Treatment of parkinson's disease through ARFGAP1 inhibition

Abstract

Methods are provided for the treatment of Parkinson's disease (PD) in patients bearing mutations in the LRRK2 gene. A therapeutically effective amount of piperazine derivative compounds are employed to inhibit the biological activity of ArfGAP1, inhibition that counteracts the deleterious effects of mutations in, or increased expression of, the LRRK2 protein.

Claims

1. A method of treating Parkinson's disease in a human subject in need thereof, comprising administering to the subject a compound having the formula: ##STR00004##

2. The method of claim 1, wherein the subject has LRRK2-mutation-induced Parkinson's Disease.

3. The method of claim 2, wherein a genome of the subject carries an autosomal dominant mutation of a LRRK2 gene.

4. A method of treating Parkinson's Disease in a human subject in need thereof, comprising administering to the subject a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound having the formula: ##STR00005##

5. The method of claim 4, wherein the subject has LRRK2-induced Parkinson's Disease.

6. The method of claim 5, wherein a genome of the subject carries an autosomal dominant mutation of a LRRK2 gene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to describe the manner in which the above-recited and other advantages and features of the disclosure can be obtained, a more particular description of the principles briefly described above will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. Understanding that these drawings depict only exemplary embodiments of the disclosure and are not therefore to be considered to be limiting of its scope, the principles herein are described and explained with additional specificity and detail through the use of the accompanying drawings in which:

(2) FIG. 1 illustrates the schematic domain structure of the LRRK2 protein. Residues 1-660 encode LRRK2-specific repeat sequences, residues 984-1278 encode the LRR domain, residues 1335-1510 encode the ROC GTPase domain, residues 1519-1795 encode the COR domain, residues 1879-2138 encode the kinase domain, and residues 2138-2527 encode the WD40 domain. The positions of mutations clearly segregating with disease are shown in red, whereas the positions of R1441H and N1437H associated with PD are highlighted in blue. The domain boundaries are indicated by the residue numbers in black.

(3) FIG. 2 illustrates experimental results in yeast cells, which demonstrate that expression of human ArfGAP1 causes a slow growth phenotype in yeast (bottom line) relative to yeast strains with empty vector.

(4) FIG. 3 illustrates the model of reciprocal regulation between ArfGAP1 and LRRK2. Increased expression of LRRK2, or mutations that increase LRRK2 kinase activity, induce cell death. ArfGAP1 binds to LRRK2, promoting hydrolysis of GTP to GDP and decreasing the kinase activity and autophosphorylation of LRRK2. LRRK2 also phosphorylates ArfGAP1, inhibiting its GAP activity. This reciprocal regulation leads to complex effects on cellular viability.

(5) FIG. 4 illustrates the results of screening 33,000 small molecule compounds against yeast gcs1 knockout strains bearing empty vector (3B-VEC) or yeast strains bearing ArfGAP1 expression vectors (3B-141).

(6) FIG. 5 illustrates the dose-dependent growth of yeast ArfGAP1 gcs1 knockout strains with six small molecules identified from the screen: chembridge ID no.'s 5420376, 5431942, 5468123, 5261344, 5429814, 5459804. The 3B-VEC point represents the growth level of gcs1 knockout yeast strain with empty vector not expressing ArfGAP1.

(7) FIG. 6 illustrates the top eight hits from the primary screen in yeast, all of which except 5468123 are phenyl piperazine-derivate molecules

DETAILED DESCRIPTION

(8) Various embodiments of the disclosure are discussed in detail below. While specific implementations are discussed, it should be understood that this is done for illustration purposes only. All references cited within this disclosure are incorporated herein. A person skilled in the relevant art will recognize that other components and configurations may be used without parting from the spirit and scope of the disclosure.

(9) Described are compositions and methods for treating Parkinson's disease through the administration of therapeutically effective amounts of substituted piperazine-derivative molecules. The treatment regime, in a preferred embodiment, is geared towards the treatment of LRRK2 mutation-induced PD through the inhibition of the ArfGAP1 protein.

