Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

Novel peptidomimetic hematoregulatory compounds.

The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation.

Conformational preferences in a benzodiazepine series of potent nonpeptide fibrinogen receptor antagonists.

Previously, we reported the direct design of highly potent nonpeptide 3-oxo-1,4-benzodiazepine fibrinogen receptor antagonists from a constrained, RGD-containing cyclic semipeptide. The critical features incorporated into the design of these nonpeptides were the exocyclic amide at the 8-position which overlaid the Arg carbonyl, the phenyl ring which maintained an extended Gly conformation, and the diazepine ring which mimicked the gamma-turn at Asp. In this paper, we investigate conformational preferences of the 8-substituted benzodiazepine analogues by examining structural modifications to both the exocyclic amide and the seven-membered diazepine ring and by studying the conformation of the benzodiazepine ring using molecular modeling, X-ray crystallography, and NMR. We found that the directionality of the amide at the 8-position had little effect on activity and the (E)-olefin analogue retained significant potency, indicating that the trans orientation of the amide, and not the carbonyl or NH groups, made the largest contribution to the observed activity. For the diazepine ring, with the exception of the closely analogous 3-oxo-2-benzazepine ring system described previously, all of the modifications led to a significant reduction in activity compared to the potent 3-oxo-1, 4-benzodiazepine parent ring system, implicating this particular type of ring system as a desirable structural feature for high potency. Energy minimizations of a number of the modified analogues revealed that none could adopt the same low-energy conformation as the one shared by the active (S)-isomer of the 3-oxo-1, 4-benzodiazepines and 3-oxo-2-benzazepines. The overall data suggest that the features contributing to the observed high potency in this series are the orientation of the 3-4 amide and the conformational constraint imposed by the seven-membered ring, both of which position the key acidic and basic groups in the proper spatial relationship.

Benzimidazole derivatives as arginine mimetics in 1,4-benzodiazepine nonpeptide vitronectin receptor (alpha v beta 3) antagonists.

In a 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists containing a benzimidazole as a novel arginine mimetic, we examined the effects of benzimidazole modifications and amide substitutions on both activity and pharmacokinetics.

Structure-activity relationships of novel hematoregulatory peptides.

Hematopoiesis is a lifelong cell renewal process regulated by a family of lineage specific hematopoietic growth factors. Several hematopoietic growth factors such as G-CSF, GM-CSF, and M-CSF have been clinically evaluated for enhancement of host defense in normal and immunocompromised patients and for the treatment of infectious diseases. This paper reports the structure-activity relationships of low molecular weight hematoregulatory peptides based on a nonapeptide (1, SK&F 107647). Like the macromolecular growth factors, these peptides modulate host defense. A molecular target for this class of compounds has not yet been identified. However, the structure-activity relationships established by this study implicate a very specific molecular recognition event that is pivotal for the biological activities of 1 and its analogues.

A check on rational drug design: crystal structure of a complex of human immunodeficiency virus type 1 protease with a novel gamma-turn mimetic inhibitor.

We have previously reported (Newlander et al., J. Med. Chem. 1993, 36, 2321-2331) the design of human immunodeficiency virus type 1 (HIV-1) protease inhibitors incorporating C7 mimetics that lock three amino acid residues of a peptide sequence into a gamma-turn. The design of one such compound, SB203238, was based on X-ray structures of reduced amide aspartyl protease inhibitors. It incorporates a gamma-turn mimetic in the P2-P1' position, where the carbonyl of the C7 ring is replaced with an sp3 methylene group yielding a constrained reduced amide. It shows competitive inhibition with Ki = 430 nM at pH 6.0. The three-dimensional structure of SB203238 bound to the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction and refined to a crystallographic R-factor (R = sigma magnitude of Fo magnitude of - magnitude of Fc magnitude of /sigma magnitude of Fo magnitude of, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively) of 0.177. The inhibitor lies in an extended conformation in the active site; however, because of the constrained geometry of the C7 ring, it maintains fewer hydrogen bonds with the protein than in most other HIV-1 protease-inhibitor complexes. More importantly, the inhibitor binds to the enzyme differently than predicted in its design, by binding with the P2-P1' alpha-carbon atoms shifted by approximately one-half a residue toward the N-terminus from their presumed positions. This study illustrates the importance of structural information in an approach to rational drug design.

Conformational study of eight-membered diazocine turn mimics by two-dimensional NMR spectroscopy.

The eight-membered ring conformations of two diazocine turn mimics, methyl-[2,5-dioxo-3-(S)-(3-omega-tosyl-guanidino-propyl)-4-methyl-octahy dro- 1,4-diazocin-1-yl]acetate (I) and methyl-[2,5-dioxo-3-(S)-(3-omega-tosyl-guanidino-propyl)-octahydro-1,4- diazocin-1-yl]acetate (II), were determined using torsion angle constraints derived from 3J(C,H) coupling constants extracted from 13C-filtered TOCSY spectra with 13C in natural abundance. For I, the torsion angle constraints derived from 3J(C,H) coupling constants were in agreement with torsion angle constraints derived from 3J(H,H) coupling constants extracted from a P.E. COSY spectrum. Similar 3J(C,H) coupling constants were found for I and II, and they shared an identical eight-membered ring conformation characterized by two cis-amide bonds and a staggered conformation of the trimethylene group in which the H3 proton is proximal to both the H6 and H8 protons.

A novel constrained reduced-amide inhibitor of HIV-1 protease derived from the sequential incorporation of gamma-turn mimetics into a model substrate.

