The History of Flow Chemistry at Eli Lilly and Company.

Flow chemistry was initially used for speed to early phase material delivery in the development laboratories, scaling up chemical transformations that we would not or could not scale up batch for safety reasons. Some early examples included a Newman Kwart Rearrangement, Claisen rearrangement, hydroformylation, and thermal imidazole cyclization. Next, flow chemistry was used to enable safe scale up of hazardous chemistries to manufacturing plants. Examples included high pressure hydrogenation, aerobic oxidation, and Grignard formation reactions. More recently, flow chemistry was used in Small Volume Continuous (SVC) processes, where highly potent oncolytic molecules were produced by fully continuous processes at about 10 kg/day including reaction, extraction, distillation, and crystallization, using disposable equipment contained in fume hoods.

Mechanistic Study of Diketopiperazine Formation during Solid-Phase Peptide Synthesis of Tirzepatide.

This study focused on investigating diketopiperazine (DKP) and the formation of associated double-amino-acid deletion impurities during linear solid-phase peptide synthesis (SPPS) of tirzepatide (TZP). We identified that the DKP formation primarily occurred during the Fmoc-deprotection reaction and post-coupling aging of the unstable Fmoc-Pro-Pro-Ser-resin active pharmaceutical ingredient (API) intermediate. Similar phenomena have also been observed for other TZP active pharmaceutical ingredient (API) intermediates that contain a penultimate proline amino acid, such as Fmoc-Ala-Pro-Pro-Pro-Ser-resin, Fmoc-Pro-Pro-Pro-Ser-resin, and Fmoc-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-resin, which are intermediates for both hybrid and linear synthesis approaches. During post-coupling aging, it is found that Fmoc deprotection can proceed in dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and acetonitrile (ACN) solvents without any piperidine addition. Density functional theory (DFT) calculations showed that a peptide that has a penultimate proline stabilizes the transition state through the C-H···π interaction during Fmoc decomposition, which causes those peptides to be more prone to cascade-deprotection reactions. Pseudo-reaction pathways are then proposed, and a corresponding macrokinetics model is developed to allow accurate prediction of the TZP peptide intermediate self-deprotection and DKP formation rate. Based on those studies, control strategies for minimizing DKP formation were further investigated and an alternative to Fmoc protection was identified (Bsmoc-protected amino acids), which eliminated the formation of the DKP byproducts. In addition, the use of oxyma additives and lower storage temperature was demonstrated to markedly improve the peptide intermediate stability to DKP degradation pathways.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0−50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3−74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Equine endometrial development during late fetal and postnatal periods.

Equine uterine development, including endometrial histogenesis, begins prenatally and is completed postnatally. Little is known about this process in the horse. Uterine tissue was acquired from 38 foals, ranging in developmental age from gestational day (GD) 300 to postnatal day (PND) 180, for assessment of endometrial histogenesis. Patterns of endometrial cell proliferation were evaluated by multispectral imaging of uterine tissue sections stained immunofluorescently for Ki-67. Labeling index (LI, % labeled cells) for Ki-67 was calculated for each endometrial cell compartment (luminal epithelium, glandular epithelium, stroma). Histologically, nascent endometrial glands were present in all pre- and postnatal uterine tissues. Overall, Ki-67 LI increased (P < 0.0001) from the pre-to postnatal periods, and was higher (P < 0.0001) in epithelium as compared to stroma. Postnatally, endometrial Ki-67 LI increased (P < 0.0001) from week 1 to week 24. Our findings confirm that, in contrast to neonatal patterns of uterine development described for domestic ungulates, equine endometrial histogenesis begins prenatally, marked by the appearance of uterine glands as early as GD 300. Epithelial proliferation associated with maturation of the equine endometrium is pronounced by postnatal week 24.

Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development.

BACKGROUND: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. OBJECTIVE: In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. RESULTS: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. CONCLUSION: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents.

Prenatal Exposures of Male Rats to the Environmental Chemicals Bisphenol A and Di(2-Ethylhexyl) Phthalate Impact the Sexual Differentiation Process.

The increasing incidence of reproductive anomalies, described as testicular dysgenesis syndrome, is thought to be related to the exposure of the population to chemicals in the environment. Bisphenol A (BPA) and di(2-ethylhexyl)phthalate (DEHP), which have hormonal and antihormonal activity, have attracted public attention due to their presence in consumer products. The present study investigated the effects of BPA and DEHP on reproductive development. Timed-pregnant female rats were exposed to BPA and DEHP by gavage from gestational days 12 to 21. Results showed that prenatal exposures to test chemicals exerted variable effects on steroidogenic factor 1 and GATA binding protein 4 protein expression and increased (P < .05) sex-determining region Y-box 9 and antimüllerian hormone protein in the infantile rat testis compared with levels in the control unexposed animals. Pituitary LHß and FSHß subunit protein expression was increased (P < .05) in BPA- and DEHP-exposed prepubertal male rats but were decreased (P < .05) in adult animals relative to control. Exposure to both BPA and DEHP in utero inhibited (P < .05) global DNA hydroxymethylation in the adult testis in association with altered DNA methyltransferase protein expression. Together the present data suggest that altered developmental programming in the testes associated with chemical exposures are related to the disruption of sexual differentiation events and DNA methylation patterns. The chemical-induced effects impact the development of steroidogenic capacity in the adult testis.