(10) In one embodiment, the therapeutically effective amount of the ArfGAP1 inhibitor has the general formula of

(11) ##STR00001##

(12) wherein n=0, 1 or 2; wherein G1 is (1) a unsaturated heterocyclic ring with five or six members, the unsaturated heterocyclic ring at least having a S or a N; or (2) a saturated carbocyclic ring with five or six members, the saturated carbocyclic ring at least being substituted with one or more of carboxylic acid or methanone, or being fused with benzene; or (3) a unsaturated bicycloheptene; or (4) a unsaturated methyl-substituted alkene with two to four Cs; and wherein G2 is (1) 4-acetophenone; or (2) 4-nitrobenzene; or (3) 2-pyridine. In a further embodiment, wherein n=1, G1 is 2-pyridine or 2-thiophene, and G2 is 4-acetophenone or 4-nitrobenzene.

(13) In another embodiment, wherein n=1, G1 is methyl-substituted butene, and G2 is 4-nitrobenzene.

(14) In another embodiment, wherein n=1, G1 is unsaturated bicycloheptene, and G2 is 4-nitrobenzene.

(15) In another embodiment, wherein n=0, G1 is indane, and G2 is 2-pyridine.

(16) In another embodiment, wherein n=0, G1 is formylcyclohexane carboxylic acid, and G2 is 4-nitrobenzene.

(17) In another embodiment, the ArfGAP1 inhibitor for treating or preventing Parkinson's disease has the general formula of:

(18) ##STR00002##

(19) wherein n=0, 1 or 2; wherein G1 is (1) a unsaturated heterocyclic ring with five or six members, the unsaturated heterocyclic ring at least having a S or a N; or (2) a saturated carbocyclic ring with five or six members, the saturated carbocyclic ring at least being substituted with one or more of carboxylic acid or methanone, or being fused with benzene; or (3) a unsaturated bicycloheptene; or (4) a unsaturated methyl-substituted alkene with two to four Cs; and wherein G2 is (1) 4-acetophenone; or (2) 4-nitrobenzene; or (3) 2-pyridine.

(20) In another embodiment, wherein n=1, G1 is 2-pyridine or 2-thiophene, and G2 is 4-acetophenone or 4-nitrobenzene.

(21) In another embodiment, wherein n=1, G1 is methyl-substituted butene, and G2 is 4-nitrobenzene.

(22) In another embodiment, wherein n=1, G1 is unsaturated bicycloheptene, and G2 is 4-nitrobenzene.

(23) In another embodiment, wherein n=0, G1 is indane, and G2 is 2-pyridine.

(24) In another embodiment, wherein n=0, G1 is formylcyclohexane carboxylic acid, and G2 is 4-nitrobenzene.

(25) In another embodiment, the ArfGAP1 inhibitor for treating or preventing Parkinson's disease has the general formula of:

(26) ##STR00003##

Administration

(27) Any suitable methods of administering a composition as described herein to a subject may be used. In these methods, the compositions can be administered to a subject by any suitable route, e.g., systemically by intravenous injection, directly to a target site, parenterally, orally, interathecally, interacranially, etc. The compositions may be administered directly to a target site by, for example, surgical delivery to an internal or external target site, or by catheter to a site accessible by a blood vessel. For example, in a method of treating PD, a composition as described herein may be delivered orally or intravenously. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously, or interathecally by peritoneal dialysis, pump infusion). For parenteral administration, the compositions are preferably formulated in a sterilized pyrogen-free form. As indicated above, the compositions described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like. The compositions described herein may be administered to mammals (e.g., rodents, humans, nonhuman primates, canines, felines, ovines, bovines) in any suitable formulation according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2000) and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (1988-1999), a standard text in this field, and in USP/NF). A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington: supra. Other substances may be added to the compositions to stabilize and/or preserve the compositions.

(28) The therapeutic methods described herein in general include administration of a therapeutically effective amount of the compositions described herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects at risk can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider. The methods and compositions herein may be used in the treatment of any other disorders or diseases relating to anemia.

