HYPERPHENYLALANINEMIA AND TREATMENTS THEREOF

Abstract

The present invention relates to compounds of formula (I) as defined in the specification for use as pharmacological chaperones having a stabilizing effect on phenylalanine hydroxylase (PAH) for the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU). The compounds include trimethoprim and analogues and derivatives thereof.

Claims

1. A method of treating hyperphenylalaninemia (HPA) in a subject comprising administering thereto an effective amount of a compound of formula (I) ##STR00013## in which X.sub.1 is C or N; R.sub.1, R.sub.2, and R.sub.3, which may be the same or different, are selected from the group consisting of H, NH.sub.2, OH, CN, NO.sub.2, SH, halogen, optionally substituted C.sub.1-C.sub.6 alkyl or C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl wherein the heteroatoms are one or more N, O or S, optionally substituted C.sub.1-C.sub.6 thiol, optionally oxidized to S(O) or S(O).sub.2, optionally substituted C.sub.1-C.sub.6 aminoalkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, (Y).sub.pC0(Z).sub.qR.sub.A and an aromatic group, optionally containing one or more heteroatoms selected from O, N and S, said aromatic group being optionally substituted, wherein Y and Z are independently selected from O and N(RB), p and q are independently 0 or 1, R.sub.A is selected from the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl or C.sub.2-C.sub.6 alkenyl, and optionally substituted C.sub.1-C.sub.6 heteroalkyl wherein the heteroatoms are one or more N, O or S, and RB is selected from H and C.sub.1-C.sub.3 alkyl or cycloalkyl; and R.sub.4 and R.sub.4 are independently selected from H, F, optionally substituted C.sub.1-C.sub.6 alkyl or C.sub.2-C.sub.6 alkenyl, optionally substituted phenyl or a 5- or 6-membered heteroaryl group or are fused to form a 3-6 membered aliphatic cyclic group which may contain one or more heteroatoms selected from N, O and S or together form ?O or ?S; or R.sub.3 and R.sub.4 are fused to form a 5- or 6-membered ring, preferably a 6-membered ring, and thereby a compound of formula IIa ##STR00014## or R.sub.1 and R.sub.4 are fused to form a 5- or 6-membered ring, preferably a 6-membered ring, and thereby a compound of formula IIb ##STR00015## in which X.sub.2 is NR.sub.10, O or S, wherein R.sub.10 is H or C.sub.1-3 alkyl which may be partially or fully fluorinated; and X.sub.3 is a single or double bond, CO, SO.sub.2 or CH.sub.2; R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9, which may be the same or different, are selected from the group consisting of H, OH, SH, halogen, CN, NO.sub.2, NH.sub.2, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.1-C.sub.6 aminoalkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl wherein the hetero atom is N, O or S, optionally substituted C.sub.1-C.sub.6 alkoxy, optionally substituted benzyloxy, optionally substituted C.sub.1-C.sub.6 thiol, optionally oxidized to S(O) or S(O).sub.2, and an aromatic group, optionally containing one or more heteroatoms selected from O, N and S, said aromatic group being optionally substituted, two of R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 may together form an optionally substituted carbocyclic or heterocyclic group; or a pharmaceutically acceptable salt or solvate thereof or a tautomer thereof .

2. The method of claim 1 wherein the substituents of any substituted C.sub.2-6 alkenyl, C.sub.1-C.sub.6 alkyl, aminoalkyl, alkoxy, thiol or heteroalkyl group, which may be the same or different, are selected from the following: halogen, CN, OH, NH.sub.2, NO.sub.2, SH, O, optionally substituted C.sub.1-C.sub.3 alkyl, optionally substituted C.sub.2-C.sub.3-alkenyl, optionally substituted C.sub.1-C.sub.3 alkoxy, optionally substituted C.sub.1-C.sub.3 thiol, optionally substituted C.sub.1-C.sub.3 aminoalkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, wherein heteroatoms are selected from N, O or S and a cyclic group which may be aromatic or aliphatic and may be a 4, 5 or 6 membered ring, optionally containing one or more heteroatoms selected from O, N and S, said cyclic group being optionally substituted.