Effect of vaccination with a multivalent modified-live viral vaccine on reproductive performance in synchronized beef heifers.

Prebreeding vaccination should provide fetal and abortive protection against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) but not impede reproduction when administered to cattle before estrus synchronization and breeding. The objective was to assess reproductive performance when naive beef heifers were vaccinated with modified-live viral (MLV) vaccine 2 days after unsynchronized estrus, and then revaccinated with MLV vaccine at 10 or 31 days before synchronized natural breeding. Sixty beef heifers naive to BVDV and BoHV-1 were randomly assigned to one of four treatment groups. Groups A and B (n = 20 per group) were vaccinated with MLV vaccine containing BVDV and BoHV-1 at 2 days after initial detected estrus, and then revaccinated 30 days later, which corresponded to 10 days (group A) or 31 days (group B) before synchronized natural breeding. Groups C and D (n = 10 per group) served as controls and were vaccinated with an inactivated vaccine that did not contain BVDV or BoHV-1 at the same time points as groups A and B, respectively. Estrous behavior was assessed using radio frequency technology. Estrus synchronization was performed, with initiation occurring at revaccination (groups A and C) or 21 days after revaccination (groups B and D). After synchronization, heifers were submitted to a bull breeding pasture for 45 days. At the end of the breeding period, heifers were assessed for pregnancy using ultrasonography. Progesterone concentrations were evaluated at estrus and 10 days after unsynchronized and synchronized estrus, at initial pregnancy check, and at the end of the study. All pregnant heifers in groups A and B and five pregnant heifers in group C were euthanized between 44 and 62 days of gestation and ovarian and conceptus tissues were assayed for BVDV and BoHV-1. Vaccination with MLV vaccine did not result in significant negative reproductive impact based on the duration of interestrus intervals, proportion of heifers exhibiting estrus within 5 days after synchronization, serum progesterone concentrations, pregnancy rates, and pregnancies in the first 5 days of the breeding season. Bovine viral diarrhea virus and BoHV-1 were not detected in luteal tissue, ovarian tissue, or fetal tissues. Use of MLV vaccine did not impede reproduction, when revaccination was performed at 10 or 31 days before synchronized natural breeding.

Discovery of potent selective bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model. Part II: optimization studies and demonstration of in vivo efficacy.

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F=78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3h after a single, 10 mg/kg, subcutaneous dose.

Discovery of potent, selective, bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model, part I: transformation of selective pyrazolodiazepinone phosphodiesterase 4 (PDE4) inhibitors into selective PDE2 inhibitors.

We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of PDE4 inhibitors, while simultaneously minimizing PDE4 activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like mode in contrast to the cAMP-like binding mode found in PDE4. Structure activity relationship studies coupled with an inhibitor bound crystal structure in the active site of the catalytic domain of PDE2 identified structural features required to minimize PDE4 inhibition while simultaneously maximizing PDE2 inhibition.

The antagonists but not partial agonists of glucocorticoid receptor ligands show substantial side effect dissociation.

A synthetic glucocorticoid receptor (GR) ligand with the efficacy of a glucocorticoid, but without the accompanying side effects, would meet an unmet medical need for the treatment of inflammatory diseases. It was hypothesized that a GR ligand that shifted helix 12 in a manner distinct from an agonist and an antagonist would confer a distinct GR conformation, resulting in differential gene expression and, ultimately, dissociation of antiinflammatory activity from side effects. A structural feature expected to interfere with helix 12 was incorporated into a nonsteroidal, tricyclic scaffold to create novel, high-affinity, and selective GR ligands that manifested a dual function in cellular assays, partial but robust agonist activity for inflammatory cytokine inhibition, and full antagonist activity for reporter gene activation. In contrast, analogs not likely to hinder helix 12 exhibited partial agonist activity for reporter gene activation. The requirement of full antagonist activity for substantial side effect dissociation was demonstrated in primary human preadipocytes, hepatocytes, and osteoblasts in which effects on adipogenesis, key genes involved in gluconeogenesis, and genes important for bone formation were examined, respectively. The dissociated GR ligands, despite lacking significant reporter gene activation, weakly recruit a limited number of coactivators such as peroxisomal proliferator-activated receptor-γ coactivator 1α. Transcriptional activation was sensitive to both peroxisomal proliferator-activated receptor-γ coactivator 1α and GR levels, providing a basis for cell-selective modulation of gene expression. The antiinflammatory activity of the dissociated ligands was further demonstrated in mouse models of inflammation. Together these results suggest that these ligands are promising candidates with robust antiinflammatory activity and likely dissociation against glucocorticoid-induced side effects.

