Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: II. Side-chain modifications

Summary The overall on- and off-rate constants for blockage of epithelial Na channels by amiloride analogs were estimated by noise analysis of the stationary Na current traversing frog skin epithelium. The (2-position) side chain structure of amiloride was varied in order to obtain structure/rate constant relationships. (1) Hydrophobic chain elongations (benzamil and related compounds of high blocking potency) increase the stability of the blocking complex (lowered off-rate), explained by attachment of the added phenyl moiety to a hydrophobic area near the site of side chain interaction with the channel protein. (2) Some other chain modifications show that the on-rate, which is smaller than a diffusion-limited rate, varies with side chain structure. In several cases this effect is not attributable to steric hindrance on encounter, and implies that the side chain interacts briefly with the channel protein (encounter complex) before the main blocking position of the molecule is attained. The encounter complex must be labile since the overall rate constants of blockage are not concentration-dependent. (3) In two cases, changes at the 2-position side chain and at other ring ligands, with known effects on the blocking rate constants, could be combined in one analog. The rate constants of blocking by the resulting compounds indicate that the structural changes have additive effects in terms of activation energies. (4) Along with other observations (voltage dependence of the rate constants and competition with the transported Na ion), these results suggest a blocking process of at least two steps. It appears that initially the 2-position side chain invades the outward-facing channel entrance, establishing a labile complex. Then the molecule is either released completely (no block) or the 6-ligand of the pyrazine ring gains access to its receptor counterpart, thus establishing the blocking complex, the lifetime of which is strongly determined by the electronegativity of the 6-ligand.     

Interactions of amiloride and other blocking cations with the apical Na channel in the toad urinary bladder

Summary A simple model of the action of amiloride to block apical Na channels in the toad urinary bladder was tested. According to the model, the positively charged form of the drug binds to a site in the lumen of the channel within the electric field of the membrane. In agreement with the predictions of the model: (1) The voltage dependence of amiloride block was consistent with the assumption of a single amiloride binding site, at which about 15% of the transmembrane voltage is sensed, over a voltage range of ±160 mV. (2) The time course of the development of voltage dependence was consistent with that predicted from the rate constants for amiloride binding previously determined. (3) The ability of organic cations to mimic the action of amiloride showed a size dependence implying a restriction of access to the binding site, with an effective diameter of about 5 angstroms. In a fourth test, divalent cations (Ca, Mg, Ba and Sr) were found to block Na channels with a complex voltage dependence, suggesting that these ions interact with two or more sites. at least one of which may be within the lumen of the pore.     

Voltage dependence of Na channel blockage by amiloride: Relaxation effects in admittance spectra

Summary Amiloride, present in the mucosal solution, causes the appearance of a distinct additional dispersion in the admittance spectrum of the apical membrane of toad urinary bladder. The parameters of this dispersion (characteristic frequency. amplitude) change with amiloride concentration and with membrane voltage. They allow the calculation of the overall rate constants for Na channel blockage by the positively charged form of amiloride. and the voltage dependence of these rate constants. The on-rate of blockage increases and the off-rate decreases when the membrane surface to which cationic amiloride has access, is made more positive. This result is suggestive of a blocking model where the cationic amidino group of amiloride, depending on its charge, senses 10 to 13% of the membrane voltage while invading the channel entrance by a single-step process, and rests at an electrical distance corresponding to 24 to 30% of membrane voltage while occupying the blocking position.     

Synthesis and characterization of methylbromoamiloride,a potential biochemical probe of epithelial Na+ channels

Summary We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na⁺ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH₃BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current ofin vitro frog skin and²²Na⁺ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH₃BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na⁺ channel.     

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.     

Structure-activity relationship in the induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells by regioisomeric phenanthrene quinones

Four regioisomeric phenanthrene (PH) quinones (Q) were investigated for their ability to induce chromosomal damage and spindle disturbances. PH 1,4-Q and PH 1,2-Q induced structural as well as numerical chromosomal aberrations, whereas the isomers PH 9,10-Q and PH 3,4-Q were virtually inactive in this respect. However, all four compounds enhanced the frequency of c-mitoses.     

Sodium flux in the apical membrane of the toad skin: Aspects of its regulation and the importance of the ionic strength of the outer solution upon the reversibility of amiloride inhibition

Summary Injection of small pulses of concentrate solutions of salts or drugs into the outer bathing fluid led to sudden increases of its solute concentration. Vigorous stirring of the outer bathing solution was used to minimize the thickness of the unstirred layer adjacent to the outer skin surface. Pulses of 1m NaCl injected into the outer compartment induced sharp increases of the SCC following a time course variable with the magnitude of the pulse and the particular condition of each skin. Comparison of the spontaneous decline of the SCC with the decline induced by a small dose of amiloride, where an increase inR was observed, indicates that the spontaneous decline cannot be explained simply as a reduction of the Na permeability of the apical membrane by self-inhibition of feedback inhibition of the apical membrane Na channels. Reduction of the driving force for Na movement into the epithelial cells must play an important role in the process. Reversibility of the amiloride inhibition of the SCC was highly dependent upon the ionic strength of the solution used to rinse and wash out the inhibitor from the outer skin surface. With H₂O, the amiloride molecules washed out slowly as compared to NaCl or KCl solutions. Na or K have the same ability to dislodge the amiloride molecules from their binding sites. This effect is apparently of a purely electrostatic nature.     