Effective Doses

(29) The compositions described herein are preferably administered to a mammal (e.g., human) in an effective amount, that is, an amount capable of producing a desirable result in a treated mammal (e.g., treating PD through administration of piperazine-derivative compounds). Such a therapeutically effective amount can be determined according to standard methods. Chemical analysis of isolated compounds, specifically piperazine-derived molecules have demonstrated a predicted ability to permeate through the blood-brain barrier for therapeutic purposes based on the following data concerning the compounds: MW: <400; Sum of (O+N): <5; PSA: <60-70 A; c Log P: <5.0; No of rotatable bonds <8; pKa: neutral or basic with pKa 7.5-10.5 (avoid acids); Non-Pgp substrate; Aqueous solubility: >60 ug/ml; Effective Permeability: >110.sup.6 cm/sec.

(30) Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. A delivery dose of a composition as described herein may be determined based on preclinical efficacy and safety.

EXAMPLES

(31) The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

(32) Screening of Small Molecules in Yeast

(33) Overexpression of heterologous proteins in the yeast Saccharomyces cerevisiae often inhibits its growth, while inhibitors of the overexpressed proteins can restore growth. These simple observations form the basis of a powerful assay to identify inhibitors of such proteins. An expression plasmid for the inducible expression of a gene of interest is introduced into a yeast strain rendered more sensitive to chemicals by deletion of efflux pumps. Protein expression is induced, cells are exposed to test chemicals, and growth is measured by optical density at 600 nm (OD600 or A600) reading.

(34) In the instant case, a S. cerevisiae gcs1 knockout strain is employed bearing a vector expressing the heterologous protein hArfGAP1 (human ArfGAP1, the human ortholog of yeast GCS1). Generally, expression of ArfGAP1 will suppress growth of gcs1 knockout yeast and thus small molecules that inhibit ArfGAP1 will lead to observable increases in growth. This in turn allows for the direct identification of small molecules that could be used to treat LRRK2-mutation induced PD.

(35) Yeast Strains Employed in Inhibitor Screening

(36) PPY17:114-3B-vec (gcs1::NatR, pdr5::HIS3, snq2::TRP1, ura3, ade2, carrying plasmid pRS315) [empty vector control strain]; PPY17:114-3B-141-A (gcs1::NatR, pdr5::HIS3, snq2::TRP1, ura3, ade2, carrying plasmid pPP16:141 pGAL1-hArfGAP1) [human ArfGAP1-expressing strain].

(37) Generation of Synthetic Complex (SC) Mix, SC Dropout Mix, and SC Selection Medium Solutions

(38) Synthetic Complete (SC) mix: 0.6-g adenine, 0.6-g uracil, 0.6-g tryptophan, 0.6-g histidine, 0.6-g arginine, 0.6-g methionine, 0.9-g tyrosine, 0.9-g lysine, 1.5-g phenylalanine, 6.0-g threonine, 3.0-g aspartic acid, 1.8-g isoleucine, 4.5-g valine, 1.8-g leucine (Sigma). All ingredients are weighed, mixed together, and a mortar and pestle is used to grind the ingredients into a homogeneous powder. The powder is stored in 50-mL Falcon tubes at room temperature.

(39) SC dropout mix: All the ingredients except for leucine are weighed, then mixed, ground, and stored as for the SC mix.

(40) SC selection medium: Yeast nitrogen base without amino acid (BD/Difco, Sparks, Md.) 0.67% SC and dropout mix 0.067%, are dissolved in water. 0.25 mL of 1N NaOH is added to every 100-mL medium to raise the pH of the solution to 6.5. The solution is autoclaved and stored at room temperature.

(41) Inhibitor Screening

(42) 1. The day before screening, the control strain containing the empty plasmid and the selected test strain bearing the plasmid with the gene of interest were inoculated into 2 mL of SC selection medium containing 2% glucose. Cells were grown overnight at 30 C. with shaking at 220 rpm. Note: it can be observed that the yeast strain expressing human expression vector and the yeast strain containing the empty vector grow at the same rate in glucose, which represses ArfGAP1 expression. However, in galactose, where ArfGAP1 expression is stimulated, the yeast strain expressing human ArfGAP1 indeed displays reduced growth relative to yeast grown with empty vector (FIG. 2).

