Scaffold repurposing of fendiline: Identification of potent KRAS plasma membrane localization inhibitors.

KRAS plays an essential role in regulating cell proliferation, differentiation, migration and survival. Mutated KRAS is a major driver of malignant transformation in multiple human cancers. We showed previously that fendiline (6) is an effective inhibitor of KRAS plasma membrane (PM) localization and function. In this study, we designed, synthesized and evaluated a series of new fendiline analogs to optimize its drug properties. Systemic structure-activity relationship studies by scaffold repurposing led to the discovery of several more active KRAS PM localization inhibitors such as compounds 12f (NY0244), 12h (NY0331) and 22 (NY0335) which exhibit nanomolar potencies. These compounds inhibited oncogenic KRAS-driven cancer cell proliferation at single-digit micromolar concentrations in vitro. In vivo studies in a xenograft model of pancreatic cancer revealed that 12h and 22 suppressed oncogenic KRAS-expressing MiaPaCa-2 tumor growth at a low dose range of 1-5 mg/kg with no vasodilatory effects, indicating their potential as chemical probes and anticancer therapeutics.

Liquid chromatographic resolution of fendiline and its analogues on a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid.

Fendiline, an effective anti-anginal drug for the treatment of coronary heart diseases, and its sixteen analogues were resolved on a CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Fendiline was resolved quite well with the separation factor (α) of 1.25 and resolution (RS) of 1.55 when a mobile phase consisting of methanol-acetonitrile-trifluoroacetic acid-triethylamine at a ratio of 80/20/0.1/0.5 (v/v/v/v) was used. The comparison of the chromatographic behaviors for the resolution of fendiline and its analogues indicated that the 3,3-diphenylpropyl group bonded to the secondary amino group of fendiline is important in the chiral recognition and the difference in the steric bulkiness between the phenyl group and the methyl group at the chiral center of fendiline is also important in the chiral recognition.

Interaction of antagonists with calmodulin: insights from molecular dynamics simulations.

We report results of 12 ns, all-atom molecular dynamics simulation (MDS) and Poisson-Boltzmann free energy calculations (PBFE) on calmodulin (CaM) bound to two molecules of trifluoperazine (TFP) and of N-(3,3, diphenylpropyl)- N'-[1- R-(3,4-bis-butoxyphenyl)-ethyl]-propylenediamine (DPD). X-ray data show very similar structures for the two complexes, yet the antagonists significantly differ with respect to their CaM binding affinities, the neutral DPD is much more potent. The goal of the study was to unravel the reason why TFP is less potent although its positive charge should facilitate binding. The electrostatic energy terms in CHARMM and binding free energy terms of the PBFE approach showed TFP a better antagonist, while inspection of hydrophobic contacts supports DPD binding. Detailed inspection of the amino acid contributions of PBFE calculations unravel that steric reasons oppose the favorable binding of TFP. Structural conditions are given for a successful drug design strategy, which may benefit also from charge-charge interactions.

2'-hydroxy-fendiline analogues as potent relaxers of isolated arteries.

Novel, 2'-hydroxy derivatives of fendiline have been synthesised and their ability to induce relaxation of isolated rat small mesenteric and coronary arteries were determined. Both derivatives examined were significantly more potent as vasodilators than fendiline itself. Similar effects were observed on both mesenteric and coronary arteries.

3-Chloro,4-methoxyfendiline is a potent GABA(B) receptor potentiator in rat neocortical slices.

Using grease-gap recording from rat neocortical slices, the GABA(B) receptor agonist baclofen elicited reversible and concentration-dependent hyperpolarizing responses (EC50=18+/-2.3 microM). The hyperpolarizations were antagonised by the GABA(B) receptor antagonist Sch 50911 [(+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid). (+)-N-1-(3-chloro-4-methoxyphenyl)ethyl-3,3-diphenylpropylamine (3-chloro,4-methoxyfendiline; 3-Cl,4-MeO-fendiline) reversibly potentiated baclofen-induced hyperpolarizing responses, which were reduced by Sch 50911, producing leftward shifts of the baclofen concentration-response curves, with a marked increase in the maximal hyperpolarization (EC50=2+/-0.5 microM). In slices preincubated with either [3H]GABA or [3H]glutamic acid, 3-Cl,4-MeO-fendiline (1 microM) potentiated the inhibitory effect of baclofen (2 microM) on the electrically evoked release of [3H]GABA and had a similar effect on the release of [3H]glutamic acid at a concentration of 0.5 microM, without affecting the basal release. These effects were blocked by Sch 50911 (10 microM). Our findings suggest that 3-Cl,4-MeO-fendiline is a potent potentiator of pre- and postsynaptic GABA(B) receptor-mediated functions.

