TRPA1 modulation by piperidine carboxamides suggests an evolutionarily conserved binding site and gating mechanism.

The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1-PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure-activity studies, thereby suggesting strategies for rational drug design.

A reversed sulfonamide series of selective RORc inverse agonists.

The identification of a new series of RORc inverse agonists is described. Comprehensive structure-activity relationship studies of this reversed sulfonamide series identified potent RORc inverse agonists in biochemical and cellular assays which were also selective against a panel of nuclear receptors. Our work has contributed a compound that may serve as a useful in vitro tool to delineate the complex biological pathways involved in signalling through RORc. An X-ray co-crystal structure of an analogue with RORc has also provided useful insights into the binding interactions of the new series.

Novel triazolo-pyrrolopyridines as inhibitors of Janus kinase 1.

The identification of a novel fused triazolo-pyrrolopyridine scaffold, optimized derivatives of which display nanomolar inhibition of Janus kinase 1, is described. Prototypical example 3 demonstrated lower cell potency shift, better permeability in cells and higher oral exposure in rat than the corresponding, previously reported, imidazo-pyrrolopyridine analogue 2. Examples 6, 7 and 18 were subsequently identified from an optimization campaign and demonstrated modest selectivity over JAK2, moderate to good oral bioavailability in rat with overall pharmacokinetic profiles comparable to that reported for an approved pan-JAK inhibitor (tofacitinib).

Structure-based discovery of C-2 substituted imidazo-pyrrolopyridine JAK1 inhibitors with improved selectivity over JAK2.

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

5-Aminopyrimidin-2-ylnitriles as cathepsin K inhibitors.

A series of pyrimidine nitrile inhibitors of Cathepsin K with reduced glutathione reactivity has been identified and Molecular Core Matching (MoCoM) has been used to quantify the effect of an amino substituent at C5.

1H-Pyrazolo[3,4-g]hexahydro-isoquinolines as selective glucocorticoid receptor antagonists with high functional activity.

Addition of the 4-fluorophenylpyrazole group to the previously described 2-azadecalin glucocorticoid receptor (GR) antagonist 1 resulted in significantly enhanced functional activity. SAR of the bridgehead substituent indicated that whereas groups as small as methyl afforded high GR binding, GR functional activity was enhanced by larger groups such as benzyl, substituted ethers, and aminoalkyl derivatives. GR antagonists with binding and functional activity comparable to mifepristone were discovered (e.g., 52: GR binding K(i) 0.7 nM; GR reporter gene functional K(i) 0.6 nM) and found to be highly selective over other steroid receptors. Analogues 43 and 45 had >50% oral bioavailability in the dog.

Elevated IGFBP-1 cause high bone turnover in growth-restricted monochorionic twins with discordant birth weight.

OBJECTIVE: To test the hypothesis that low birth weight twins have a higher risk of osteoportotic fracture in later life, we investigated the association between fetal IGF axis and type-1 collagen markers of bone turnover in monochorionic (MC) twins with or without discordant birth weight of >or=20%. METHODS: Maternal and cord bloods were collected from gestational age matched MC twins of discordant (n = 16) and concordant birth weights (n = 16). The samples were assayed for cross linked carboxyl terminal telopeptide (ICTP, a marker of bone resorption) and pro-peptide (PICP, a marker of bone formation) of type I collagen, IGF-1, and IGFBP-1 by radio-immunoassay. RESULTS: The growth-restricted twins (IUGR) of discordant group had higher fetal IGFBP-1 and ICTP (P < 0.001) levels, while PICP (P < 0.001) was lower than the co-twins with normal weight (AGA). In contrast, cord blood levels of IGF-1, IGFBP-1, ICTP, and PICP in concordant twin pairs were comparable to AGA twins. The concordant and AGA twins had a positive correlation between ICTP and PICP levels (y = 23x - 711; r = 0.84; P < 0.001; n = 48) but no such association was found in IUGR twins. Instead, IGFBP-1 levels in IUGR twins had a negative association with PICP (r = 0.81; P < 0.001; n = 16) and a positive correlation with ICTP (r- = 0.51; P < 0.05; n = 16). No such association was found in concordant and AGA twins. CONCLUSION: These data suggest that growth-restricted twins had high bone turnover, due to elevated IGFBP-1. This association seems to be independent of maternal and genetic factors.

Comparative study of perinatal outcome of dichorionic and trichorionic iatrogenic triplets.

