Binding kinetics redefine the antagonist pharmacology of the corticotropin-releasing factor type 1 receptor.

Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics on CRF1 receptor antagonist pharmacology was investigated by measuring the association rate constant (k1), dissociation rate constant (k₋1), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k₋1/k1), 170-fold range of k₋1, and 13-fold range of k1. The k₋1 values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k₋1 t(1/2) of 1.6-7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k1, approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF1 receptor antagonists.

A Long-Acting PYY3-36 Analog Mediates Robust Anorectic Efficacy with Minimal Emesis in Nonhuman Primates.

The gut hormone PYY3-36 reduces food intake in humans and exhibits at least additive efficacy in combination with GLP-1. However, the utility of PYY analogs as anti-obesity agents has been severely limited by emesis and rapid proteolysis, a profile similarly observed with native PYY3-36 in obese rhesus macaques. Here, we found that antibody conjugation of a cyclized PYY3-36 analog achieved high NPY2R selectivity, unprecedented in vivo stability, and gradual infusion-like exposure. These properties permitted profound reduction of food intake when administered to macaques for 23 days without a single emetic event in any animal. Co-administration with the GLP-1 receptor agonist liraglutide for an additional 5 days further reduced food intake with only one animal experiencing a single bout of emesis. This antibody-conjugated PYY analog therefore may enable the long-sought potential of GLP-1/PYY-based combination treatment to achieve robust, well-tolerated weight reduction in obese patients.

Studies on the structure-activity relationship of bicifadine analogs as monoamine transporter inhibitors.

Compounds with various activities and selectivities were discovered through structure-activity relationship studies of bicifadine analogs as monoamine transporter inhibitors. The norepinephrine-selective 2-thienyl compound S-6j was efficacious in a rodent pain model.

Metabolic fuels, neuropeptide Y, and estrous behavior in Syrian hamsters.

Of the various environmental factors influencing reproduction, food availability plays a particularly significant role, and an insufficient supply of oxidizable metabolic fuels inhibits reproduction in female mammals. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. However, the specific neuroendocrine alterations that mediate the suppression of estrous behavior are unknown. Several conditions that inhibit female sexual behavior are thought to be associated with altered neuropeptide Y (NPY) activity in the brain. Intracerebroventricular (ICV) infusion of NPY inhibits estrous behavior in ovariectomized steroid-primed rats and hamsters. Furthermore, food-deprived rats have an increase in NPY mRNA in the arcuate nucleus (ARC) of the hypothalamus. Unlike rats, studies in Syrian hamsters have failed to detect any alterations in ARC NPY mRNA following food deprivation. Here we show that ARC NPY immunoreactivity and mRNA is increased in food-deprived hamsters but not in hamsters given other metabolic challenges that inhibit estrous behavior. These findings support the hypothesis that NPY contribute to, but not be critical for, the nutritional inhibition of sexual receptivity.

Post-conception reproductive competition in cooperatively breeding common marmosets.

Common marmosets are cooperatively breeding monkeys that exhibit high female reproductive skew. Subordinate females usually fail to breed as a consequence of ovulation suppression and inhibition of sexual behavior, and, even when they do breed, typically rear fewer infants than dominants. We evaluated possible mechanisms of post-conception reproductive competition by comparing hormonal profiles across pregnancy, pregnancy outcomes, infant survivorship, and behavior in laboratory-housed families containing one (N=9) or two (N=7) breeding females. Breeding females in plurally breeding groups did not exhibit well-defined dominance relationships and rarely engaged in escalated aggression with one another. No significant differences were found among singly breeding mothers, plurally breeding mothers, and plurally breeding daughters in urinary chorionic gonadotropin or estradiol sulfate concentrations during pregnancy, fetal biparietal diameter, frequency of spontaneous abortion, frequency of stillbirths, number of live-born infants per litter, or infant mortality rates. When females gave birth while another female in the family was pregnant, however, their infants were highly likely to be killed. The perpetrator was definitively identified in only one family, in which a pregnant female killed her daughter's infant. These results are consistent with observations of free-living common marmosets and suggest that breeding females do not regularly influence one another's pregnancy outcomes, but that they may commonly kill each other's infants, especially during their own pregnancy. Our findings further suggest that infanticide by breeding females may have selected for the evolution of reproductive restraint in subordinate female marmosets.