C7 mimetics, designed to lock three amino acid residues of a peptide chain into a gamma-turn conformation, were introduced sequentially between the P3 to P2' positions of a model HIV-1 protease substrate I (resulting in compounds II-IV) to probe its conformational requirements in binding to HIV-1 protease. Of these, compound IIIa with the C7 mimetic replacing Asn-Tyr-Pro, corresponding to the P2 through P1' positions of substrate, was found to be an inhibitor with a Ki of 147 microM. Reduction of the amide bond in the C7 mimetic of IIIa resulted in a novel constrained reduced-amide mimetic VIa with a Ki of 430 nM. This corresponds to over a 300-fold improvement in inhibitory activity over the original C7 mimetic. The inhibitory activity of mimetic VIa was in addition found to be 44-fold better than a similar linear reduced-amide containing inhibitor V. The synthesis of these mimetics are described.

Inhibition of neuronally induced relaxation of canine lower esophageal sphincter by opioid peptides.

Opioid peptides have profound effects on gut motility. To assess their actions on enteric neurons regulating sphincteric smooth muscle, the ability of several opioid agonists to antagonize the neuronally induced relaxation of canine lower esophageal sphincter smooth muscle was examined. Opioid peptides selective for mu (FK 33-824) or delta [( D-Pen2,D-Pen5]enkephalin) receptors produced a concentration dependent inhibition of electrical field stimulation (EFS)-induced relaxation. In contrast, neither kappa (ketocycloclazine) or sigma (SK & F 10047) opioid agonists were potent inhibitors of EFS-induced relaxation. This inhibition was relatively selective for opioid agonists since BHT 933 (alpha 2 adrenoceptor agonist) and SK & F 89124 (D2 dopamine agonist) did not inhibit EFS-induced relaxation. Furthermore, naloxone antagonized the effects of both FK 33-824 and DPDPE. These functional data suggest that opioid receptors are present on sphincteric intrinsic inhibitory neurons and that stimulation of these neuronal receptors can regulate lower esophageal sphincter relaxation.

Structure-activity relationships of novel vasopressin antagonists containing C-terminal diaminoalkanes and (aminoalkyl)guanidines.

We report the synthesis and biological activity of a series of analogues of the vasopressin antagonists [Pmp1,D-Tyr(Et)2,Val4]arginine-vasopressin (1) and [Pmp1,D-Tyr(Et)2,Val4,desGly9]arginine-vasopressin (2), where part or all of the tripeptide tail has been replaced by a simple alkyldiamine [NH(CH2)nNH2] or (aminoalkyl)guanidine [NH(CH2)nNHC(= NH)NH2] in order to examine the effects that variation of the length and orientation of the tripeptide tail have on renal vasopressin (V2) receptor antagonist activity. The results show that the entire tripeptide tail (Pro-Arg-Gly-NH2) can be replaced by an alkyldiamine or an (aminoalkyl)guanidine, compounds 15 and 16, respectively, indicating that there is no orientational requirement for the basic functional group coming off the cyclic hexapeptide ring. Also, there seems to be an "optimal" distance between the basic functional group and the hexapeptide ring since receptor affinity of the antagonists begins to fall off when the basic functional group is too close (compound 13) or extends too far (compounds 8-10) from the hexapeptide ring. These results suggest all that is necessary for retention of antagonist affinity and potency is a basic functional group, amine or guanidine, extended an optimal distance from the hexapeptide ring.

Cyclic enkephalin analogues containing alpha-amino-beta-mercapto-beta,beta-pentamethylenepropionic acid at positions 2 or 5.

Analogues of the highly potent and delta-receptor-selective enkephalins 1-4 were prepared with alpha-amino-beta-mercapto-beta,beta-pentamethylenepropionic acid (Apmp) replacing the beta,beta-dimethylcysteine (Pen) at positions 2 or 5. The peptides 5-8 were prepared by employing D,L-Apmp and, following oxidative cyclization, the resulting diastereomeric peptides were separated and purified by preparative high performance liquid chromatography. Compounds 7 and 8, with D- or L-Apmp substituted at position 5 are approximately 5 orders of magnitude more potent in the MVD assay than analogues 5 or 6 with D- or L-Apmp at position 2. While displaying less delta-receptor selectivity than the corresponding Pen-containing compounds, 7 and 8 are an order of magnitude more potent. All the analogues showed diminished delta-receptor selectivity in the rat brain binding assay. Compounds 7 and 8 displayed delta-receptor affinity comparable to the corresponding Pen-containing analogues.

Passive smoking on commercial airline flights.

In-flight exposure to nicotine, urinary cotinine levels, and symptom self-reports were assessed in a study of nine subjects (five passengers and four attendants) on four routine commercial flights each of approximately four hours' duration. Urine samples were collected for 72 hours following each flight. Exposures to nicotine measured during the flights using personal exposure monitors were found to be variable, with some nonsmoking areas attaining levels comparable to those in smoking sections. Attendants assigned to work in nonsmoking areas were not protected from smoke exposure. The type of aircraft ventilation was important in determining the levels of in-flight nicotine exposure. The environmental tobacco smoke levels that occurred produced measurable levels of cotinine (a major metabolite of nicotine) in the urine of passengers and attendants. Passengers who experienced the greatest smoke exposure had the highest levels of urinary cotinine. Changes in eye and nose symptoms between the beginning and end of the flights were significantly related both to nicotine exposure during the flight and to the subsequent urinary excretion of cotinine. In addition, subjects' perceptions of annoyance and smokiness in the airplane cabin were also related to in-flight nicotine exposure and urinary excretion measures.