Intracellular electrolyte concentrations in the frog skin epithelium: Effect of vasopressin and dependence on the Na concentration in the bathing media

Summary The intracellular electrolyte concentrations of the frog skin epithelium have been determined in thin freeze-dried cryosections using the technique of electron microprobe analysis. Stimulation of the transepithelial Na transport by arginine vasopressin (AVP) resulted in a marked increase in the Na concentration and a reciprocal drop in the K concentration in all epithelial cell layers. The effects of AVP were cancelled by addition of amiloride. It is concluded from these results that the primary mechanism by which AVP stimulates transepithelial Na transport is an increase in the Na permeability of the apical membrane. However, also some evidence has been obtained for an additional stimulatory effect of AVP on the Na pump. In mitochondria-rich cells and in gland cells no significant concentration changes were detected, supporting the view that these cells do not share in transepithelial Na transport. Furthermore, the dependence of the intracellular electrolyte concentrations upon the Na concentration in the outer and inner bathing solution was evaluated. Both in control and AVP-stimulated skins the intracellular Na concentration showed saturation already at low external Na concentrations, indicating that the self-inhibition of transepithelial Na transport is due to a reduction of the permeability of the apical membrane. After lowering the Na concentration in the internal bath frequently a Na increase in the outermost and a drop in the deeper epithelial layers was observed. It is concluded that partial uncoupling of the transport syncytium occurs, which may explain the inhibition of the transepithelial Na transport and blunting of the AVP response under this condition.     

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium,and by other divalent ions

Summary Direct inhibitory effects of Ca²⁺ and other ions on the epithelial Na⁺ channels were investigated by measuring the amiloride-blockable²²Na⁺ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol. 81:643–666, 1983) we found that the presence of micromolar concentrations of Ca²⁺ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive²²Na⁺ uptake could not be blocked by Ca²⁺ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba²⁺, Sr²⁺, or VO²⁺, but not by Mg²⁺. The Ca²⁺ inhibition constant was a strong function of pH, and varied from 0.04 m at pH 7.8 to >10 m at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of²²Na⁺ uptake in vesicles that contained 0.5 m Ca²⁺. This Ca²⁺ activity produced a maximal inhibition of²²Na⁺ uptake at pH7.4 but had no effect at pH7.0. The tracer fluxes measured in the absence of Ca²⁺ were pH independent over this range. The data is compatible with the model that Ca²⁺ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca²⁺ from binding to this site but does not by itself inhibit transport. The fact that the apical Na⁺ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca²⁺, rather than, or in addition to changes in the cytoplasmic level of free Ca²⁺.     

Structure-activity relationship studies on a series of piperazinebenzylalcohols and their ketone and amine analogs as melanocortin-4 receptor ligands

A series of piperazinebenzylalcohols were prepared and studied to compare with their ketone and amine analogs as MC4R antagonists. Several benzylalcohols such as 14a and 14g displayed low nanomolar binding affinities (K_i < 10 nM), and high selectivities over other melanocortin receptor subtypes.     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

Structure-activity relationship studies on a novel class of antiproliferative agents derived from Lavendustin A. Part I: Ring A modifications

The potent antiproliferative agent SDZ LAP 977, which has shown efficacy in a clinical proof of concept study in actinic keratosis patients, has been previously demonstrated to block the cell cycle in mitosis. In the present study, we further explored the mode of action: SDZ LAP 977 binds to the "colchicine binding site" on tubulin and, thus, inhibits tubulin polymerization in vitro. Moreover, we established structure-activity relationships for the effect of modifications in the 2,5-dimethoxyphenyl moiety ("ring A") of the molecule on in vitro antiproliferative activity.     

Nonsteroidal anti-inflammatory drugs: I. A study of “structure-efficacy of the anti-inflammatory effect relationship activity”

Using the computer system SARD-21 (Structure Activity Relationship &; Design) structural features typical for the high-and low-effective nonsteroidal anti-inflammatory drugs (NSAIDs) have been recognized and the influence of these features on the anti-inflammatory properties have been evaluated. This information has been used for generation of the model for prediction of anti-inflammatory effectiveness of pharmaceutical preparations with 76% and 81% levels of recognition by two methods. The recognized structural parameters may be successfully used for creation of new highly effective NSAIDs, and also for modification of structures of known NSAIDs for the increase of effectiveness of their anti-inflammatory action.     