Characterization of thien-2-yl 1S,2R-milnacipran analogues as potent norepinephrine/serotonin transporter inhibitors for the treatment of neuropathic pain.

Thien-2-yl 1S,2R-milnacipran analogues were synthesized and characterized as norepinephrine/serotonin transporter inhibitors. These compounds possessed higher potencies than 1S,2R-milnacipran (2R-1) while maintaining low molecular weight and moderate lipophilicity, which are the important features for the pharmacological and pharmacokinetic characteristics of milnacipran (1). Thus, compound 5c exhibited IC50 values of 2.3 and 32 nM, respectively, at NET and SERT, which were more than 10-fold better than those of 1 (NET IC50 = 77 nM, SERT IC50 = 420 nM). Moreover, 5c achieved the same efficacy as 1, but with much lower doses, in a rodent spinal nerve ligation pain model. In addition, 5c displayed desirable pharmacokinetic properties in several species, including high oral availability and significant brain penetration.

Food deprivation inhibits estrous behavior in hormone-treated Syrian hamsters despite elevated estradiol levels.

Estradiol and progesterone (P) induce female mammalian reproductive behaviors, which are, in turn, sensitive to food availability. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. While this suppression of estrous behavior may be due to alterations in neural steroid receptor levels, it is also possible that decreased levels of circulating estradiol could be involved in mediating this suppression. Ovariectomized Syrian hamsters given varying doses of estradiol benzoate (EB) and P were tested to determine whether increasing doses of sex steroids would overcome the suppressive effects of food deprivation on estrous behavior. As expected, lordosis duration decreased in food-deprived animals. Increasing the levels of EB, but not P, increased lordosis duration in the food-deprived animals so that animals who were given 20 microg of EB had lordosis durations significantly longer than food-deprived hamsters that received 1.5 microg and 2.5 microg EB. Following an injection of 2.5 microg of EB, food-deprived hamsters actually had higher circulating levels of estradiol than ad libitum-fed animals. Therefore, increasing circulating levels of estradiol can increase lordosis durations in fasted animals; however, the suppression of estrous behavior occurs despite increased circulating estradiol levels in ovariectomized, steroid-treated animals. The most parsimonious explanation for this phenomenon is a deprivation-induced reduction in neural responsiveness to estradiol.

Disinhibition of female sexual behavior by a CRH receptor antagonist in Syrian hamsters.

Several conditions that inhibit female sexual behavior are thought to be associated with altered corticotropin-releasing hormone (CRH) activity in the brain. The present experiments examined the hypothesis that endogenous CRH receptor signaling mediates the inhibition of estrous behavior by undernutrition and in other instances of sexual dysfunction. Intracerebroventricular (ICV) infusion of CRH or urocortin inhibited estrous behavior in ovariectomized steroid-primed hamsters. Conversely, ICV infusion of the CRH receptor antagonist astressin prevented the suppression of estrous behavior by food deprivation or by ICV administration of neuropeptide Y. Astressin treatment also induced sexual receptivity in nonresponders, animals that do not normally come into heat when treated with hormones, and this effect persisted in subsequent weekly tests in the absence of any further astressin treatment. Activation of the hypothalamo-pituitary-adrenocortical axis was neither necessary nor sufficient to inhibit estrous behavior, indicating that this phenomenon is due to other central actions of CRH receptor agonists. This is the first direct evidence that CRH receptor signaling may be a final common pathway by which undernutrition and other conditions inhibit female sexual behavior.