Treatment or prophylaxis of diseases caused by pilus-forming bacteria

A method for identifying a potentially therapeutically useful substance capable of interacting with a periplasmic molecular chaperone, thereby preventing, inhibiting or enhancing the interaction between a periplasmic molecular chaperone and a pilus subunit, the method comprising at least one of the following steps:

• 1) testing a candidate substance in an assay in which the possible prevention, inhibition or enhancement by the substance of the interaction between the periplasmic molecular chaperone and the pilus subunit is determined by
• adding the substance to a system comprising the periplasmic molecular chaperone or an analogue thereof in an immobilized form and the pilus subunit or an equivalent thereof in a solubilized form and determining the change in binding between the pilus subunit or equivalent thereof and the periplasmic molecular chaperone or analogue thereof caused by the addition of the substance, or
• adding the substance to a system comprising the pilus subunit or an equivalent thereof in an immobilized form and the periplasmic molecular chaperone or an analogue thereof in a solubilized form and determining the change in binding between the pilus subunit or equivalent thereof and the periplasmic molecular chaperone or analogue thereof caused by the addition of the substance, or
• adding the substance to a system comprising the pilus subunit or an equivalent thereof as well as the periplasmic molecular chaperone or an analogue thereof in solubilized form and determining the change in binding between the pilus subunit or equivalent thereof and the periplasmic molecular chaperone or analogue thereof caused by the addition of the substance, or
• adding the substance to a system comprising the pilus subunit or an equivalent thereof as well as the periplasmic molecular chaperone or an analogue thereof in solubilized form and measuring the change in binding energy caused by the addition of the substance, and identifying the substance as potentially therapeutically useful if a significant change in the binding energy between the pilus subunit or equivalent thereof and the periplasmic molecular chaperone or analogue thereof is observed,
• and identifying the substance as potentially therapeutically useful if a significant change in the binding or binding energy between the pilus subunit or equivalent thereof and the periplasmic molecular chaperone or analogue thereof is observed;
• 2) testing a candidate substance in an assay in which the possible prevention, inhibition or enhancement of the interaction between the periplasmic molecular chaperone and the pilus subunit is determined by
• adding the substance to a system comprising living tissue-adhering pilus-forming bacteria followed by determination of the growth rate of the bacteria, a reduction in growth rate compared to a corresponding system wherein the substance has not been added being indicative of prevention, inhibition or enhancement of the binding between the periplasmic molecular chaperone and the pilus subunit, or
• adding the substance to a system comprising living tissue-adhering pilus-forming bacteria followed by a determination of the tissue adhesion of the bacteria, a reduction in tissue adhesion compared to a corresponding system wherein the substance has not been added being indicative of prevention, inhibition or enhancement of the binding between the periplasmic molecular chaperone and the pilus subunit,
• and identifying the substance as potentially therapeutically useful if a reduction in growth rate or tissue adhesion is observed after the addition of the substance;
• and
• 3) administering, to an experimental animal, a substance which has been established in vitro to prevent, inhibit or enhance the interaction between a periplasmic molecular chaperone and a pilus subunit, the experimental animal being inoculated with tissue-adhering pilus-forming bacteria before, simultaneously with or after the administration of the substance, and electing as a substance suitably capable of interacting with a periplasmic molecular chaperone, a substance preventing and/or curing and/or alleviate disease caused by the bacteria.

A method for identifying and/or designing a substance, X, capable of interacting with a periplasmic molecular chaperone, e.g. binding to the periplasmic molecular chaperone, with a predicted binding free energy equal to or better than a predetermined threshold value, the method comprising

• 1) selecting a substance, A, which could potentially interact with a site in the periplasmic molecular chaperone, and providing a 3-dimensional structural representation thereof,
• 2) predicting the binding free energy between the substance A and the site in the periplasmic molecular chaperone or a site analogous to such a site,
• 3) if the predicted binding free energy between the substance A and the site in the periplasmic molecular chaperone or the analogous site is equal to or better than the predetermined threshold value, then identifying the substance A as the substance X,
• 4) if the predicted binding free energy between the substance A and the site in the periplasmic molecular chaperone or the analogous site is not equal to or better than a predetermined threshold value, then modifying the 3-dimensional structural representation and predicting the binding free energy between the thus modified substance, B, and the site in the periplasmic molecular chaperone or the analogous site, and
• 5) repeating step 4 until the predicted binding free energy determined between the resulting substance, X, and the site in the periplasmic molecular chaperone or the analogous site is equal to or better than the predetermined threshold value.

