in cells transfected with plasmid that expressed microRNA-122a or microRNA-328. Disruption of the corresponding miRNA response element (MRE) within CYP3A4 3'UTR led to a 2- to 3-fold increase in luciferase activity. Immunoblot analyses indicated that CYP3A4 protein was down-regulated over 30% by miR-27b and mmu-miR-298 in LS-180 and PANC1 cells. The decrease in CYP3A4 protein expression was associated with significantly decreased CYP3A4 mRNA levels, as determined by quantitative real-time PCR (qPCR) analyses. Likewise, interactions of miR-27b or mmu-miR-298 with VDR 3'UTR were supported by luciferase reporter assays. The mmu-miR-298 MRE site is well conserved within the 3'UTR of mouse, rat, and human VDR. Down-regulation of VDR by the two miRNAs was supported by immunoblot and qPCR analyses. Furthermore, overexpression of miR-27b or mmu-miR-298 in PANC1 cells led to a lower sensitivity to cyclophosphamide. Together, these findings suggest that CYP3A4 gene expression may be regulated by miRNAs at both the transcriptional and posttranscriptional level.

Testosterone and structurally related anabolic steroids have been used to treat hypogonadism, muscle wasting, osteoporosis, male contraception, cancer cachexia, anemia, and hormone replacement therapy in aging men or age-related frailty; while antiandrogens may be useful for treatment of conditions like acne, alopecia (male-pattern baldness), hirsutism, benign prostatic hyperplasia (BPH) and prostate cancer. However, the undesirable physicochemical and pharmacokinetic properties of steroidal androgen receptor (AR) ligands limited their clinical use. Nonsteroidal AR ligands with improved pharmacological and pharmacokinetic properties have been developed to overcome these problems. This review focuses on the pharmacokinetics, metabolism, and pharmacology of clinically used and emerging nonsteroidal AR ligands, including antagonists, agonists, and selective androgen receptor modulators.Testosterone and structurally related anabolic steroids have been used to treat hypogonadism, muscle wasting, osteoporosis, male contraception, cancer cachexia, anemia, and hormone replacement therapy in aging men or age-related frailty; while antiandrogens may be useful for treatment of conditions like acne, alopecia (male-pattern baldness), hirsutism, benign prostatic hyperplasia (BPH) and prostate cancer. However, the undesirable physicochemical and pharmacokinetic properties of steroidal androgen receptor (AR) ligands limited their clinical use. Nonsteroidal AR ligands with improved pharmacological and pharmacokinetic properties have been developed to overcome these problems. This review focuses on the pharmacokinetics, metabolism, and pharmacology of clinically used and emerging nonsteroidal AR ligands, including antagonists, agonists, and selective androgen receptor modulators.

Testosterone use as anabolic therapy is controversial. Here, meta-analyses show that testosterone increases skeletal muscle mass and strength in androgen-deficient young men, older men and men with chronic illness; these data provide a compelling rationale for the development of selective androgen receptor modulators that mimic testosterone's effects without its side effects.

Compound S4 [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] is a novel nonsteroidal selective androgen receptor modulator that demonstrates tissue-selective androgenic and anabolic effects. The purpose of this in vitro study was to identify the phase I metabolites, potential species differences in metabolism, and the cytochromes P450 (P450s) involved in the phase I metabolism of S4 using 14C-S4, recombinant P450s, and other liver enzyme preparations from human, rat, and dog. The major phase I metabolism pathways of S4 in humans were identified as deacetylation of the B-ring acetamide group, hydrolysis of the amide bond, reduction of the A-ring nitro group, and oxidation of the aromatic rings, with deacetylation being the predominant pathway observed with most of the enzyme preparations tested. Among the major human P450 enzymes tested, CYP3A4 appeared to be one of the major phase I enzymes that could be responsible for the phase I metabolism of S4 [Km= 16.1 μM, Vmax= 1.6 pmol/(pmol · min)] in humans and mainly catalyzed the deacetylation, hydrolysis, and oxidation of S4. In humans, the cytosolic enzymes mainly catalyzed the hydrolysis reaction, whereas the microsomal enzymes primarily catalyzed the deacetylation reactions. Similar phase I metabolic profiles were observed in rats and dogs as well, except that the amide bond hydrolysis seemed to occur more rapidly in rats. In summary, these results showed that the major phase I reaction of S4 in human, rat, and dog is acetamide group deacetylation.

N-Acetyltransferase (NAT) is one of the major phase II enzymes involved in drug metabolism. Both species differences and polymorphism are observed in NAT expression. During the preclinical development of a novel selective androgen receptor modulator, S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (S4), we also observed species differences in S4 metabolism due to the interaction between the deacetylation metabolite M1 and NAT, which converted M1 back to S4 both in vitro and in vivo. During incubation with human liver cytosol or rat liver S9 fraction in the presence of acetyl-CoA, more than 50% of M1 (2 μM) was converted back to S4, but this conversion was not observed in the incubation with dog liver S9 fraction or human liver microsome. In vivo pharmacokinetic experiments showed that M1 could be rapidly converted back to S4 in rats, but a similar conversion was not observed in dogs. When S4 was administered, the formation of M1 was only observed in dogs due to the absence of NAT expression. Simultaneous fitting of the concentration-time profiles of both S4 and M1 showed that more than 50% of S4 was deacetylated to M1 in dogs after i.v. administration of S4, whereas more than 80% of M1 was converted to S4 in rats after i.v. administration of M1. Considering the polymorphism in NAT expression, the interaction between M1 and NAT may raise concerns for drug-drug interactions during clinical applications of S4. The observed species differences suggested that interspecies scaling might not be applicable for predicting the metabolism and disposition of S4 in humans.

