EP1563063A2 - Calmodulin independent activation of nitric oxide synthase by nadph analogs - Google Patents

Abstract

Methods for activation of a constitutive nitric oxide synthase are described, as are agents which activate a constitutive nitric oxide synthase (e.g., NADPH analogs), methods of identifying such agents, and methods of treatment of diseases or conditions associated with nitric oxide production by a constitutive nitric oxide synthase, by administering an agent which activates the constitutive nitric oxide synthase.

Description

CALMODULL INDEPENDENT ACTIVATION OF NITRIC OXIDE SYNTHASE BY NADPH ANALOGS

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/424,653, filed November 7, 2002. The entire teachings of the above application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Nitric oxide (NO), a small molecule which is highly toxic at moderate concentrations, is a key messenger in mammalian physiology. NO is produced in humans by related enzymes wliich comprise the nitric oxide synthase (NOS) family. Two of these enzymes, endothelial NOS and neuronal NOS, are constitutively expressed (the "constitutive" NOS isoforms, or cNOS); the third, immune NOS, is inducible.

Endothelial NOS (eNOS) produces NO which controls vascular tone (hence blood pressure), dilates the airways, and controls numerous processes dependent on local dilation of blood vessels, such as gas exchange in lungs, penile erection, and renal function. Brain or neuronal NOS (bNOS or nNOS) produces NO which functions as a neurotransmitter. It is implicated in neural potentiation and bran development, and also controls peristalsis in the gut. eNOS and nNOS are constitutive enzymes controlled by mtracellular calcium and the regulatory protein, calmodulin (CaM). The general control mechanism in these constitutive NOS isoforms is the calcium/calmodulin dependent switching of interdomain electron transfer, which requires the CAM binding site and the autoinhibitory element of the FMN binding domain (Salerno, J. et al, J. Biol Chem. 272:29169 (1997)) and which correlates with the presence of additional sequence elements in the FAD binding domain and C terminal. Removal of the C terminal tail has been reported to produce a truncated, constitutively active eNOS (Roman, L.J. et al, Chemical Reviews 102:1119 (2002)). Recently, electron transfer through the reductase domains of NOS has been reported to be calmodulin independent in the absence of NADPH in rapid kinetics experiments (Daff, S. et al, Nitric Oxide 6:366 (2002)).

SUMMARY OF THE INVENTION

The present invention pertains to agents which activate a constitutive nitric oxide synthase (eNOS, nNOS) by inhibitor displacement, such as by displacing NADP⁺/NADPH; by having binding domain overlap on the nitric oxide synthase with NADPH; by filling the adenine portion of the pyridine nucleotide binding site on the nitric oxide synthase, without initiating inhibition of electron transfer; or by preventing binding of NADPH to the nitric oxide synthase. Representative agents which activate a constitutive NOS by inhibitor displacement include NADPH analogs (e.g., 2' AMP; 5' AMP; 2'5'ADP; ADP, ATP). The invention also pertains to methods of identifying agents that modulate activity of a constitutive nitric oxide synthase, by assaying the ability of the agents to displace an inhibitor (e.g.,

NADPVNADPH); as well as to methods of identifying agents that modulate activity of a constitutive nitric oxide synthase, by assaying the ability of the agents to compete with NADPH for binding to the constitutive nitric oxide synthase. The invention additionally pertains to methods of altering activity of a constitutive nitric oxide synthase, by contacting the nitric oxide synthase with an agent as described. The invention further pertains to methods of treating a disease modulated by production of nitric oxide by a constitutive nitric oxide synthase in an individual, by administering to the individual an effective amount of an agent as described. The agents and methods of the invention provide a means for activating a constitutive NOS in a manner that differs from other previously known methods of activating constitutive NOS, and thereby broaden the scope of available activators for these important enzymes as well as the scope of therapeutics for NO-mediated diseases and conditions.

DETAILED DESCRIPTION OF THE INVENTION The present invention pertains to the discovery of the ability of an NADPH analog to hyperactivate NOS, particularly eNOS. As described herein, abnormalities were noted in the activity of certain eNOS preparations. It was observed that affinity chromatography purified eNOS was active in the absence of calmodulin (CaM) prior to dialysis. Removal of eluent 2' AMP, an NADPH analog, removed the CaM independent activity and restored calcium/CaM control. Other potential causes for the CaM independent activation (buffer, pH, high ionic strength) were eliminated. 2'AMP is a competitive inhibitor with respect to NADPH, suggesting that displacement of NADPH/NADP⁺ from its binding site produced a state in which the reductase domains were competent to support catalysis by electron transfer. The results were confirmed and extended using mictotiter Griess activity assays of NO production in 96 well plates. 2'AMP not only activated eNOS in the absence of calmodulin, but produced hyperactivation of eNOS in the presence of calcium/CaM. Specifically, when the initial concentrations of NADPH and 2'AMP were equal, the activity of eNOS was 2-3 times that of eNOS activated by CaM alone. These effects appeared to correlate with changes in the optical spectra of the enzyme in the charge transfer region.

