Discovery of NXT-10796, an orally active, intestinally restricted EP4 agonist prodrug for the treatment of inflammatory bowel disease.

A property-focused optimization strategy was employed to modify the carboxylic acid head group of a class of EP4 agonists in order to minimize its absorption upon oral administration. The resulting oxalic acid monohydrazide-derived carboxylate isostere demonstrated utility as a class of prodrug showing colon-targeted delivery of parent agonist 2, with minimal exposure observed in the plasma. Oral administration of NXT-10796 demonstrated tissue specific activation of the EP4 receptor through modulation of immune genes in the colon, without modulation of EP4 driven biomarkers in the plasma compartment. Although further in depth understanding of the conversion of NXT-10796 is required for further assessment of the developability of this series of prodrugs, using NXT-10796 as a tool molecule has allowed us to confirm that tissue-specific modulation of an EP4-modulated gene signature is possible, which allows for further evaluation of this therapeutic modality in rodent models of human disease.

Total Synthesis of Aflastatin A.

The total syntheses of aflastatin A and its C3-C48 degradation fragment (6a, R = H) have been accomplished. The syntheses feature several complex diastereoselective fragment couplings, including a Felkin-selective trityl-catalyzed Mukaiyama aldol reaction, a chelate-controlled aldol reaction involving soft enolization with magnesium, and an anti-Felkin-selective boron-mediated oxygenated aldol reaction. Careful comparison of the spectroscopic data for the synthetic C3-C48 degradation fragment to that reported by the isolation group revealed a structural misassignment in the lactol region of the naturally derived degradation product. Ultimately, the data reported for the naturally derived aflastatin A C3-C48 degradation lactol (6a, R = H) were attributed to its derivative lactol trideuteriomethyl ether (6c, R = CD3). Additionally, the revised absolute configurations of six stereogenic centers (C8, C9, and C28-C31) were confirmed.

Novel pyridone EP4 agonists featuring allylic alcohol ω-chains.

Novel prostaglandin E2 receptor 4 (EP4) agonists featuring a pyridone core and an allylic alcohol ω-chain were discovered. These agonists were shown to be selective over EP1, EP2 and EP3. Analogs harboring a 4-carboxylic acid phenethyl α-chain displayed improved potency over those containing an n-heptanoic acid chain. Key SAR relationships were also identified.

Crohn's Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70.

Nod2 is a cytosolic, innate immune receptor responsible for binding to bacterial cell wall fragments such as muramyl dipeptide (MDP). Upon binding, subsequent downstream activation of the NF-κB pathway leads to an immune response. Nod2 mutations are correlated with an increased susceptibility to Crohn's disease (CD) and ultimately result in a misregulated immune response. Previous work had demonstrated that Nod2 interacts with and is stabilized by the molecular chaperone Hsp70. In this work, it is shown using purified protein and in vitro biochemical assays that the critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the ability to bind bacterial ligands. A limited proteolysis assay and luciferase reporter assay reveal regions of Hsp70 that are capable of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino acid subset of Hsp70 that stabilizes the CD-associated variants of Nod2 and restores a proper immune response upon activation with MDP was identified. This work suggests that CD-associated Nod2 variants could be stabilized in vivo with a molecular chaperone.

In vitro characterization of MK-1439, a novel HIV-1 nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC50) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC95) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of <3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent.

Discovery and optimization of indazoles as potent and selective interleukin-2 inducible T cell kinase (ITK) inhibitors.

There is evidence that small molecule inhibitors of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signaling cascade, could represent a novel asthma therapeutic class. Moreover, given the expected chronic dosing regimen of any asthma treatment, highly selective as well as potent inhibitors would be strongly preferred in any potential therapeutic. Here we report hit-to-lead optimization of a series of indazoles that demonstrate sub-nanomolar inhibitory potency against ITK with strong cellular activity and good kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of the complexes.

Design, synthesis and structure-activity relationships of a novel class of sulfonylpyridine inhibitors of Interleukin-2 inducible T-cell kinase (ITK).

Starting from benzylpyrimidine 2, molecular modeling and X-ray crystallography were used to design highly potent inhibitors of Interleukin-2 inducible T-cell kinase (ITK). Sulfonylpyridine 4i showed sub-nanomolar affinity against ITK, was selective versus Lck and its activity in the Jurkat cell-based assay was greatly improved over 2.

Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses.

The optimization of a novel series of non-nucleoside reverse transcriptase inhibitors (NNRTI) led to the identification of pyridone 36. In cell cultures, this new NNRTI shows a superior potency profile against a range of wild type and clinically relevant, resistant mutant HIV viruses. The overall favorable preclinical pharmacokinetic profile of 36 led to the prediction of a once daily low dose regimen in human. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK).