OBJECTIVE: The purpose of this study was to compare the perinatal outcome of dichorionic and trichorionic triplets who were conceived by assisted reproductive techniques. STUDY DESIGN: In this retrospective study, the maternal, neonatal, and chorionicity data were collected from 106 sets of trichorionic triamniotic and 34 sets of dichorionic triamniotic triplet pregnancies who were conceived by assisted reproductive techniques between January 1986 and December 2000. Perinatal and neonatal data were evaluated in relation to chorionicity and intertriplet birth weight discordance. RESULTS: The dichorionic triamniotic triplets have an 8-fold higher risk of perinatal death than trichorionic triamniotic gestations (odds ratio, 7.9; 95% CI, 4.4-14.0; P < .001). This is attributed to a higher risk of very low birth weight (P < .01), delivery at < 30 weeks of gestation (P < .001), and premature rupture of membrane (P < .001) in dichorionic triamniotic triplets compared with trichorionic triamniotic pregnancies. Twin-twin transfusion syndrome (odds ratio, 11.5; 95% CI, 4.8-27.7; P < .001), delivery at < 30 weeks of gestation (odds ratio, 40.5; 95% CI, 16.9-97; P < .01), premature rupture of membrane (odds ratio, 6.7; 95% CI, 3.8-11.9; P < .01), and nulliparity (odds ratio, 3.1; 95% CI, 1.6-6.1; P < .05) had independent effects on perinatal loss rate. CONCLUSION: The dichorionic triplets have an 8 times higher perinatal mortality rate than trichorionic triamniotic pregnancies.

Perinatal outcome of spontaneously conceived triplet pregnancies in relation to chorionicity.

OBJECTIVE: The purpose of this study was to determine the perinatal outcome of spontaneously conceived triplet pregnancies in relation to chorionicity. STUDY DESIGN: In this retrospective study, maternal, neonatal, and chorionicity data were collected from 88 sets of triplet pregnancies that were delivered at >20 weeks of gestation from 3 tertiary referral centers in the United Kingdom. The data were collected between January 1986 and December 2000. There were 49 sets of trichorionic triamniotic triplet pregnancies and 39 sets of triplet pregnancies with a monochorionic pair (ie, dichorionic triamniotic triplets). The outcome of each pregnancy was assessed in relation to chorionicity. RESULTS: The overall perinatal mortality rate was 151.5 per 1000 total births, with dichorionic triamniotic triplets having a 5.5-fold higher risk than trichorionic triamniotic gestations (odds ratio, 5.5; 95% CI, 2.5-12.2). The dichorionic triamniotic triplets have a higher risk of delivery at < 30 weeks of gestation (odds ratio, 4.6; 95% CI, 1.6-11.8; P < .05) and birth weight of < 1000 g (odds ratio, 53.6; 95% CI, 17.5-164; P < .05) than those of trichorionic triamniotic pregnancies. The neonatal morbidity in terms of respiratory distress syndrome (P < .001), anemia (P < .01), and intraventricular hemorrhage (P < .001) were higher in dichorionic triamniotic compared with trichorionic triamniotic triplets. The premature rupture of membrane (odds ratio, 7.5; 95% CI, 3.5-15.7) and twin-twin transfusion syndrome (odds ratio, 14.9; 95% CI, 6.6-4) were independent risk factors for perinatal death. CONCLUSION: In spontaneously conceived triplets, the incidence of dichorionicity was 44%. The dichorionic triamniotic triplets have a 5.5-fold higher risk of adverse perinatal outcome predominantly because of twin-twin transfusion syndrome and premature rupture of membranes.

Prevalence of cranial scan abnormalities in preterm twins in relation to chorionicity and discordant birth weight.

OBJECTIVE: The purpose of this study was to determine the incidence of ultrasonographically detected cerebral white matter lesions (WMLs) in preterm twins at birth in relation to chorionicity, discordant weight and twin-twin transfusion syndrome (TTTS). METHODS: In this retrospective study, perinatal, neonatal, and cranial scan data of 85 monochorionic (MC) and 94 dichorionic (DC) twin pregnancies (341 infants) delivered between 24 and 34 weeks of gestation were collected. Data were analysed according to chorionicity, discordant birth weight (>20%), single intrauterine death and TTTS. RESULTS: The cerebral WML was seen in 14% of preterm twins. Monochorionic infants had higher risks of WML than DC twin (odds ratio 7.1; 95% CI 3.28-15.8). In MC group, discordant weight (37%), TTTS (38%), single intrauterine death (67%) had higher incidence of cerebral WML than concordant weight infants (7%). Similarly, incidence of WML was higher in DC discordant compared with concordant weight infants (13% versus 2%; P < 0.05). CONCLUSION: Monochorionic infants had a seven-fold higher incidence of cerebral WML than DC infants. Discordant birth weight, TTTS and survivor of co-twin demise are an independent risk of cerebral white matter lesion.

Placenta as a link between amino acids, insulin-IGF axis, and low birth weight: evidence from twin studies.