3. The method of claim 2 wherein the substituents of any substituted C.sub.2-3 alkenyl, C.sub.1-3 alkyl, aminoalkyl, alkoxy, thiol or heteroalkyl group are selected from: halogen, NH.sub.2, OH, SH, O, CN and NO.sub.2

4. The method of claim 1, wherein R.sub.1, R.sub.2 and R.sub.3 are selected from NH.sub.2 and OH, preferably wherein one or more of R.sub.1, R.sub.2 and R.sub.3 is NH.sub.2.

5. The method of claim 1, wherein R.sub.2 is NH.sub.2, and either both of R.sub.1 and R.sub.3 are NH.sub.2, or one of R.sub.1 or R.sub.3 is NH.sub.2 and the other is H; or wherein the compound is a compound of formula (IIa) or (IIb), and R.sub.2 is NH.sub.2 and R.sub.1 or R.sub.3 is OH.

6. (canceled)

7. The method of claim 1, wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are selected from methoxy, SCH.sub.3, OH, Cl, F, or methyl, or two of R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 together form OCH.sub.2O, OCH.sub.2CH.sub.2O or phenyl.

8. The method of claim 7, wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are selected from methoxy and Cl, or two of R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 together form OCH.sub.2O.

9. The method of claim 1 wherein R.sub.4 is H.

10. The method of claim 1 wherein R.sub.4 is H or fused to R.sub.1 or R.sub.3.

11. The method of claim 1 wherein 1, 2 or 3, preferably 2 or 3 of R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are C.sub.1-C.sub.3 alkoxy, most preferably methoxy.

12. The method of claim 1 wherein two of R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 together form a 5- or 6-membered ring, preferably with 1 or 2 heteroatoms, most preferably with 2 oxygen atoms.

13. The method of claim 1 wherein X.sub.3 is C(?O).

14. The method of claim 1, wherein X.sub.1 is N and X.sub.2 is NH.

15. The method of claim 1, wherein the compound is selected from ##STR00016## ##STR00017##

16. The method of claim 1, wherein the hyperphenylalaninemia (HPA) is phenylketonuria (PKU).

17. An in vitro method of stabilising phenylalanine hydroxylase (PAH) which comprises contacting PAH with a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt or solvate thereof.

18. (canceled)

19. A pharmaceutical pack comprising, not in admixture but for simultaneous or sequential administration, a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt or solvate thereof and a further agent effective in the treatment of HPA.

20. A composition comprising a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt or solvate thereof and a further agent effective in the treatment of HPA.

21. A pack as claimed in claim 19, wherein the further agent is BH.sub.4 or an analogue or precursor thereof.

22. (canceled)

23. (canceled)

24. The method of claim 1, wherein the compound has essentially no inhibitory effect on dihydrofolate reductase (DHFR).

25. The method of claim 1, wherein the treated condition is mild HPA, mild PKU or classic PKU in a subject exhibiting ?10% of wild-type PAH activity.

26. A composition as claimed in claim 20, wherein the further agent is BH.sub.4 or an analogue or precursor thereof.

Description

[0077] The present invention is described in the following non-limiting examples and figures:

[0078] FIG. 1. Effect of compounds 1-7, compounds III and IV (from Pey et al., 2008) and of BH.sub.2 on PAH immunoreactive protein level and on PAH activity determined on lysates of HEK293 cells permanently expressing human wild-type PAH. (A) The cells were grown for 24 h in normal medium and then 0.04 mg/ml (average 116.7 ?M) of the separated compounds (with 1% DMSO) was added. The controls contained 1% DMSO. After 8 h further incubation at these conditions the cells were harvested and PAH protein was determined by Western blot and activity measured in clarified lysates. (B) PAH activity when the compounds were added together with the BH.sub.4 precursor BH.sub.2 (200 ?M). BH.sub.2 (200 ?M) alone, at the same conditions, had a stimulating effect of 24.8% on PAH protein and 21.7% on PAH activity.

[0079] FIG. 2. Effect of Compound 53 and BH.sub.2 on the immunoreactive hPAH protein level (upper panels) and PAH activity (lower panels) of HEK293 EBNA cells transiently expressing the indicated PKU mutants. A concentration of 200 ?M for Compound 53 and BH.sub.2 was used and the upper panels show as well the original blotting membranes. (*) Significant differences with respect to the mutant-1% DMSO control, p<0.05.