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.

Depicting and defining sleep disturbed breathing associated with vagal nerve stimulation.

BACKGROUND: Vagal nerve stimulators (VNS), which have been approved for management of refractory epilepsy and depression, induce unique disturbances of breathing during sleep (SDBVNS) that are not captured well using standard criteria. The primary purpose of this retrospective study was to compare AASM definitions with alternative criteria to more accurately measure SDBVNS We also sought to assess outcome variables that may be clinically relevant and response to positive airway pressure therapy. METHODS: We analyzed the electronic medical records and comprehensive polysomnography results of all adult subjects with active VNS for epilepsy who were referred to the sleep center for suspected sleep apnea (2015-2020). We compared standard AASM criteria for defining apneas/hypopnea index (AHIAASM) with three novel scoring criteria for hypopnea according to degree of oxygen desaturation associated with VNS events: AHIVNS0 (none required); AHIVNS2 (2% required); and AHIVNS3 (3% required). RESULTS: Twenty-six subjects were included in the final analysis with 35 PSGs (14 females/12 males). The mean age was 33.6 years and mean body mass index (BMI) of 32.2 kg/m2. AHIAASM measured ≥ 15/hour in 7 (26.9%) subjects versus 21 (80.8%) by AHIVNS0; 15 (70.0%) by AHIVNS2; and 5 (19.2%) by AHIVNS3. Clinically significant hypoxemia was not present. The mean time SpO2<89% was 7 (20.8) minutes. Oximetry tracings often showed a desaturation pattern that resembled a sawfish rather than sawtooth. Arousals specifically linked to VNS activation were not elevated (2.9/hour). The baseline AHIVNS0 was 27.7/hour with a lowest AHIVNS0 on PAP of 27.9/hr. CONCLUSIONS: AASM scoring criteria significantly underestimated the degree of VNS induced respiratory disturbances. VNS events were not associated with increased arousals or significant hypoxemia. PAP therapy was an ineffective treatment in this population. This study adds to the increasing body of evidence of sleep disordered breathing related to VNS and questions the clinical significance of this finding.

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor.

Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.

Aminoalkylation of [1.1.1]Propellane Enables Direct Access to High-Value 3-Alkylbicyclo[1.1.1]pentan-1-amines.

Bicyclo[1.1.1]pentanes are effective bioisoteres for aromatic rings, tert-butyl groups, and alkynes. Here we report the first method to synthesize 3-alkylbicyclo[1.1.1]pentan-1-amines directly from [1.1.1]propellane via sequential addition of magnesium amides and alkyl electrophiles. The mild reaction conditions tolerate a variety of important functional groups and enable efficient incorporation of several pharmaceutically relevant amines onto the bicyclo[1.1.1]pentane scaffold. This method's utility is highlighted by its ability to significantly streamline the syntheses of several important bicyclo[1.1.1]pentan-1-amine building blocks.

MF498 [N-{[4-(5,9-Diethoxy-6-oxo-6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)-3-methylbenzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide], a selective E prostanoid receptor 4 antagonist, relieves joint inflammation and pain in rodent models of rheumatoid and osteoarthritis.

Previous evidence has implicated E prostanoid receptor 4 (EP4) in mechanical hyperalgesia induced by subplantar inflammation. However, its role in chronic arthritis remains to be further defined because previous attempts have generated two conflicting lines of evidence, with one showing a marked reduction of arthritis induced by a collagen antibody in mice lacking EP4, but not EP1-EP3, and the other showing no impact of EP4 antagonism on arthritis induced by collagen. Here, we assessed the effect of a novel and selective EP4 antagonist MF498 [N-{[4-(5,9-diethoxy-6-oxo-6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)-3-methylbenzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide] on inflammation in adjuvant-induced arthritis (AIA), a rat model for rheumatoid arthritis (RA), and joint pain in a guinea pig model of iodoacetate-induced osteoarthritis (OA). In the AIA model, MF498, but not the antagonist for EP1, MF266-1 [1-(5-{3-[2-(benzyloxy)-5-chlorophenyl]-2-thienyl}pyridin-3-yl)-2,2,2-trifluoroethane-1,1-diol] or EP3 MF266-3 [(2E)-N-[(5-bromo-2-methoxyphenyl)sulfonyl]-3-[5-chloro-2-(2-naphthylmethyl)phenyl]acrylamide], inhibited inflammation, with a similar efficacy as a selective cyclooxygenase 2 (COX-2) inhibitor MF-tricyclic. In addition, MF498 was as effective as an nonsteroidal anti-inflammatory drug, diclofenac, or a selective microsomal prostaglandin E synthase-1 inhibitor, MF63 [2-(6-chloro-1H-phenanthro[9,10-d]imidazol-2-yl)isophthalonitrile], in relieving OA-like pain in guinea pigs. When tested in rat models of gastrointestinal toxicity, the EP4 antagonist was well tolerated, causing no mucosal leakage or erosions. Lastly, we evaluated the renal effect of MF498 in a furosemide-induced diuresis model and demonstrated that the compound displayed a similar renal effect as MF-tricyclic [3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone], reducing furosemide-induced natriuresis by approximately 50%. These results not only suggest that EP4 is the major EP receptor in both RA and OA but also provide a proof of principle to the concept that antagonism of EP4 may be useful for treatment of arthritis.