Insulin treatment prevents diabetes-induced alterations in astrocyte glutamate uptake and GFAP content in rats at 4 and 8 weeks of diabetes duration.

Rat astrocyte function is changed by diabetes mellitus relative to the nondiabetic state and we believe that altered function contributes to the central nervous system symptoms manifested by individuals with diabetes. We report here a comparison of astrocyte glutamate uptake and GFAP expression in streptozotocin-induced type 1 diabetic rats and insulin-treated diabetic rats at 4 and 8 weeks following diabetes onset. In glial plasmalemmal vesicle (GPV) preparations from treated rats, insulin prevented the increase observed in untreated, diabetic rats of both sodium-dependent and sodium-independent glutamate uptake. We determined by immunoblotting and immunohistochemistry that insulin treatment prevented the decrease of GFAP expression detected in the cerebral cortex, hippocampus, and cerebellum of untreated, diabetic rats. These observations indicate that insulin effects on astrocyte function are significant in managing diabetes-induced central nervous system pathology.

Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial phenylalanyl-tRNA synthetase.

A series of benzyl phenyl ethers (BPEs) is described that displays potent inhibition of bacterial phenylalanyl-tRNA synthetase. The synthesis, SAR, and select ADMET data are provided.

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus.

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

Examination of edge effects with different storage conditions of preplated dimethyl sulfoxide nanospots in ChemLib 1,536- and 3,456-well assay-ready plates.

For ultra-high-throughput screening, 10-30 nl of compound dissolved in 75% dimethyl sulfoxide (DMSO)/25% water (vol/vol) is spotted into 1,536- and 3,456-well ChemLib plates (Aurora Biotechnologies, Carlsbad, CA) and stored appropriately for a short time before screening. Although this practice eliminates the compound plating bottleneck, plated volumes of DMSO slowly evaporate from assay wells if plates are not properly stored in the interim. Since many assays are sensitive to DMSO concentrations, even slight evaporation may cause intra-plate variation and thus decrease assay quality. Using a cytochrome P450 3A4 Vivid Blue assay (Invitrogen, Carlsbad), we investigated the rate, pattern, and quantity of evaporation over a 1-year time frame to identify best practices for long-term (i.e., 6 months or greater) storage of assay-ready compound plates. Our findings regarding evaporation at plate edges indicate that nanospots preplated in ChemLib 1,536- or 3,456-well plates are best stored at -80 degrees C, in a bag, with or without the outer evaporation wells filled or at -20 degrees C, in a bag, with evaporation wells filled.

Simultaneous screening of multiple bacterial tRNA synthetases using an Escherichia coli S30-based transcription and translation assay.

The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.

Effect of administering oxytocin or cloprostenol in the periovulatory period on pregnancy outcome and luteal function in mares.

Mares (n = 37) were treated from 4h after breeding through 2 days post-ovulation with oxytocin or cloprostenol. Oxytocin (20 units i.m.) was administered every 6 h and cloprostenol (250 mcg i.m.) daily. Luteal function was impaired for several days following treatment, however, lower progesterone levels among cloprostenol treated mares in this study did not result in decreased pregnancies. Pregnancy outcome at 15 days post-ovulation was not different between the oxytocin (13/18) and cloprostenol (13/19) treatment groups, respectively (P = 0.80). The results of this study indicate cloprostenol can be used to treat post-breeding mares through the second day following ovulation without decreasing pregnancy outcome.

Luteal function in mares following administration of oxytocin, cloprostenol or saline on day 0, 1 or 2 post-ovulation.

Mares (n = 30) were treated in the post-ovulatory period with saline, oxytocin, or cloprostenol (Clo). Dose, administration frequency and treatment day (Day 0, 1 or 2 post-ovulation) were evaluated. Interovulatory interval of control cycles was 22.7 (+/-0.36) days with a range of 20.6 (+/-1.44) to 23.8 (+/-1.39) days among all treatment groups. Mares treated with two micro-doses of cloprostenol on Day 2 post-ovulation had the shortest interovulatory interval. This group also had the lowest mean circulating progesterone concentrations on Days 3-7 and 13, and was the slowest group to reach concentrations of 5 ng/ml. Repeated administration of cloprostenol over 24 h in the early post-ovulatory period may more effectively impair luteal function than single doses. This could negatively affect pregnancy outcome but may be effective for lysing the early post-ovulatory luteal structure when mares are not bred.