Current evidence suggests that reduced placental transport of amino acids regulates fetal growth. We determined the association between fetal nutrition and the insulin-IGF axis by measuring the plasma concentrations of amino acids, insulin, IGF-I and IGF-binding protein-1 (IGFBP-1) in maternal and cord blood from gestational age-matched dichorionic (DC) twins with (n = 10) and without discordant birth weights (n = 10). In the growth-restricted (IUGR) twins, fetal concentrations of total essential (P < 0.01), nonessential (P < 0.01), and branched chain amino acids (P < 0.01) were lower than those in the appropriate for gestational age co-twins and concordant twin pairs. The IUGR twins had lower fetal concentrations of insulin (P < 0.001) and IGF-I (P < 0.05) and higher concentrations of IGFBP-1 (P < 0.01) than their appropriate for gestational age co-twins. In the discordant group, fetal IGFBP-1 had a negative association with fetal insulin (r = 0.71; P < 0.001), total essential amino acids (r = 0.78; P < 0.001), and branched chain amino acids (r = 0.64; P < 0.01). There was a positive correlation between total essential amino acids (r = 0.63; P < 0.001) and branched chain amino acids (r = 0.58; P < 0.01) and plasma insulin. However, there were no associations among fetal insulin, IGFBP-1 and nonessential amino acids. These data demonstrate the link between the reduction in certain essential and nonessential amino acids and alterations in fetal circulating levels of insulin and IGFBP-1, in growth-restricted twins.

Perinatal outcome following amniotic septostomy in chronic TTTS is independent of placental angioarchitecture.

OBJECTIVES: To determine whether the vascular anatomy of monochorial placenta influences the success of amniotic septostomy for the treatment of chronic mid-trimester twin-twin transfusion syndrome, we report placental anastomoses and perinatal data of 13 pregnancies treated by amniotic septostomy in combination with amnioreduction (AR). The placental anastomoses were delineated postnatally by perfusion studies. Perinatal outcome was also evaluated in relation to umbilical artery Doppler waveform of the donor twin. RESULTS: The median gestational age at septostomy was 21 weeks (range 18 to 25.5 weeks). Amniotic septostomy in combination with single AR procedure successfully resolved polyhydramnios in all cases. The median gestational age at delivery and the septostomy to delivery interval were 27 weeks (range 20 to 34 weeks) and 4 weeks (range 0.3 to 13.6 weeks), respectively. Of the 26 fetuses, 10 died in utero and four died within a week of life, with a combined survival rate of 46%. There was no relation between the clinical outcome and angioarchitecture of the placenta. However, pregnancy loss was higher in the donor twin with absent end-diastolic flow umbilical artery Doppler waveform than those with end-diastolic flow (85 vs 17%; p < 0.001). CONCLUSION: This study suggests that although amniotic septostomy is a promising method for the correction of oligohydramnios and/or polyhydramnios, perinatal survival rate does not depend on angioarchitecture of the placenta. Instead, umbilical artery Doppler waveform of the donor twin may be a better marker for survival rate.

Influence of vasopressin in the pathogenesis of oligohydramnios-polyhydramnios in monochorionic twins.

OBJECTIVES: The pathophysiology of oligohydramnios-polyhydramnios in monochorionic (MC) twins complicated by chronic twin-twin transfusion syndrome (TTTS) is poorly understood. We hypothesise that oliguria and oligohydramnios in the donor twin of chronic TTTS, occurs due to antidiuretic and vasoconstrictive activity of vasopressin (AVP). METHODS: We measured AVP levels in maternal, fetal and amniotic fluid samples obtained in utero and at birth from 44 MC twins with (n=27) or without chronic TTTS (n=17). Concentrations of AVP in pg/ml were determined by immuno-radiometry assay. RESULTS: In donor fetuses, plasma and amniotic fluid AVP levels were higher than those of the recipient twins in utero (P<0.001) and at birth (P<0.001). No such differences were found between the non-TTTS twins. The plasma AVP concentrations were higher in the recipient fetuses with severe hydrops than those without hydrops (2.8+/-0.7 pg/ml versus 0.3+/-0.3 pg/ml; P<0.05). Maternal AVP levels were comparable between the TTTS and non-TTTS groups. In the non-TTTS twins, both plasma and amniotic fluid AVP levels were higher than those of the recipient twins (P<0.001) but lower than those of the donor twins (P<0.001). There was a significant association between amniotic fluid and plasma AVP levels both in the TTTS (r=0.78; P<0.001) and non-TTTS (r=0.70; P<0.01) infants. CONCLUSIONS: Vasopressin concentrations in the donor twins were three times higher than their co-twins which suggests that oligohydramnios may occur as a consequence of AVP mediated reduction in fetal urine output.