[0080] FIG. 3. The effect of Compound 53 on the activity of hPAH. Effect of Compound 53 on (A) the Phe dependent specific activity of full-length tetrameric wild-type hPAH, assayed at 100 ?M BH.sub.4, (B) the BH.sub.4-dependent activity of full-length hPAH, assayed with 1 mM L-Phe, and (C) the BH.sub.4-dependent activity of truncated dimeric hPAH ?N102/?C24, assayed with 1 mM L-Phe. (D) A summary of the calculated steady state enzyme-kinetic parameters. Values are expressed as the average of three independent experiments?SD. The concentration of Compound 53 in the assays was 137.8 ?M and 2% DMSO. **Significant Differences with respect to control, p<0.01.

[0081] FIG. 4. Analogues of Compound 53 identified through similarity search of commercially available compounds. 11 selected compound analogues of Compound 53 grouped based on the number of atom substitutions: group 1 analogues (?3 atom substitutions) are shown in row 1; group 2 analogues (>3 atom substitutions) in row 2; group 3 (different scaffold) in row 3.

[0082] FIG. 5. Selected compounds from the ChEMBL database with the same scaffold as Compound 53, but with substitutions notably on the pyrimidine ring, yielding reduced affinity for DHFR.

[0083] FIG. 6. Effect of Compound 53 derivatives on PAH activity in lysates from HEK293 cells permanently expressing human wild-type PAH, cultured in the presence of an average compound concentration of 144.9 ?M, with 1% DMSO. The dashed line represents the normalized activity of the control (1% DMSO), also used as reference for the activity values of the compounds.

[0084] FIG. 7. A selection of further compounds (analogues of Compound 53.10) for use according to the present invention, chaperone activity has been confirmed by DSF testing.

[0085] FIG. 8. A selection of further compounds (analogues of Compound 53.10) for use according to the present invention.

[0086] FIG. 9. A selection of further compounds (analogues of Compound 53) for use according to the present invention.

[0087] FIG. 10. A selection of further compounds (analogues of Compound 53.10) for use according to the present invention.

EXAMPLES

Example 1

Materials And Methods

Materials

Compounds

[0088] Compounds were ordered from TimTec and Sigma Aldrich (purity >90% for the different batches), prepared at concentrations of 4 mg/mL in 100% DMSO. Compounds III and IV from Pey et al., 2008, supra, were obtained from Maybridge (Maybridge Ltd., UK)

Enzymes

[0089] Tetrameric full length wild-type (WT) human PAH (hPAH) and truncated ?N102/?C24-hPAH corresponding to a dimeric form containing the catalytic domain of known crystal structure were recombinantly expressed in E. coli fused to maltose-binding protein and purified by amylose-affinity chromatography and further cleaved and isolated to homogeneity essentially as described [Martinez, A., et al., Biochem. J., 1995. 306: p. 589-597]. Protein concentration was measured in a NanoDrop spectrophotometer (Thermo Scientific) using the absorbance at 280 nm and the theoretical molar extinction coefficient of 49780 M.sup.?1 cm.sup.?1 for WT-PAH and 46675 M.sup.?1 cm.sup.?1 for ?N102/?C24-hPAH.

Assay of PAH Activity

[0090] PAH activity on isolated recombinant enzymes was measured at 37? C. for 1 min with quantification of L-Tyr formed by HPLC with fluorimetric detection, essentially as described [Martinez et al. supra] with the indicated concentrations of the compounds. In order to better eliminate the PAH inhibitors, we further adapted the assay: reaction mixtures containing 0.25 ?g of hPAH, 100 mM NaHepes pH 7.0, 20 mM NaCl, 0.04 mg/ml catalase, 50 ?M ferrous ammonium sulphate, 1 mM L-Phe, 0.05% bovine albumin serum, 2% DMSO and 0.04 mg/ml compound were dispensed in a 96-well PCR plates (Roche) and incubated for 40 min at 42? C. After an equilibration of the plate at 37? C. for 3 min the reaction was initiated by adding 75 ?M BH.sub.4 (Schircks Laboratories, Jona, Switzerland) in 5 mM DTT and stopped after 1 min by adding 50 ?l of 2% (v/v) acetic acid in ethanol (all concentrations referred to a final reaction volume of 50 ?l) prior to determination of L-Tyr produced by HPLC with fluorimetric detection (?.sub.excitation=274 nm; ?.sub.emission=304 nm). Controls with 2% DMSO without compound were routinely assayed as references for data processing. A BRAVO automated liquid handling platform was used in these assays.