Structure-activity relationships and pharmacokinetic parameters of quinoline acylsulfonamides as potent and selective antagonists of the EP(4) receptor.

A new series of EP(4) antagonists based on a quinoline acylsulfonamide scaffold have been identified as part of our on-going efforts to develop treatments for chronic inflammation. These compounds show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. Acceptable pharmacokinetic profiles have also been demonstrated across a series of preclinical species.

Enantioselective total synthesis of callipeltoside A: two approaches to the macrolactone fragment.

The enantioselective total synthesis of callipeltoside A is described. Two syntheses of the macrolactone subunit are included: the first relies upon an Ireland-Claisen rearrangement to generate the trisubstituted olefin geometry and the second utilizes an enantioselective vinylogous aldol reaction for this purpose. Enantioselective syntheses of the sugar and chlorocyclopropane side chain fragments are also disclosed. The relative and absolute stereochemistry of this natural product was determined by fragment coupling with the two enantiomers of the side chain fragment.

Bacterial Derived Carbohydrates Bind Cyr1 and Trigger Hyphal Growth in Candida albicans.

The dimorphic yeast Candida albicans is the most common pathogenic fungus found in humans. While this species is normally commensal, a morphological switch from budding yeast to filamentous hyphae allows the fungi to invade epithelial cells and cause infections. The phenotypic change is controlled by the adenylyl cyclase, Cyr1. Interestingly, this protein contains a leucine-rich repeat (LRR) domain, which is commonly found in innate immune receptors from plants and animals. A functional and pure LRR domain was obtained in high yields from E. coli expression. Utilizing a surface plasmon resonance assay, the LRR was found to bind diverse bacterial derived carbohydrates with high affinity. This domain is capable of binding fragments of peptidoglycan, a carbohydrate polymer component of the bacterial cell wall, as well as anthracyclines produced by Streptomyces, leading to hyphae formation. These findings add another dimension to the human microbiome, taking into account yeast-bacteria interactions that occur in the host.

Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTßR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

The effect of NOD2 on the microbiota in Crohn's disease.

Recent advancements toward the treatment of Crohn's disease (CD) indicate great promise for long-term remission. CD patients suffer from a complex host of dysregulated interactions between their innate immune system and microbiome. The most predominant link to the onset of CD is a genetic mutation in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (NOD2). NOD2 responds to the presence of bacteria and stimulates the immune response. Mutations to NOD2 promote low diversity and dysbiosis in the microbiome, leading to impaired mucosal barrier function. Current treatments suppress the immune response rather than enhancing the function of this critical protein. New progress toward stabilizing NOD2 signaling through its interactions with chaperone proteins holds potential in the development of novel CD therapeutics.

Fragment Molecular Orbital Method Applied to Lead Optimization of Novel Interleukin-2 Inducible T-Cell Kinase (ITK) Inhibitors.

Inhibition of inducible T-cell kinase (ITK), a nonreceptor tyrosine kinase, may represent a novel treatment for allergic asthma. In our previous reports, we described the discovery of sulfonylpyridine (SAP), benzothiazole (BZT), indazole (IND), and tetrahydroindazole (THI) series as novel ITK inhibitors and how computational tools such as dihedral scans and docking were used to support this process. X-ray crystallography and modeling were applied to provide essential insight into ITK-ligand interactions. However, "visual inspection" traditionally used for the rationalization of protein-ligand affinity cannot always explain the full complexity of the molecular interactions. The fragment molecular orbital (FMO) quantum-mechanical (QM) method provides a complete list of the interactions formed between the ligand and protein that are often omitted from traditional structure-based descriptions. FMO methodology was successfully used as part of a rational structure-based drug design effort to improve the ITK potency of high-throughput screening hits, ultimately delivering ligands with potency in the subnanomolar range.