These results are supported by the hypothesis that NADPH/NADP⁺ produce a "conformational lock" in eNOS that inhibits electron transfer (Daff, S. et al, Nitric Oxide 6:366 (2002)). hihibitors which fill the adenine portion of the pyridine nucleotide binding site can potentially activate electron transfer by displacement of NADPH/NADP⁺. Since NADPH reduction of eNOS, particularly eNOS, is not close to rate limiting, a wide range of inhibitor concentrations can be tolerated before inhibition of electron donation by NADPH becomes more important than activation by removal of the charge transfer complex. As a result of these discoveries, agents can be identified which can be used to activate constitutive NOS isoforms. The activation occurs by inhibitor displacement, such as by displacement of the reductant NADP⁺ NADPH. For example, in one embodiment, the agent can be an agent which has binding domain overlap on the NOS enzyme with NADPH (e.g., an agent which fills the adenine portion of the pyridine nucleotide binding site), but which does not initiate inhibition of electron transfer (as does NADPH). In another embodiment, the agent can be an agent which prevents binding of NADPH. hi addition, methods are now available for activating constitutive NOS activity by administration of the agents, as well as methods for treating diseases or conditions associated with NO production by constitutive NOS isoforms by administration of the agents. The invention additionally pertains to use of the agents, as described herein, for the manufacture of a medicament for the treatment of diseases or conditions associated with NO production by constitutive NOS isoforms.

Isolating and Identifying New NOS Activators and Inhibitors Based on the discoveries described herein, it is now possible to identify agents which modulate (increase or decrease) the activity of a constitutive NOS. Agents which "increase" the activity are those which activate or promote the activity of the NOS. Agents which "decrease" the activity are those which inactivate, interfere with, minimize or prevent the activity of the enzyme. Agents of the invention can modulate NOS activity independently of calmodulin (CaM) activation; that is, whether or not CaM is associated with the NOS, it is the agent, rather than the CaM, that modulates the NOS activity.

In the methods of the invention, agents of interest (the "test agent") are assessed for an ability to modulate the activity of a constitutive NOS. Screens for agents are performed in a manner so that it can determined whether the test agent is competing with NADPH for interaction with NOS (e.g., displacing the inhibitor, such as displacing NADPH/NADP⁺). Thus, an excess of NADPH is not desirable; rather, the amount of NADPH in assays to screen for agents of interest should be varied, in order to facilitate detection o£ competition between the test agent and NADPH.

Bearing in mind these considerations, assays can be used to determine whether an agent modulates NOS activity. A sample of the agent to be tested (the "test agent") is contacted with a sample of a constitutive NOS, thereby generating a test sample (herein referred to as a "synthase sample"). After incubation of the synthase sample under conditions appropriate for activity of the enzyme (e.g., in the presence of NADPH), the level of NOS activity is measured by standard methods (e.g., by measurement of production of NO). The level of NOS activity is then measured and compared to the amount of activity in a control sample under the same conditions but in the absence of the test agent. If the level of activity in the synthase test sample is different from the level of activity of a control sample of the NOS under the same conditions but in the absence of the test agent, then the agent modulates the activity of the NOS. Similar assays can be used to determine whether an agent modulates the activity of one constitutive NOS isoform without modulating the activity of other constitutive NOS isoform, by comparing the level of activity of each isoform in the presence of the agent. Additional description of assays for determining whether an agent modulates NOS activity can be found, for example, in U.S. Patent 6,150,500, the entire teachings of which are incorporated herein by reference.

Agents Agents of the invention include agents that activate a constitutive nitric oxide synthase (NOS) by inhibitor displacement, such as by displacement of the reductant NADP7NADPH. For example, in one embodiment, the agent can be an agent which has binding domain overlap on the NOS enzyme with NADPH (e.g., an agent which fills the adenine portion of the pyridine nucleotide binding site), but which does not initiate inliibition of electron transfer (as does NADPH). In another embodiment, the agent can be an agent which prevents binding of NADPH. Representative agents include NADPH analogs, hi one particular embodiment, the agent is an analog of the adenosi e part of NADPH; in another particular embodiment, the agent is an analog having a phosphate at the 2' position. Representative NADPH analogs include 2'AMP; 5' AMP; 2'5'ADP; ADP; ATP.