[0091] The steady-state kinetic parameters were estimated with 0.25 ?g hPAH (0.965 ?M subunit), 1 mM L-Phe and different BH.sub.4 concentrations in the range 0-400 ?M, and with 100 ?M BH.sub.4 and L-Phe (0-1 mM). The saturation curves were fitted to hyperbolic (for BH.sub.4) or sigmoidal (for L-Phe) kinetic models with SigmaPlot v. 9.0. (SPSS). Kinetic parameters are presented as mean?SEM obtained from nonlinear regression analysis.

[0092] A similar assay was used to measure PAH activity in cells lysates using 1 mM L-Phe and 200 ?M BH.sub.4 with 10 min reactions and about 20-90 ?g of total protein. Free amino acids and contaminants of low molecular weight were previously removed from the extracts using Zebra Desalt Spin columns (Pierce Biotechnology). Under these conditions, PAH activity was linear to the amount of protein in the extracts.

Expression of WT and Mutant PAH in Eukaryotic Cells

[0093] Human embryonic kidney cells (HEK293; Life Technologies?) permanently expressing WT-hPAH, prepared with the Flp-In system (Life Technologies?) were kindly provided by Per Knappskog, Haukeland University Hospital. About 1 million cells were grown in DMEM medium containing 4.5 g/l glucose, 10% (v/v) fetal calf serum, 0.25 mg/ml gentamycine, 2 mM L-glutamine and 50 ?g/ml hygromycin B, and after 24 h medium was changed and the test compounds were added at a concentration of 0.02 mg/ml (1% DMSO). Eight hours later, the cells were harvested, and the pellets frozen in dry ice and stored at ?80? C. Control experiments revealed no significant effects of 1% DMSO either on cell growth and survival or on PAH activity and immunoreactivity (data not shown). To prepare the cell extracts, the frozen cell pellets were thawed in TBS (+protease inhibitor) buffer and cells were homogenized with 0.1% TX-100, incubated 15 minutes at 4? C. and extracts were clarified by centrifugation 20000 g for 15 min at 4? C. Protein concentrations were normalized using a Direct Detect Spectrometer (Merck Millipore).

[0094] The 7 selected compounds as well as the first generation pharmacological chaperones (Compounds III and IV) were further analyzed in the presence and absence of BH.sub.2 (200 ?M), added as precursor of BH.sub.4, to account for additive effects. A higher concentration of compound was also used 0.04 mg/ml (average of 116.7 ?M) at otherwise same experimental conditions.

[0095] For transient expression of WT and mutant PAH, HEK293 EBNA (Epstein-Barr virus) cells, with low endogenous PAH expression, were grown as above. Cells were grown for 19 h, when they were transfected transiently transfected with 1 pg of pcDNA3-PAH vector (WT or mutant PAH constructs R261Q, R252W, 165T and R68S) using the Lipofectamine system (Life Technologies?) as described by the manufacturer. Compound 53, with and without BH.sub.2 (200 ?M) was added after 5 h of transfection at 200 ?M concentration in 1% DMSO, and further incubated for 24 h, at which time the cells were harvested and stored at ?80? C. In all cases a parallel negative control with only 1% DMSO was included. Cell lysates were prepared as indicated above.