Statistical properties of ion channel records. Part I: relationship to the macroscopic current

Macroscopic ion channel current can be derived by summation of the stochastic records of individual channel currents. In this paper, we present two probability density functions of single channel records that can uniquely determine the macroscopic current regardless of other statistical properties of records or the stochastic model of channel gating (presented often with stationary Markov models). We show that H(t), probability density function of channel opening events (introduced explicitly in this paper), and D(t), probability density function of the open duration (sometimes has named dwell time distribution as well), determine the normalized macroscopic current, G(t), through G(t) = P(t) - H(t) * Q(t) where P(t) is the cumulative density function of H(t), Q(t) is the cumulative density function of D(t), * is the symbol of convolution integral and G(t) is the macroscopic current divided by the amplitude of single channel current and the number of single channel sweeps. Compared to other equations for the macroscopic current, here the macroscopic current is expressed only in terms of the statistical properties of single channel current and not the stochastic model of ion channel gating or a conditioned form of macroscopic current. Single channel currents of an inactivating BK channel were used to validate this relationship experimentally too. In this paper, we used median filters as they can remove the unwanted noise without smoothing the transitions between open and closed states (compare to low pass filters). This filtering leads to more accurate measurement of transition times and less amount of missed events.     

Isoforms of Na,K-ATPase inArtemia saline: I. Detection by FITC binding and time course

Summary Partially purified Na,K-ATPase from whole nauplii at various stages of development, analyzed by SDS-PAGE, reveals a polydisperse and two subunits (denoted 1 and 2). In the absence of Ca²⁺, ATP-inhibitable fluorescein isothiocyanate (FITC) labeling is restricted to the subunit of this enzyme, even in crude naupliar homogenates. The intensity of the -specific fluorescent signal (i.e., the sum of the yield from both isoforms) is proportional to Na,K-ATPase activity during development. FITC-labeled subunits were detected at 8 hr of development prior to the detection of measurable Na,K-ATPase activity. The 2/1 ratio changed from an initial value of 1.25 to a peak of 1.75 at 32 hr of development, then reverted to a ratio of 1.25 by 42 hr, and remained constant thereafter. Pulse chase studies with³⁵S-methionine indicated that the developmental increase in enzyme activity is coincident with amino acid incorporation into the subunits, implying that enzyme synthesis is active during enzyme accumulation.During the tenure of an Educational Commission for Foreign Medical Graduates Visiting Associate Professorship.     

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Summary Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na⁺ and in the absence of K⁺ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.Deceased (September 13, 1990).     

Antitumor agents 279. Structure-activity relationship and in vivo studies of novel 2-(furan-2-yl)naphthalen-1-ol (FNO) analogs as potent and selective anti-breast cancer agents

In our ongoing modification study of neo-tanshinlactone (1), we discovered 2-(furan-2-yl)naphthalen-1-ol (FNO) derivatives 3 and 4 as a new class of anti-tumor agents. To explore structure-activity relationships (SAR) of this scaffold, 18 new analogs, 6-12 and 14-24, were designed and synthesized. The C11-esters 7 and 12 displayed broad anti-tumor activity (ED₅₀ 1.1-4.3 μg/mL against seven cancer cell lines), while C11-hydroxymethyl 14 showed unique selectivity against the SKBR-3 breast cancer cell line (ED₅₀ 0.73 μg/mL). Compounds 15 and 22 displayed potent and selective anti-breast tumor activity (ED₅₀ 1.7 and 0.85 μg/mL, respectively, against MDA-MB-231). The SAR results demonstrated that the substitutions from the ring-opened lactone ring C of 1 are critical to the anti-tumor potency as well as the apparent tumor-tissue type selectivity. Treatment with 3 in Brca1^f11/f11p53^f5&6/f5&6Cre^c mice models significantly inhibited the proliferation of mammary epithelial cells and branching of mammary glands.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement

To investigate Na⁺ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na⁺-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na⁺ in the presence of various Mg²⁺ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na⁺ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na⁺ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na⁺ ions and followed by the conformational transition E₁/E₂ of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na⁺ ions are bound. Received: 8 October 1998/Revised: 29 December 1998     

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 1: Initial structure-activity relationship studies of a hit from a high throughput screening

The P2X3 receptor is primarily expressed in the peripheral sensory nerves, and therefore, antagonists of this receptor may be useful for the treatment of chronic pain. Pyrrolinone derivatives have been identified as a novel class of P2X3 receptor antagonists. A lead structure with moderate activity was discovered through a high-throughput screening assay. A structure-activity study led to the discovery of several P2X3 receptor antagonists. Compound 34 showed potent and specific antagonistic activity and analgesic efficacy.