Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors.

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole "linker" between the pyrimidine or pyridine "core" ring and the "tail" ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.

KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 µM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Improved homology modeling of the human & rat EP4 prostanoid receptors.

BACKGROUND: The EP4 prostanoid receptor is one of four GPCRs that mediate the diverse actions of prostaglandin E2 (PGE2). Novel selective EP4 receptor agonists would assist to further elucidate receptor sub-type function and promote development of therapeutics for bone healing, heart failure, and other receptor associated conditions. The rat EP4 (rEP4) receptor has been used as a surrogate for the human EP4 (hEP4) receptor in multiple SAR studies. To better understand the validity of this traditional approach, homology models were generated by threading for both receptors using the RaptorX server. These models were fit to an implicit membrane using the PPM server and OPM database with refinement of intra and extracellular loops by Prime (Schrödinger). To understand the interaction between the receptors and known agonists, induced-fit docking experiments were performed using Glide and Prime (Schrödinger), with both endogenous agonists and receptor sub-type selective, small-molecule agonists. The docking scores and observed interactions were compared with radioligand displacement experiments and receptor (rat & human) activation assays monitoring cAMP. RESULTS: Rank-ordering of in silico compound docking scores aligned well with in vitro activity assay EC50 and radioligand binding Ki. We observed variations between rat and human EP4 binding pockets that have implications in future small-molecule receptor-modulator design and SAR, specifically a S103G mutation within the rEP4 receptor. Additionally, these models helped identify key interactions between the EP4 receptor and ligands including PGE2 and several known sub-type selective agonists while serving as a marked improvement over the previously reported models. CONCLUSIONS: This work has generated a set of novel homology models of the rEP4 and hEP4 receptors. The homology models provide an improvement upon the previously reported model, largely due to improved solvation. The hEP4 docking scores correlates best with the cAMP activation data, where both data sets rank order Rivenprost>CAY10684 > PGE1 ≈ PGE2 > 11-deoxy-PGE1 ≈ 11-dexoy-PGE2 > 8-aza-11-deoxy-PGE1. This rank-ordering matches closely with the rEP4 receptor as well. Species-specific differences were noted for the weak agonists Sulprostone and Misoprostol, which appear to dock more readily within human receptor versus rat receptor.

Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist.

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo.

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.

Regulation of syntaxin1A-munc18 complex for SNARE pairing in HEK293 cells.

The formation and dissolution of SNARE protein complexes is essential for Ca(2+)-triggered fusion of neurotransmitter-filled vesicles at the presynaptic membrane. Among the pre-synaptic SNARE proteins, the activation of the Q-SNARE syntaxin1A is a critical event for SNARE complex formation. Activation requires syntaxin1A to transit from a munc18-bound non-interacting state to one competent for SNARE binding. The molecular mechanisms that regulate this transition remain unclear. The propensity of syntaxin1A to promote voltage-dependent steady-state inactivation of N-type Ca(2+) channels and accelerate their entry into inactivation was used in a heterologous cell expression system to elucidate regulation of syntaxin1A protein-protein interactions. We report that coexpression of munc18 eliminated the promoting effect of syntaxin1A on inactivation. This effect of munc18 was completely disrupted by coexpression of munc13-1, but not munc13-2 or munc13-3. Also, since expression of munc13-1 with syntaxin1A resulted in an inactivation phenotype identical to that of munc18 with syntaxin1A, the action of munc13-1 on the munc18-syntaxin1A complex was functionally unique and did not result from competitive binding interactions. Furthermore, munc13 expressed with syntaxin1A and munc18 promoted redistribution of a cytosolic SNAP25 mutant to the membrane, a result indicative of syntaxin1A-SNAP25 SNARE pairing. These data demonstrate an important role of munc13 to control the protein-protein interactions of syntaxin1A in vivo, and support munc13 as critical to dissociating syntaxin1A-munc18 complexes and making syntaxin1A available for SNARE interactions.

Facilitation of Ca(2+)-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells.

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1-V12, Rac1-L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin-permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1-N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21-activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1-V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1-V12-mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin-A inhibited secretion and eliminated the secretory effects of Rac1-V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca2+ influx, partly through regulation of cytoskeletal organization.

Synergistic effects of Munc18a and X11 proteins on amyloid precursor protein metabolism.

X11 proteins have been shown to modulate metabolism of the amyloid precursor protein (APP) and to reduce the secretion of beta-amyloid peptides (Abeta) that are associated with Alzheimer's disease. Whereas X11alpha interacts with APP via its phosphotyrosine-binding domain, recent reports indicate that additional regulatory interactions involve the N terminus of X11. Here we report that the syntaxin-1a-binding protein Munc18a, which interacts with the Munc18a-interacting domain (MID) at the N terminus of X11, strongly regulates the actions of X11 on APP metabolism. When co-expressed with X11alpha, Munc18a potentiated the retention of APP and suppression of Abeta secretion by X11alpha. As a result, the constitutive release of Abeta40 was nearly abolished. Experiments using N terminus deletion mutants of X11alpha/beta and the MID-deficient X11gamma revealed that the majority of the regulatory effect by Munc18a occurred independent of a direct interaction of Munc18a with X11, although the presence of X11 was required. Munc18a expression induced a small increase in beta-secretase activity, whereas it also intensified the reduction in Abeta40 secretion by X11alpha. These data indicate that Munc18a in concert with X11 acts to suppress gamma-secretase processing. We conclude that Munc18a acts through direct and indirect interactions with X11 proteins and powerfully regulates APP metabolism and Abeta secretion.