Western Blot Analyses

[0096] Western blot analyses were performed on either cell extracts from permanently expressing or transiently transfected HEK293 cell lysates after SDS-PAGE (10% acrylamide) with 5-10 ?g total protein in each lane. Proteins were transferred into a PVDF membrane and subsequently blocked and incubated with a polyclonal mouse anti-human PAH (PH8; Merck Millipore) at 1 ?g/ml, neomycin phosphotransferase II (Merck Millipore #06-747) and GAPDH (Abcam, #ab9485) as primary antibodies. Neomycin phosphotransferase II was used as a marker of the plasmid transfection efficiency, and GAPDH was used as loading controls. Goat anti-mouse and goat anti-rabbit HRP conjugate (Bio-Rad) were used as secondary antibodies. Lastly, membranes were developed by chemiluminescence (ECL; Amersham), and immunoquantification in a Fluor-S Multilmager (Bio-Rad) using ImageLab v.5.1 software.

Derivatization/Selection of Related Structures to Compound 53

[0097] Similarity search for Compound 53 analogues was performed using the R-package Chemlnf and OpenBabel [O'Boyle, N. M., et al., J Cheminform, 2011. 3: p. 33] over a selection of small molecule libraries of purchasable compounds obtained from the chemical vendors Vitas-Lab, Sigma Aldrich, Otava and MolPort. All compounds showing a Tanimoto coefficient of >0.5. towards compound 53 were selected for manual inspections.

Differential Scanning Fluorimetry (DSF)

[0098] DSF was used to monitor the thermal denaturation of recombinant WT-hPAH protein in the presence of the fluorescent dye SYPRO Orange (Sigma-Aldrich). The experiments were carried out in a LightCycler 480 Real-Time PCR (RT-PCR) instrument (Roche Applied Science, Indianapolis, Ind.). In each well 49 ?l of a hPAH protein solution containing 0.1 mg/ml (1.93 ?M subunit) in 20 mM NaHepes pH 7.0, 200 mM NaCl and 5?SYPRO Orange, were dispensed with the help of a multichannel pipette. Next, in a BRAVO automated liquid handling platform (Agilent Technologies, Santa Clara, Calif.) 1 ?l of each test compound was added to a final volume of 50 ?l/well, with final concentrations of 4% DMSO and 0.04 mg/ml compound. Plates were incubated at room temperature (RT) for 30 min before loading them into the LightCycler and starting the data acquisition. The thermal denaturation was monitored by following the expected increase in fluorescence intensity of the extrinsic probe SYPRO Orange (instrument filter settings: ?.sub.exc=465 nm; ?.sub.em=610 nm) as a consequence of the unfolding/denaturation of the protein. Melting curves were registered from 20 to 95? C. at a scan rate of 2.4 ? C./min and the experimental data obtained allowed the extraction of values of T.sub.m (midpoint melting temperature) by fitting, smoothing, normalization and analysis of the aforementioned unfolding curves using in-house software. T.sub.m represents the temperature at which the fraction of unfolded (or folded) protein is 50% and it is calculated as the intersection between denaturation curve and fraction of unfolding .sub.XU=0.5. Control experiments with 4% DMSO were performed in the same way.

[0099] The T.sub.m-values for hPAH in the presence of each compound was compared to the value for the control without compound but with 4% DMSO, and the shifts in Tm (?T.sub.m) were calculated. ?T.sub.m (=T.sub.m Compound?T.sub.m DMSO control).

Results

Expression Studies in HEK293 Cells Permanently Expressing hPAH

[0100] We analyzed the effect of 20 compounds on the cell growth of HEK293 expressing hPAH at 0.02 mg/ml, as well as on the immunoquantified levels of hPAH protein and activity. Many of the compounds provoked the detachment of the cells and we thus only selected 7 evidently non-toxic compounds with the best profiles to be further analyzed at a higher concentration, 0.04 mg/ml. In addition, we also examined if the compounds provided additive or synergetic effects with BH.sub.4. We used BH.sub.2 as precursor of BH.sub.4 since the addition of BH.sub.4 directly to the cellular medium is toxic, while BH.sub.2 is taken up and intracellularly converted to BH.sub.4.

[0101] The compounds were analyzed at an average concentration of 116.7 ?M and BH.sub.2 at 200 ?M. Control experiments with 1% DMSO were also performed. As observed in FIG. 1A, one of the compounds, Compound 53, had a very large stimulating effect on PAH protein and activity, especially the latter. This effect was larger than that of compounds III and IV, first generation pharmacological chaperones from Pey et al., 2008 (supra). Furthermore, the effect of compound B with BH.sub.2 was additive (FIG. 1B). Finally, this compound at a concentration of 68.9 ?M did not have large effect on TH and TPH2 activity respect to 2% DMSO controls.