(43) 2. The next day, 1 mL of overnight culture was transferred to a microfuge tube and centrifuged at 4700g for 5 min. The supernatant was discarded, and the pellet was washed with sterile water and centrifuged at 4700g for 5 min to eliminate traces of glucose.

(44) 3. The plates containing the small molecules to be screened was removed from the freezer and thawed at room temperature for approximately 30-60 min.

(45) 4. The pellet was suspended in 1-mL sterile water and the A 600 was measured. Cell were diluted to A 600=0.01 in appropriate SC liquid selection medium containing 2% galactose. 10 mL of diluted test cells were prepared for each 96-well plate to be tested. A lower volume of control cells was required.

(46) 5. 100 L of test cells was transferred to all but four wells of the sterile 96-well plates using a dispensing eight-channel pipettor. 100-L medium without yeast was added to two control wells and 100-L medium with control cells was added to two wells.

(47) 6. A control 96-well plate was prepared. To columns 1-4 (32 wells), 100-L control cells diluted to A 600=0.008 was added. To columns 5-8, 100 L test cells diluted to A 600=0.01 was added, and to columns 9-12, 100-L medium without cells was added.

(48) 7. The chemicals (small molecule library) were transferred from the storage plates to plates containing yeast using a hand-held pinning tool or a robotic pinning tool. The pinning tool was cleaned and disinfected by dipping and shaking the pins in 10% bleach for 10 s, followed by dipping and shaking in 70% ethanol for 10 s, followed by air drying or drying over a flame. When the pins were cool, the pinning tool was dipped into the chemical storage plate, the pinning tool was then removed carefully without touching the edges of the well and dipped into the test plates without touching the edges of the wells. The pins were removed in the same manner. The pins were washed and disinfected and the process was repeated until all the chemicals had been transferred to the test plates.

(49) 8. The plates were placed in the humidifier box and incubated at 30 C. for 40-42 h.

(50) 9. Stacks of five plates were shaken on a vortexer at low speed (e.g., setting 4 of a Genie 2 Vortex mixer) for 90 s to resuspend the yeast cells, and the A 600 was measured using a 96-well plate reader.

(51) Growth Restoration Calculation

(52) 1. Control plate: The average A 600 of test wells (columns 1-4), control wells (columns 5-8), and medium-only wells (columns 9-12) was calculated.

(53) 2. The A 600 of treated control and human ArfGAP1 expressing yeast strains was plotted. See FIG. 4. The % growth restoration for each compound tested was calculated using the formula: % Growth restoration=(Test & chemTest)/(ControlTest)100, where Test & chem is the A 600 reading of a well containing the test strain treated with a chemical, Test is the average A 600 of test cells not treated with chemicals determined from the control plate, and Control is the average A 600 of control cells determined from the control plate.

(54) 3. The wells showing highest levels of growth restoration were selected as actives. Active chemicals for secondary assays were selected if they were obvious outliers and/or showed >50% growth restoration.

(55) Active Chemical Confirmation

(56) 1. Active wells were visually inspected in an inverted microscope to ensure that the increased A 600 reading was indeed due to an increased number of yeast cells rather than being due to compound precipitation or contamination by other microorganisms.

(57) 2. To confirm the primary screening results, the activity of each active chemical was retested at various concentrations against both the test and control strains (FIG. 5). The EC 50 for each active compound was established and compounds were selected that combined high potency and low toxicity. Overall, eight compounds were isolated from the screen from among 33,000 small molecules (FIG. 6). Seven of these molecules had a signature phenyl piperazine structure.

(58) 3. Note: The active compounds could also be tested against a test strain for an unrelated gene that also causes growth inhibition when overexpressed in yeast. Chemicals that restore growth by general mechanisms, such as interference with the activity of the GAL1 promoter, should also restore growth inhibited by any gene.