A Dual Inhibitor of DYRK1A and GSK3ß for ß-Cell Proliferation: Aminopyrazine Derivative GNF4877.

Loss of ß-cell mass and function can lead to insufficient insulin levels and ultimately to hyperglycemia and diabetes mellitus. The mainstream treatment approach involves regulation of insulin levels; however, approaches intended to increase ß-cell mass are less developed. Promoting ß-cell proliferation with low-molecular-weight inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) offers the potential to treat diabetes with oral therapies by restoring ß-cell mass, insulin content and glycemic control. GNF4877, a potent dual inhibitor of DYRK1A and glycogen synthase kinase 3ß (GSK3ß) was previously reported to induce primary human ß-cell proliferation in vitro and in vivo. Herein, we describe the lead optimization that lead to the identification of GNF4877 from an aminopyrazine hit identified in a phenotypic high-throughput screening campaign measuring ß-cell proliferation.

Selective DYRK1A Inhibitor for the Treatment of Type 1 Diabetes: Discovery of 6-Azaindole Derivative GNF2133.

Autoimmune deficiency and destruction in either ß-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting ß-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human ß-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).

Liver-Targeted Toll-Like Receptor 7 Agonist Combined With Entecavir Promotes a Functional Cure in the Woodchuck Model of Hepatitis B Virus.

Current therapeutics for chronic infection with hepatitis B virus (HBV) rarely induce functional cure due to the immunotolerant status of patients. Small molecule agonists targeting toll-like receptor 7 (TLR7) have been shown to elicit a functional cure in animal models of HBV but sometimes with poor tolerability due to immune-related toxicities. In an effort to increase the therapeutic window of TLR7 agonists to treat chronic hepatitis B (CHB), we developed an oral TLR7 agonist, APR002, designed to act locally in the gastrointestinal tract and liver, thus minimizing systemic exposure and improving tolerability. Here, we describe the pharmacokinetic/pharmacodynamic (PK/PD) profile of APR002 in mice and uninfected woodchucks as well as the safety and antiviral efficacy in combination with entecavir (ETV) in woodchucks with CHB. Treatment of woodchucks chronically infected with woodchuck hepatitis virus (WHV) with weekly oral doses of APR002 was well-tolerated. While APR002 and ETV single agents did not elicit sustained viral control, combination therapy resulted in durable immune-mediated suppression of the chronic infection. These woodchucks also had detectable antibodies to viral antigens, enhanced interferon-stimulated gene expression, and loss of WHV covalently closed circular DNA. Conclusion: APR002 is a novel TLR7 agonist exhibiting a distinct PK/PD profile that in combination with ETV can safely attain a functional cure in woodchucks with chronic WHV infection. Our results support further investigation of liver-targeted TLR7 agonists in human CHB.

Stable Nanoemulsions for the Delivery of Small Molecule Immune Potentiators.

Adjuvants are required to enhance immune responses to typically poorly immunogenic recombinant antigens. Toll-like receptor agonists (TLRa) have been widely evaluated as adjuvants because they activate the innate immune system. Currently, licensed vaccines adjuvanted with TLRa include the TLR4 agonist monophosphoryl lipid, while additional TLRa are in clinical development. Unfortunately, naturally derived TLRa are often complex and heterogeneous entities, which brings formulation challenges. Consequently, the use of synthetic small-molecule TLRa has significant advantages because they are well-defined discrete molecules, which can be chemically modified to modulate their physicochemical properties. We previously described the discovery of a family of TLR7 agonists based on a benzonaphthyridine scaffold. In addition, we described how Alum could be used to deliver these synthetic TLRa. An alternative adjuvant approach with enhanced potency over Alum are squalene containing oil-in-water emulsions, which have been included in licensed influenza vaccines, including Fluad (MF59 adjuvanted) and Pandemrix (AS03 adjuvanted). Here, we describe how to enable the co-delivery of a TLR7 agonist in a squalene-based oil-in-water emulsion, for adjuvant evaluation.

Incorporation of Phosphonate into Benzonaphthyridine Toll-like Receptor 7 Agonists for Adsorption to Aluminum Hydroxide.

Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby enhancing co-delivery with antigen.

Strategies for designing synthetic immune agonists.