A novel compound of the general formula:

• 31
• wherein
• V1 is O, S, SO, SO2, CH2, C(OH)H, CO or CS;
• W1 is O, S, SO2, SO3, CH2 or NH;
• R1 is H;
• C1-24 alkyl, C1-24 alkenyl or C1-24 alkynyl, which alkyl, alkenyl and alkynyl may be substituted with one or more substituents independently selected from OH, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO2NH2 and —SO2NH2;
• acyl;
• or —(CH2CH2O)s—H, wherein s=1,2,3;
• R2 is a group of the formula
• 32
• wherein
• A is —CH—(CH2)n—, or —CH═CH—(CH2)n−1— (n>0) or
• 33
• B is —(CH2)m— or ═CH—(CH2)m−1— (m>0);
• X2 is N, CH or C (when B is ═CH—(CH2)m−1—;
• and
• Y2 is O, S, NH, H2 or H (n=1);
• and
• 4>m+n>0, n<3, and m<3;
• or R2 is a group of the formula
• 34
• wherein
• A is —CH—(CH2)n—, or —CH═CH—(CH2)n−1— (n>0) or
• 35
• B is —(CH2)m— or ═CH—(CH2)m−1— (m>0) ;
• and
• X′2 is O, NH, CH2 or S (when p=0);
• N or CH (p=1);
• or C (when p=1 and B is ═CH—(CH2)m−1—);
• V2, Z2 and W2 are independently H, OH, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO2NH2, —SO2NH2, or V2 and Z2, or Z2 and W2 together form —NHC(O)NH—, —C(O)NHC(O)—, —NHS(O2)NH—, —C(O)NHO—, —C(S)NHO—, —S(O2)NHO—, or —S(O2)NHC(O)—;
• 4>m+n>0, n<3, and m<3;
• or R2 is a group —W5—(C1-5 alkyl or C2-5 alkenyl or C2-5 alkynyl) wherein W5 is a bond or is selected from —O—, —S—, —SO2—, and —NHC(O)—, and the C1-5 alkyl, C2-5 alkenyl or C2-5 alkynyl moiety may be substituted with up to three groups selected independently from OH, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO2NH2 and —SO2NH2;
• —Z1—R3 is —SO2(OH), —PO(OH)2, —OSO2(OH), —NHSO2(OH), —SPO(OH)2, —CH2COOH, tetrazol-5-yl or tetrazol-5-ylmethyl, or salts thereof;
• or Z1 is —O—, —S—, —NH—, or —CH2—, and R3 is a group of the formula:
• 36
• D is —CH2—, —CO—, —SO2—, —NH—SO2—, —NH—CO—, —O—PO(OH)— or a salt thereof;
• Z3 is H, OH, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO2NH2, —SO2NH2, —SO2(OH), —PO(OH)2, —OSO2(OH), —NHSO2(OH), —COOH, tetrazolyl-5-yl or tetrazolyl-5-ylmethyl or a salt thereof,
• with the proviso that when D is —CH2—, —CO—, —SO2—, —NHSO2— or —NHCO—, then Z3 is —SO2(OH), —PO(OH)2, —OSO2(OH), —NHSO2(OH), —COOH, tetrazolyl-5-yl or tetrazolyl-5-ylmethyl or a salt thereof;
• X3 and Y3 independently are H, NO2, SO2NH2, CONH2, CF3 or F;
• and
• U4-W4 is —CHCH—, —CH2CH2—, —C(OH)CH—, —CHC(OH)—, —CH(OH)CH2—, —CH2CH(OH)—, —CH(OH)CH(OH)—, —C(O)NH—, —NHC(O)—;
• Y1 is —O— or —S—;
• R4 is H or, when Y1 is S, S(CH2)qN(R9)3+ and q is an integer 2-4, where R9 is H or CH3;
• R5 is H;
• C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl, and the C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl moiety may be substituted with OH, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO2NH2 or —SO2NH2;
• or aryl, aryl(C1-2)alkyl, heterocyclyl, or heterocyclyl(C1-2)alkyl which may optionally be substituted in the aryl or heterocyclyl moieties with one, two or three substituents selected independently from OH, F, Cl, NH2, CONH2, NHCOH, and SO2NH2;
• X1 is —O—, —S— or —NH—;
• R6 is H or, when X1 is NH, acyl, HOCNH-Val-Met-, HOCNH-Ile-(S,S)-dioxo-methionyl- or HOCNH-Val-(pyran-4-on-2-yl)-alanyl-;
• or a salt thereof.