N-Acetyltransferase (NAT) is one of the major phase II enzymes involved in drug metabolism. Both species differences and polymorphism are observed in NAT expression. During the preclinical development of a novel selective androgen receptor modulator, S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (S4), we also observed species differences in S4 metabolism due to the interaction between the deacetylation metabolite M1 and NAT, which converted M1 back to S4 both in vitro and in vivo. During incubation with human liver cytosol or rat liver S9 fraction in the presence of acetyl-CoA, more than 50% of M1 (2 µM) was converted back to S4, but this conversion was not observed in the incubation with dog liver S9 fraction or human liver microsome. In vivo pharmacokinetic experiments showed that M1 could be rapidly converted back to S4 in rats, but a similar conversion was not observed in dogs. When S4 was administered, the formation of M1 was only observed in dogs due to the absence of NAT expression. Simultaneous fitting of the concentration-time profiles of both S4 and M1 showed that more than 50% of S4 was deacetylated to M1 in dogs after i.v. administration of S4, whereas more than 80% of M1 was converted to S4 in rats after i.v. administration of M1. Considering the polymorphism in NAT expression, the interaction between M1 and NAT may raise concerns for drug-drug interactions during clinical applications of S4. The observed species differences suggested that interspecies scaling might not be applicable for predicting the metabolism and disposition of S4 in humans.

Compound S4 [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] is a novel nonsteroidal selective androgen receptor modulator that demonstrates tissue-selective androgenic and anabolic effects. The purpose of this in vitro study was to identify the phase I metabolites, potential species differences in metabolism, and the cytochromes P450 (P450s) involved in the phase I metabolism of S4 using 14C-S4, recombinant P450s, and other liver enzyme preparations from human, rat, and dog. The major phase I metabolism pathways of S4 in humans were identified as deacetylation of the B-ring acetamide group, hydrolysis of the amide bond, reduction of the A-ring nitro group, and oxidation of the aromatic rings, with deacetylation being the predominant pathway observed with most of the enzyme preparations tested. Among the major human P450 enzymes tested, CYP3A4 appeared to be one of the major phase I enzymes that could be responsible for the phase I metabolism of S4 [Km = 16.1 µM, Vmax = 1.6 pmol/(pmol · min)] in humans and mainly catalyzed the deacetylation, hydrolysis, and oxidation of S4. In humans, the cytosolic enzymes mainly catalyzed the hydrolysis reaction, whereas the microsomal enzymes primarily catalyzed the deacetylation reactions. Similar phase I metabolic profiles were observed in rats and dogs as well, except that the amide bond hydrolysis seemed to occur more rapidly in rats. In summary, these results showed that the major phase I reaction of S4 in human, rat, and dog is acetamide group deacetylation.

The present study aimed to identify selective androgen receptor modulators (SARMs) with in vivo pharmacological activity. We examined the in vitro and in vivo pharmacological activity of four chiral, nonsteroidal SARMs synthesized in our laboratories. In the in vitro assays, these compounds demonstrated moderate to high androgen receptor (AR) binding affinity, with Kivalues ranging from 4 to 37 nM, and three of the compounds efficaciously stimulated AR-mediated reporter gene expression. The compounds were then administered subcutaneously to castrated rats to appraise their in vivo pharmacological activity. Androgenic activity was evaluated by the ability of these compounds to maintain the weights of prostate and seminal vesicle, whereas levator ani muscle weight was used as a measure of anabolic activity. The maximal response (Emax) and dose for half-maximal effect (ED50) were determined for each compound and compared with that observed for testosterone propionate (TP). Compounds S-1 and S-4 demonstrated in vivo androgenic and anabolic activity, whereas compounds S-2 and S-3 did not. The activities of S-1 and S-4 were tissue-selective in that both compounds stimulated the anabolic organs more than the androgenic organs. These two compounds were less potent and efficacious than TP in androgenic activity, but their anabolic activity was similar to or greater than that of TP. Neither S-1 nor S-4 caused significant luteinizing hormone or follicle stimulating hormone suppression at doses near the ED50value. Thus, compounds S-1 and S-4 were identified as SARMs with potent and tissue-selective in vivo pharmacological activity, and represent the first members of a new class of SARMs with selective anabolic effects.

A high-performance field asymmetric waveform ion mobility spectrometry (FAIMS)-IMS-MS platform was developed and applied to explore the conformational diversity of the singly and doubly charged bradykinin (BK + H+)+and (BK + 2H+)2+ions. With pure N2as the FAIMS carrier gas, more than ten conformers of (BK + H+)+can be resolved using FAIMS-IMS, as compared to only four conformers resolved using either FAIMS or IMS alone. Interestingly, multiple conformers of (BK + H+)+were found to have completely different values of FAIMS compensation voltage (CV), while their IMS drift times were essentially the same, which were also proven experimentally to not result from the structural annealing by the collisional heating in the ion funnel. The separations in the FAIMS and IMS dimensions are substantially orthogonal, and the overall resolving power of two-dimensional FAIMS-IMS separation is largely proportional to the product of the separation resolving powers of FAIMS and IMS. Using a gas mixture of N2/He to further improve the resolving power of the FAIMS separation, the total resolving powers of the combined FAIMS and IMS separation were estimated to be about 1020 and 1400 for (BK + H+)+and (BK + 2H+)2+ions, respectively, which are significantly higher than the resolving power of any ion mobility-based separation techniques demonstrated so far. The combined FAIMS-IMS can thus be a much more powerful technique to explore the structural diversity of biomolecules.

Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication.

For Abstract see ChemInform Abstract in Full Text.