[0102] From this point we decided to focus on Compound 53 as the best hit for further characterizations and hit optimization.

Expression Studies in HEK293 Cells Transiently Expressing PKU Mutants

[0103] We studied the transient expression of the PKU mutants I65T-, R68S-, R252W- and R261Q-PAH in cells for 24 hours in the absence or presence of 200 ?M of compound 53, without and with 200 ?M BH.sub.2. These 4 PKU mutations represent 4 different phenotypic groups in PKU patients, and allele recurrence: R68S is a mild mutation (frequency>2% of the alleles), 165T is mild-moderate (frequency=3.9% of the alleles), R261Q is highly variable (mild-moderate-severe) and very frequent (9.2% of alleles), and R252W is a severe mutation (frequency=2.5% of the alleles). Cells were harvested, and steady-state PAH immunoreactive protein at 51 kDa, corrected for loading (GAPDH; 38 kDa) and expression controls (Neo;30 kDa) and PAH activity were measured in soluble cell extracts (FIG. 2).

[0104] Except for R252W-PAH, which showed an activity below the assay detection limit, the DMSO-controls of the PKU mutants presented measureable immunoreactive levels (25-70% of WT) and activities (25-50% of WT). In the presence of compound 53 both hPAH protein levels and PAH activity of cells expressing mutants I65T and R261Q were greatly increased. BH.sub.2 also increased protein and activity of the same cells, and trends for additive effects for Compound 53 and BH.sub.2 were also observed. An effect of Compound 53 on the activity of cells expressing R68S was also measured, but neither the compound or BH.sub.4 affected the severe mutant R252W, although we observed a little stimulation of the protein content when cells expressing this mutant were grown with both compound and cofactor analogue together (FIG. 2A).

Effect of Compound 53 on the hPAH Activity

[0105] Once we demonstrated the good effect of Compound 53 in stimulating PAH protein and activity in cells expressing hPAH (both WT and PKU mutants) we studied in more detail the effect of the compound on the steady state enzyme kinetics (FIG. 3). For the DMSO control enzyme S.sub.0.5(L-Phe) and K.sub.m(BH.sub.4) values are in accordance with those obtained in previous studies for the full length enzyme, although the activity values and V.sub.max are higher. As we can observe in the phenylalanine titration graph (FIG. 3A,D), the compound does not affect the kinetic parameters for the substrate (V.sub.max and S.sub.0.5), or the characteristic positive cooperativity for L-Phe also remains unmodified. On the other hand, we observed a small inhibitory effect of the compound in the BH.sub.4-concentracion curve, resulting in decreased V.sub.max and K.sub.m for the cofactor (FIG. 3B,D), indicative of an uncompetitive inhibition. The results suggest that the compound does not affect the binding of L-Phe or the activating regulatory conformational change caused by the substrate. The uncompetitive mechanism towards BH.sub.4 indicates that compound 53 binds tighter to the transition state than to the free enzyme and the sequestering of the enzyme-BH.sub.4 complex results in apparent decrease of K.sub.m by the Chatelier principle.

[0106] When the effect of the compound is tested with a truncated form of the enzyme, i.e. the dimeric hPAH ?N102/?C24, lacking both the regulatory domain and the tetramerization motif, compound 53 did not change the V.sub.max or K.sub.m-values for BH.sub.4 (FIG. 3C,D), indicating no effect on the binding of BH.sub.4 in this enzyme form.