Enhancing the immune system is a validated strategy to combat infectious disease, cancer and allergy. Nevertheless, the development of immune adjuvants has been hampered by safety concerns. Agents that can stimulate the immune system often bear structural similarities with pathogen-associated molecular patterns found in bacteria or viruses and are recognized by pattern recognition receptors (PRRs). Activation of these PRRs results in the immediate release of inflammatory cytokines, up-regulation of co-stimulatory molecules, and recruitment of innate immune cells. The distribution and duration of these early inflammatory events are crucial in the development of antigen-specific adaptive immunity in the forms of antibody and/or T cells capable of searching for and destroying the infectious pathogens or cancer cells. However, systemic activation of these PRRs is often poorly tolerated. Hence, different strategies have been employed to modify or deliver immune agonists in an attempt to control the early innate receptor activation through temporal or spatial restriction. These approaches include physicochemical manipulation, covalent conjugation, formulation and conditional activation/deactivation. This review will describe recent examples of discovery and optimization of synthetic immune agonists towards clinical application.

Pancreas-Specific Delivery of ß-Cell Proliferating Small Molecules.

Our research groups recently described a series of small-molecule inducers of ß-cell proliferation that could be used to increase ß-cell mass. To mitigate the risk of nonspecific proliferation of other cell types, we devised a delivery strategy built on the tissue specificity observed in the experimental ß-cell imaging agent (+)-dihydrotetrabenazine (DTBZ). The ß-cell proliferator agent aminopyrazine (AP) was covalently linked with (+)-DTBZ to afford conjugates that retain both the proliferation activity and binding affinity for vesicular monoamine transporter-2 (VMAT2). In vivo mouse tissue distribution studies of a prototypical AP-DTBZ conjugate showed 15-fold pancreas exposure over plasma. Tissue-to-plasma ratios in liver and kidneys were two- and five-fold, respectively. This work is the first demonstration of enhanced delivery of ß-cell-proliferating molecules to the pancreas by leveraging the intrinsic tissue specificity of a ß-cell imaging agent.

A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells.

Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8(+) T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8(+) T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8(+) T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8(+) T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production.

Rational design of small molecules as vaccine adjuvants.

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

Topical SMIP-7.7, a toll-like receptor 7 agonist, protects against genital herpes simplex virus type-2 disease in the guinea pig model of genital herpes.

BACKGROUND: Development of more effective therapies for genital herpes simplex virus type-2 (HSV-2) infections remains a priority. The toll-like receptors (TLR) are attractive targets for the immunomodulation of primary and recurrent genital herpes infection. The guinea pig model of genital HSV-2 disease was therefore used to evaluate the efficacy of a new TLR-7 agonist, SMIP-7.7. METHODS: The effects of SMIP-7.7 at concentrations between 0.90% and 0.09% were compared to the vehicle control or Aldara(®) (3M Health Care Limited, Northridge, CA, USA) as treatment for genital HSV-2 infections. Following intravaginal inoculation of Hartley guinea pigs with 10(6) pfu HSV-2 (MS strain), animals were treated intravaginally beginning at 36 h post-infection. Animals were evaluated for acute disease, acute virus replication, recurrent disease and shedding, as well as infection of the dorsal root ganglia. RESULTS: Treatment with SMIP-7.7 significantly decreased mean total lesion scores during primary infection (all doses, P<0.01 compared with vehicle control, and similar to Aldara(®)). Vaginal virus titres were reduced in treated animals compared with vehicle control (P<0.001 for each treatment versus vehicle control on day 4). Treatment with SMIP-7.7 also significantly decreased the number of recurrent lesion days, the number of days with recurrent virus shedding and the infection of the dorsal root ganglia compared to the vehicle control, and was similar to Aldara(®). As opposed to Aldara(®), SMIP-7.7 did not induce fever or weight loss during treatment. CONCLUSIONS: SMIP-7.7 improves the outcome of primary and recurrent HSV-2 disease comparable to Aldara(®) but without some of the side effects associated with Aldara(®).

Biarylimidazoles as inhibitors of microsomal prostaglandin E2 synthase-1.

Microsomal prostaglandin E(2) synthase (mPGES-1) represents a potential target for novel analgesic and anti-inflammatory agents. High-throughput screening identified several leads of mPGES-1 inhibitors which were further optimized for potency and selectivity. A series of inhibitors bearing a biaryl imidazole scaffold exhibits excellent inhibition of PGE(2) production in enzymatic and cell-based assays. The synthesis of these molecules and their activities will be discussed.

Applying chemical tools to the discovery of novel regenerative medicine.

Regeneration is the process of restoring cells, tissues and structures that are lost during disease, injury or aging. Most regenerative processes involve stem or progenitor cells. However, regeneration mediated by stem or progenitor cells in adult mammalian systems is limited. Hence, small molecules that promote stem cell proliferation and/or differentiation are highly sought after, either as molecular tools to further understand stem cell biology or as drug leads for development of novel regenerative medicine.:

A genome-wide overexpression screen in yeast for small-molecule target identification.