Methods of Altering NOS Activity

The agents that activate a constitutive NOS, as described herein, can be used to activate a constitutive NOS isoform. To activate the constitutive NOS, the NOS of interest (eNOS or nNOS, or both) is contacted with an agent as described herein, under conditions for interaction between the agent and the NOS of interest. More than one agent can be used concurrently, if desired.

Methods of Treatment

The agents that activate a constitutive NOS can also be used to activate the constitutive NOS isoform in vivo. In a preferred embodiment, the agent is used to activate a constitutive NOS isoform in a mammal, such as a human, in order to treat a disease or condition associated with NO production. The term, "treatment" as used herein, refers not only to ameliorating symptoms associated with the disease or condition, but also preventing or delaying the onset of the disease or condition, and also lessening the severity or frequency of symptoms of the disease or condition. For example, in certain methods of the invention, one or more agent(s) that activate a constitutive NOS (e.g., an agent described herein) is administered to an individual. The agent can be administered in dosage formulations containing conventional, non-toxic, physiologically- acceptable carrier(s) or excipient(s) The carrier and composition can be sterile. The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc. Methods of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral, intranasal, subcutaneous, rectal, buccal, vaginal, intraurethral, by inhalation spray, or via an implanted reservoir. Other suitable methods of introduction can also include rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.

The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings or other mammals of interest. For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubihzing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The agent may be incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.

Agents described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc. The agents are administered in a therapeutically effective amount. The amount of agents which will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The therapeutically effective amount can be administered in a single dose, or a series of doses separated by appropriate intervals, such as hours, days, or weeks. The term "single dose," as used herein, can be a solitary dose, and can also be a sustained release dose, such as by a controlled-release dosage formulation or a continuous infusion. Other drugs can also be administered in conjunction with the agent, and more than one agent that activates a constitutive NOS can be administered at the same time.

In certain embodiments of the invention, an agent that activates a constitutive NOS is administered in order to treat a condition modulated by production of nitric oxide by the cNOS (e.g., hypertension, atherosclerosis, diabetes, or acute asthma for eNOS). In another embodiment of the invention, an agent which activates a cNOS can be used as a means for treating male erectile dysfunction; in a preferred embodiment, the agent is administered intraurethrally to limit systemic side effects.

The invention is further illustrated by the following Exemplification, which is not intended to be limiting in any way. EXEMPLIFICATION

It was noted that affinity chromatography purified eNOS is active in the absence of calmodulin prior to dialysis. Removal of eluent 2' AMP, an NADPH analog, removed CaM independent activity and restored Ca⁺²/ CaM control. 2' AMP is a competitive inhibitor with respect to NADPH, suggesting that displacement of NADPH/NADP⁺ from its binding site produced a state in which the reductase domains were competent to support catalysis by electron transfer.

These results were confirmed and extended. Titration of cNOS with 2'AMP not only activated cNOS in the absence of calmodulin, but produced hyperactivation of eNOS in the presence of Ca⁺²/ CaM. Specifically, when the initial concentrations of NADPH and 2' AMP were equal, eNOS activity was 3-5 times that of eNOS activated by CaM alone. These effects appeared to correlate with changes in the optical spectra of the enzyme in charge transfer region.

These results supported the hypothesis that NADPH/NADP⁺ produces a 'conformational lock' in cNOS that inhibits electron transfer (Daff, S. et al, Nitric Oxide 6:366 (2002)). Inhibitors which fill the adenine portion of the pyridine nucleotide binding site can potentially activate electron transfer by displacement of NADPH/NADP⁺. Since NADPH reduction of cNOS, particularly eNOS, is not close to rate limiting, a wide range of inhibitor concentrations can be tolerated before inhibition of electron donation by NADPH becomes more important than activation by removal of the charge transfer complex. These effects are closely related to loss of control in C terminally truncated cNOS and effects of C terminal phosphorylation (Lane, P. and Gross, S.S., J Biol. Chem. 277(21): 19087-94 (2002)), which involve adjacent sites. The initial observations were the result of activity assays of partially purified column fractions. NOS holoenzymes were purified using a 2'5'ADP affinity column; undialyzed eNOS column fractions from this step were essentially calmodulin independent, and were at least 50% as active as purified eNOS preparations (data not shown). The major factors which differentiated the purified control fractions from the undialyzed fractions which has CaM independent activity, were high salt and the presence of an NADPH antagonist, 2'AMP, which was used to elute the column. Although very high ionic strength can partially activate NOS, the salt concentrations associated with the purification were not high enough to produce NOS activation by themselves.