Derivatization of Compound 53

[0107] Compound 53 is a known antibacterial agent, trimethoprim (TMP). It binds to bacterial variants of dihydrofolate reductase (DHFR) with nM affinity and is thus a potent inhibitor for this enzyme. To avoid inhibition of DHFR using Compound 53 for PKU treatment we set out to identify compound analogues with reduced affinity for DHFR while retaining PAH efficacy. As a first step we aimed at characterizing the chemical features of Compound 53 imperative for the stabilization of PAH. An initial search of commercially available compounds was carried out as described above. All compounds showing a Tanimoto coefficient >0.5 towards Compound 53 were selected for manual inspections. A final selection of 11 compounds (with identifiers c53.1-c53.11) was made with the objective of determining the efficacy of individual atoms in the structure as well as determining the efficacy of both ring structures of Compound 53 to stabilize PAH (FIG. 4). Of the 11 selected compounds, 3 compounds carried the same chemical scaffold with only minor changes to the structure (?3 atom substitutions; group 1); 4 compounds retained the chemical scaffold, but included a larger number of atom substitutions (>3 atom substitutions; group 2); and another 4 compounds obtained a different chemical scaffold but keeping certain chemical features (group 3).

[0108] After initial testing of these first 11 analogues we identified additionally 13 compounds of a particularly potent analogue (c53.10). These compounds share the same scaffold as Compound 53, but contain a fused ring system on the pyrimidine ring (see FIGS. 7 and 8).

[0109] The ChEMBL database contains bioactivity data for large numbers of small molecules. We utilized this data set to identify the chemical features imperative for DHFR inhibition for compounds with the same scaffold as Compound 53. We found that all compounds similar to Compound 53 with an unmodified pyrimidine ring showed strong inhibition of bacterial DHFR. This is supported by the X-ray structure of DHFR in complex with Compound 53 (PDB ID 1DG5) where Asp27 forms a double H-bond with the pyrimidine ring of Compound 53. Consequently, compounds with substitutions of specific H-bond donor atoms of the pyrimidine ring show weaker affinity towards DHFR (see FIG. 5 for selected compounds). We identified 5 compounds with the same chemical scaffold as Compound 53 with substitutions on the pyrimidine ring affecting the affinity towards DHFR. Notably, Compound 53 with one nitrogen of the pyrimidine substituted to a carbon atom (CHEMBL 128600; FIG. 5) shows an IC50 value of 10 mM (the absent methyl group elsewhere in the compound is not believed to be material to binding affinity).

Example 2

Screening and Affinity Determination by SPR

[0110] For detailed determination of binding affinity of Compound 53 and derivatives (FIG. 4) SPR was performed on a Biacore T200 (GE Healthcare Life Sciences) at a temperature of 25? C. A hPAH solution at 0.08 ?g/?l in a final volume of 200 ?l in acetate buffer pH 5.0 was immobilized onto series S sensor chip CM5 by the standard amine coupling procedure at a flow rate of 30 ?l/min and PBS-P+ buffer as running buffer aiming for RU 20000. After the coupling reaction the surface of the sensor chip was washed to eliminate unbound species with HBS buffer (0.01 M NaHepes pH 7.4, 0.15 M NaCI) at steady flow of 20 ?l/min for 1 hour until the baseline drift was around 0.05 RU/s; note that an early injection of 40 ?l dithiothreitol (DTT) 10 mM in HBS allowed for this reduced washing time.

[0111] For the screening, compounds were assayed at a concentration of 200 ?M, with 5% DMSO in duplicates, including negative controls with 5% DMSO, positive controls, with 200 ?M BH.sub.2 and solvent correction was also performed. For K.sub.D calculations, a compound concentration 0-500 ?M (in 5% DMSO) were applied at otherwise analogous experimental design and analyses were performed using Biacore T200 Evaluation Software, version 2.0 (GE Healthcare Life Sciences).

Results

[0112] All the responses from the compounds of the screening were subjected to blank subtraction (negative control 5% DMSO), molecular weight adjustment and solvent correction to account for differences in bulk response. Next a cut-off value for the screening was selected based on the response of the positive control of our experiments: RU BH.sub.2+3*SD (2.88+3*1.79=8.27). In this manner, from the 11 derivatives of Compound 53 tested (see FIG. 4) we weeded out those compounds with a lower response than 8.27, namely #53.2, #53.9 and #53.11.

[0113] In addition and for the rest of the compounds which continued our workflow, binding affinities were determined and the results are shown in the following table. The selected conditions to measure affinity are selected for high throughput measurements, but the affinities are overestimated though the relative K.sub.D for the different compounds are reliable.