We describe a multicopy gene suppression screen of drug sensitivity in Saccharomyces cerevisiae that facilitates the identification of cellular targets of small molecules. An array of yeast transformants harboring a multicopy yeast genomic library was screened for resistance to growth inhibitors. Comparison of array growth patterns for several such inhibitors allowed the differentiation of general and molecule-specific genetic suppressors. Specific resistance to phenylaminopyrimidine (1), an inhibitor identified from a kinase-directed library, was associated with the overexpression of Pkc1 and a subset of downstream kinases. Components of two other pathways (pheromone response/filamentous growth and Pho85 kinase) that genetically interact with the PKC1 MAPK signaling cascade were also identified. Consistent with the suppression screen, inhibitor 1 bound to Pkc1 in yeast cell lysate and inhibited its activity in vitro. These results demonstrate the utility of this approach for the rapid deconvolution of small-molecule targets.

Design, synthesis, and activity of HDAC inhibitors with a N-formyl hydroxylamine head group.

Histone deacetylases (HDAC) are promising targets for cancer chemotherapy. HDAC inhibitors are thought to act in part by disrupting normal cell cycle regulation, resulting in apoptosis and/or differentiation of transformed cells. Several HDAC inhibitors, which contain hydrophobic tails and the Zn(2+) chelator hydroxyamic acid as a head group, are potent inhibitors of HDACs both in vitro and in vivo. In this study, a related class of compounds with a N-formyl hydroxylamino head group has been synthesized and their ability to inhibit HDACs have been assayed in biochemical and cellular assays. These compounds were found to have comparable activities to suberoylanilide hydroxyamic acid (SAHA) in HDAC enzymatic assays and histone hyperacetylation cellular assays.

Development and characterization of nonpeptidic small molecule inhibitors of the XIAP/caspase-3 interaction.

Elevated expression of inhibitor of apoptosis protein (IAP) family members in various types of cancers is thought to provide a survival advantage to these cells. Thus, antiapoptotic functions of IAPs, and their potential as novel anticancer targets have attracted considerable interest. Among the IAPs, the X chromosome-linked inhibitor of apoptosis protein (XIAP) is regarded as the most potent suppressor of mammalian apoptosis through direct binding and inhibition of caspases. A high-throughput biochemical screen of a combinatorial chemical library led to the discovery of a novel nonpeptidic small molecule that has the ability to disrupt the XIAP/caspase-3 interaction. The activity of this nonpeptidic small molecule inhibitor of the XIAP/caspase-3 interaction has been characterized both in vitro and in cells. Molecules of this type can be used to conditionally inhibit the cellular function of XIAP and may provide insights into the development of therapeutic agents that act by modulating apoptotic pathways.

One-pot two-step microwave-assisted reaction in constructing 4,5-disubstituted pyrazolopyrimidines.

[reaction: see text] A microwave-assisted reaction was developed to facilitate the construction of 4,5-disubstituted pyrazolopyrimidines. This one-pot two-step process involves a sequential S(N)Ar displacement of the C4 chloro substituent with various anilines and amines, followed by a Suzuki coupling reaction with different boronic acids. Using microwave irradiation leads to high product conversion, low side product formation, and shorter reactions.

Synthetic small molecules that control stem cell fate.

In an attempt to better understand and control the processes that regulate stem cell fate, we have set out to identify small molecules that induce neuronal differentiation in embryonic stem cells (ESCs). A high-throughput phenotypic cell-based screen of kinase-directed combinatorial libraries led to the discovery of TWS119, a 4,6-disubstituted pyrrolopyrimidine that can induce neurogenesis in murine ESCs. The target of TWS119 was shown to be glycogen synthase kinase-3beta (GSK-3beta) by both affinity-based and biochemical methods. This study provides evidence that GSK-3beta is involved in the induction of mammalian neurogenesis in ESCs. This and such other molecules are likely to provide insights into the molecular mechanisms that control stem cell fate, and may ultimately be useful to in vivo stem cell biology and therapy.

A versatile linkage strategy for solid-phase synthesis of N,N-dimethyltryptamines and beta-carbolines.

Various tryptamines are captured by a vinylsulfonylmethyl polystyrene resin, generating a safety-catch linkage. Beta-carbolines can be formed from 4 by a Pictet-Spengler reaction with the introduction of R(1). Tryptamine 4 can also be derivatized by acylation or copper-mediated coupling to introduce R(2). If X = Br, Suzuki coupling can be used to introduce R(3). After derivatization, the indole derivatives are activated with methyl iodide and released under mild basic condition. [reaction: see text]