Titration of eNOS with 2'AMP in the presence of 'fully' activating levels of Ca⁺²/CaM was performed using a Greiss assay (see, e.g., the Griess Reagent System, Promega Corporation, Madison, WI; and manufacturer's instructions for use). Calcium stoichometries were between two and four. The titration produced an additional activation corresponding to an apparent Kd of ~lmM. This corresponded closely to the concentration of NADPH, and suggested that the effect is due to competition of ligands with similar binding constants (NADPH and 2'AMP).

The effect of 2'AMP on the optical spectra of reductase cofactors in eNOS was also examined for eNOS as isolated and reduced with 1 mM NADPH. eNOS only, eNOS NADPH, eNOS NADPH with 2'AMP, eNOS NADPH with 2'AMP and CAM, and NADPH 2'AMP were compared. It was found that sequential addition of 2'AMP and calmodulin caused additional steady state reduction of flavin cofactors, most readily seen below 500 nm, and also a decrease in long wavelength species (data not shown). It was also noted that 2'AMP derivatives functioned as better activators than 3' derivatives, and AMP -based ligands were more efficient than GMP -based ligands. Cyclic AMP, cyclic GMP, GDP and GTP did not function as activators, and 3'5' ADP was only a very weak activator.

How do the NOS control elements work together? It is clear that regions of interest are not always close together on the reductase. This is most easily understood in terms of common effects on domain alignment, rather than direct interactions between all the elements involved. In view of the conformation of the enzyme, it is difficult to dock NOS oxygenase domains with a reductase domain model based on NADPH P450 reductase to obtain a close enough distance for electron transfer. A conformational shuttle in which the FMN binding domain moves significantly may be a necessary feature of electron transfer (e.g., Ghosh, D.K. and Salerno, J.C., Front. Biosci. δ:dl93-209 (2003)). Multiple constraints may be necessary to interrupt this shuttle. The teachings of all references cited are hereby incorporated herein in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

CLALMSWhat is claimed is:

1. An agent which activates a constitutive nitric oxide synthase by inhibitor displacement.

2. An agent of Claim 1, wherein the agent displaces NADP7NADPH

3. An agent of Claim 1, wherein the agent is an agent which has binding domain overlap on the nitric oxide synthase with NADPH.

An agent of Claim 3, wherein the agent fills the adenine portion of the pyridine nucleotide binding site, but does not initiate inhibition of electron transfer.

5. An agent of Claim 1, wherein the agent prevents binding of NADPH to the nitric oxide synthase.

6. A method of altering activity of a constitutive nitric oxide synthase, comprising contacting the nitric oxide synthase with an agent of any one of Claims 1-5.

7. A method of Claim 6, wherein the constitutive nitric oxide synthase is endothelial nitric oxide synthase.

8. A method of Claim 6, wherein the constitutive nitric oxide synthase is neuronal nitric oxide synthase.

9. A method of altering activity of a constitutive nitric oxide synthase, comprising contacting the nitric oxide synthase with an NADPH analog.

10. A method of Claim 9, wherein the NADPH analog is selected from the group consisting of: 2' AMP, 5' AMP, 2'5'ADP, ADP and ATP.

11. A method of treating a disease modulated by production of nitric oxide by a constitutive nitric oxide synthase in an individual, comprising administering to the individual an effective amount of an agent of any one of Claims 1-5.

12. A method of treating a disease modulated by production of nitric oxide by a constitutive nitric oxide synthase in an individual, comprising administering to the individual an effective amount of an NADPH analog.

13. A method of Claim 12, wherein the NADPH analog is selected from the group consisting of: 2' AMP, 5' AMP, 2'5'ADP, ADP and ATP.

14. A method of identifying an agent that modulates activity of a constitutive nitric oxide synthase, comprising assaying the ability of the agent to displace an inhibitor.

15. A method of identifying an agent that modulates activity of a constitutive nitric oxide synthase, comprising assaying the ability of the agent to compete with NADPH for binding to the constitutive nitric oxide synthase.