Calcimimetic and calcilytic drugs: just for parathyroid cells?

The cell surface calcium receptor (Ca2+ receptor) is a particularly difficult receptor to study because its primary physiological ligand, Ca2+, affects numerous biological processes both within and outside of cells. Because of this, distinguishing effects of extracellular Ca2+ mediated by the Ca2+ receptor from those mediated by other mechanisms is challenging. Certain pharmacological approaches, however, when combined with appropriate experimental designs, can be used to more confidently identify cellular responses regulated by the Ca2+ receptor and select those that might be targeted therapeutically. The Ca2+ receptor on parathyroid cells, because it is the primary mechanism regulating secretion of parathyroid hormone (PTH), is one such target. Calcimimetic compounds, which active this Ca2+ receptor and lower circulating levels of PTH, have been developed for treating hyperparathyroidism. The converse pharmaceutical approach, involving calcilytic compounds that block parathyroid cell Ca2+ receptors and stimulate PTH secretion thereby providing an anabolic therapy for osteoporosis, still awaits clinical validation. Although Ca2+ receptors are expressed throughout the body and in many tissues that are not intimately involved in systemic Ca2+ homeostasis, their physiological and/or pathological significance remains speculative and their value as therapeutic targets is unknown.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

Crystallization and preliminary diffraction analysis of Ca(2+)-calmodulin-drug and apocalmodulin-drug complexes.

Ca(2+)-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3"-(beta-chloroethyl)-2",4"-dioxo-3,5"- spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4- di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 A and differ in unit cell parameters from each other as well as from crystals of Ca(2+)-calmodulin or Ca(2+)-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca(2+)-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca(2+)-free calmodulin crystals diffracting-beyond 2.5 A resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca(2+)-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies.

Dissimilar mechanisms of action of anticalmodulin drugs: quantitative analysis.

A novel molecule from the arylalkylamine family of drugs, KHL-8430, has been identified as a potent and specific inhibitor of calmodulin activity. The effect of this drug on calmodulin-mediated enzymatic actions has been analyzed to exemplify how to model the mechanism of action of a functional calmodulin antagonist. The approach used includes both binding and enzyme kinetic studies. In both types of experiments, the effects of drugs on calmodulin-phosphofructokinase [ATP:D[fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11] and calmodulin-phosphodiesterase (3':5' cyclic nucleotide phosphodiesterase, EC 3.6.1.3) interactions have been investigated. We have found that KHL-8430, in contrast to trifluoperazine, a classical anticalmodulin drug, competes with neither phosphofructokinase nor phosphodiesterase for calmodulin binding, yet it liberates phosphofructokinase from calmodulin inhibition and phosphodiesterase from calmodulin stimulation. The anticalmodulin activity occurs at lower KHL-8430 than trifluoperazine concentrations. These findings might establish the functional importance of these differences in the specificity of these drugs. The synthesis of the data suggests that (i) whereas trifluoperazine antagonizes both phosphofructokinase and phosphodiesterase binding to calmodulin, KHL-8430 interacts with calmodulin complexed with enzymes; (ii) KHL-8430 binds to the calmodulin-phosphofructokinase complex with an affinity constant of 0.8 microM, whereas the binding constant of trifluoperazine is 2.5 microM (iii) within the ternary complex the dimeric form of the kinase preserves activity that is otherwise inactive; and (iv) the binding of trifluoperazine and KHL-8430 to calmodulin exhibits negative cooperativity. The approach used in this study makes it possible to screen for the calmodulin antagonist effect of other drugs as well.