TABLE-US-00001 TABLE 1 Compound ID. K.sub.D (?M) ? SER c53-TMP 58.5 ? 35.8 c53.1 104 ? 14 c53.3 860 ? 1377 c53.4 45.2 ? 9.1 c53.5 370 ? 228 c53.6 103 ? 48.8 c53.7 6.9 ? 4.9 c53.8 c53.10 22.9 ? 2.7

[0114] Interestingly, as highlighted in bold in the table, we were able by our similarity search to find three derivatives that presented a lower K.sub.D value than our initial hit Compound 53 (c53). In other words, c53.4, c53.7 and c53.10, according to these results, would bind with higher affinity towards hPAH with possibly enhanced potency as pharmacological chaperones.

[0115] Due to the high potency and alternative structure of c53.10, we identified and bought additionally 13 c53.10 analogues. 7 of these (FIG. 7) have been tested using DSF and all show a ?T.sub.m value of ?5? C.

[0116] Binding affinities were determined for some further compounds of the invention (derivatives of compounds 53 and 53.10; as shown in FIGS. 7, 9 and 10). The results are shown in Table 2. The methodology used was the same as discussed above.

TABLE-US-00002 TABLE 2 Compound ID. K.sub.D (?M) ? SER c53.14 24.7 ? 12.1 c53.16 76.3 ? 26.4 c53.17 11.0 ? 3.7 c53.19 37.2 ? 23.7 c53.20 23.2 ? 11.7 c53.22 30.1 ? 6.5 c53.26 62.1 ? 30.7 c53.29 191 ? 20 c53.31 55.9 ? 6.7 c53.32 102 ? 11 c53.33 182 ? 32 c53.35 23.7 ? 5.0 c53.40 11.6 ? 1.6 c53.41 12.6 ? 6.4

Example 3

Expression Studies in HEK293 Cells Permanently Expressing hPAH

[0117] Human embryonic kidney cells (HEK293; Life Technologies?) permanently expressing WT-hPAH, prepared with the Flp-In system (Life Technologies?) were kindly provided by Per Knappskog, Haukeland University Hospital. About 1 million cells were grown in DMEM medium containing 4.5 g/l glucose, 10% (v/v) fetal calf serum, 0.25 mg/ml gentamycine, 2 mM L-glutamine and 50 ?g/ml hygromycin B, and after 24 h medium was changed and the compounds were added at a concentration of 0.04 mg/ml (1% DMSO). Eight hours later, the cells were harvested, and the pellets frozen in dry ice and stored at ?80? C. Control experiments revealed no significant effects of 1% DMSO either on cell growth and survival or on PAH activity and immunoreactivity (data not shown). To prepare the cell extracts, the frozen cell pellets were thawed in TBS (+protease inhibitor) buffer and cells were homogenized with 0.1% TX-100, incubated 15 minutes at 4? C. and extracts were clarified by centrifugation 20000 g for 15 minutes at 4? C. Protein concentrations were normalized using a Direct Detect Spectrometer (Merck Millipore).

Results

[0118] Five Compound 53 derivatives were further analyzed in HEK293 cell cultures to observe how the activity of hPAH was altered by the presence of these compounds (FIG. 6).

[0119] Even though all the derivatives studied increased hPAH with respect to the control, it was c53.4 and c53.10 that reached levels comparable to c53 (around 40% increase).

Example 4

[0120] Further tests on derivatives of Compounds 53 and 53.10 to assess enzyme activity were carried out in the same way as set out in Example 3, with the results set out below in Table 3. Some natural variance is seen between the results in FIG. 6 and Table 3 for those compounds which were tested in both experiments.

TABLE-US-00003 TABLE 3 Compound ID PAH activity-cells (%) c53 148 ? 16 c53.1 130 ? 8 c53.3 112 ? 10 c53.4 165 ? 28 c53.6 120 ? 12 c53.7 119 ? 5 c53.10 155 ? 13 c53.14 175 ? 23 c53.16 146 ? 10 c53.17 206 ? 10 c53.19 170 ? 17 c53.20 206 ? 5 c53.22 154 ? 16 c53.26 109 ? 5 c53.29 155 ? 7 c53.31 125 ? 8