Azepines and piperidines with dual norepinephrine dopamine uptake inhibition and antidepressant activity.

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

Identification of C-2 hydroxyethyl imidazopyrrolopyridines as potent JAK1 inhibitors with favorable physicochemical properties and high selectivity over JAK2.

Herein we report on the structure-based discovery of a C-2 hydroxyethyl moiety which provided consistently high levels of selectivity for JAK1 over JAK2 to the imidazopyrrolopyridine series of JAK1 inhibitors. X-ray structures of a C-2 hydroxyethyl analogue in complex with both JAK1 and JAK2 revealed differential ligand/protein interactions between the two isoforms and offered an explanation for the observed selectivity. Analysis of historical data from related molecules was used to develop a set of physicochemical compound design parameters to impart desirable properties such as acceptable membrane permeability, potent whole blood activity, and a high degree of metabolic stability. This work culminated in the identification of a highly JAK1 selective compound (31) exhibiting favorable oral bioavailability across a range of preclinical species and robust efficacy in a rat CIA model.

Identification of imidazo-pyrrolopyridines as novel and potent JAK1 inhibitors.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as rheumatoid arthritis (RA), by specific targeting of the JAK1 pathway. Examination of the preferred binding conformation of clinically effective, pan-JAK inhibitor 1 led to identification of a novel, tricyclic hinge binding scaffold 3. Exploration of SAR through a series of cycloamino and cycloalkylamino analogues demonstrated this template to be highly tolerant of substitution, with a predisposition to moderate selectivity for the JAK1 isoform over JAK2. This study culminated in the identification of subnanomolar JAK1 inhibitors such as 22 and 49, having excellent cell potency, good rat pharmacokinetic characteristics, and excellent kinase selectivity. Determination of the binding modes of the series in JAK1 and JAK2 by X-ray crystallography supported the design of analogues to enhance affinity and selectivity.

Discovery and optimization of C-2 methyl imidazopyrrolopyridines as potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2.

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

Synthesis and SAR of novel 2-arylthiazolidinones as selective analgesic N-type calcium channel blockers.

A series of new N-type (Ca(v)2.2) calcium channel blockers derived from the 'hit' structures 2-(3-bromo-4-fluorophenyl)-3-(2-pyridin-2-ylethyl)thiazolidin-4-one 9 and its 2-[4-(4-bromophenyl)pyridin-3-yl]-3-isobutyl analogue 10 is described. Extensive SAR studies using a range of synthetic approaches resulted in novel, patented compounds with IC50 values of up to 0.2 microM in an in vitro IMR32 assay, and selectivities for N/L of up to 30-fold. The new compounds described have potential in treatment of neuropathic pain.

Use of an in situ disulfide cross-linking strategy to study the dynamic properties of the cytoplasmic end of transmembrane domain VI of the M3 muscarinic acetylcholine receptor.

The ligand-induced activation of G protein-coupled receptors (GPCRs) is predicted to involve pronounced conformational changes on the intracellular surface or the receptor proteins. A reorientation of the cytoplasmic end of transmembrane domain VI (TM VI) is thought to play a key role in GPCR activation and productive receptor/G protein coupling. Disulfide cross-linking studies with solubilized, Cys-substituted mutant versions of bovine rhodopsin and the M3 muscarinic acetylcholine receptor suggested that the cytoplasmic end of TM VI is conformationally highly flexible, even in the absence of activating ligands (Farrens, D. L., et al. (1996) Science 274, 768-770; Zeng, F. Y., et al. (1999) J. Biol. Chem. 274, 16629-16640). To test the hypothesis that the promiscuous disulfide cross-linking pattern observed in these studies was caused by the use of solubilized receptor proteins endowed with increased conformational flexibility, we employed a recently developed in situ disulfide cross-linking strategy that allows the detection of disulfide bonds in Cys-substituted mutant M3 muscarinic receptors present in their native membrane environment. Specifically, we used membranes prepared from transfected COS-7 cells to analyze a series of double Cys mutant M3 receptors containing one Cys residue within the sequence K484(6.29) to S493(6.38) at the cytoplasmic end of TM VI and a second Cys residue at the cytoplasmic end of TM III (I169C(3.54)). This analysis revealed a disulfide cross-linking pattern that was strikingly more restricted than that observed previously with solubilized receptor proteins, both in the absence and in the presence of the muscarinic agonist, carbachol. Carbachol stimulated the formation of disulfide bonds in only two of the 10 analyzed mutant muscarinic receptors, I169C(3.54)/K484C(6.29) and I169C(3.54)/A488C(6.33), consistent with an agonist-induced rotation of the cytoplasmic end of TM VI. These findings underline the usefulness of analyzing the structural and dynamic properties of GPCRs in their native lipid environment.