A novel compound of the general formula

• 37
• wherein
• X is O, P, P(O), S, SO, SO2, CH2, C(OH)H, or a group NQ11, wherein Q11 is H, OH, C1-24 acyl or C1-24 alkyl;
• Z11 is a bond, O, CH2, S, SO, SO2, or a group NQ12, wherein Q12 is H, C1-24 acyl or C1-24 alkyl;
• R11 is H;
• C1-24 alkyl, C1-24 alkenyl, or C1-24 alkynyl, which may be substituted with one ore more substituents independently selected from —OH, —COOH, —F, —Cl, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO3NH2 and —SO2NH2;
• acyl;
• or —(CH2CH2O)s—H, wherein s=1, 2, or 3;
• or R11 is CH═CH—(CH2)n′—Q13 or —(CH2)n′—Q13, wherein Q13 is an aryl or a heteroaryl group substituted with OH, COOH, F, Cl, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO3NH2 and —SO2NH2, and wherein n′≧0;
• R12 and R13 are independently OH, H, F, Cl, OW11, or O(CO)W11, wherein W11 is C1-24 alkyl, C1-24 alkenyl or C1-24 alkynyl, or an aryl or a heteroaryl group substituted with —OH, —COOH, —F, —Cl, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO3NH2 and —SO2NH2;
• Z12 is a bond, O, S, or CH2;
• R14 is —(CH2)n″—Q14, wherein Q14 is an aryl group or a heteroaryl group substituted with —OH, —COOH, —F, —Cl, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO3NH2 and —SO2NH2, and wherein n″=0, 1, 2, or 3;
• Z13 is a bond, O, CH2, S, SO, SO2, NQ14Q15, wherein Q14 is H, C1-24 acyl or C1-24 alkyl, and Q15 is CO or —C(O)W12, wherein W12 is O or NW13, wherein W13 is H, OH, C1-24 acyl or C1-24 alkyl;
• R15 is H;
• C1-24 alkyl, C1-24 alkenyl or C1-24 alkynyl, which alkyl, alkenyl and alkynyl may be substituted with one or more substituents independently selected from, —OH, —COOH, —F, —Cl, —CONH2, —CSNH2, —CONHOH, —CSNHOH, —NHCHO, —NHCONH2, —NHCSNH2, —NHSO3NH2 and —SO2NH2;
• acyl or —(CH2C)H2Os—H, wherein s=1, 2, or 3;
• or R15 is CH═CH—(CH2)n′—Q13, or —(CH2)n′—Q13, wherein Q3 is as defined above and wherein n′≧0;
• or a salt thereof.

A novel pyranoside or a salt thereof, which is selected from the group consisting of

• Ethyl 2,3-O-Dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside;
• Ethyl 6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside;
• Methylglycolyl 6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-β-D-glucohexopyranoside;
• 2-(Hydroxy)ethyl 4-O-benzyl-β-D-glucopyranoside;
• Sodium glycolyl 4-O-benzyl-β-D-glucohexopyranoside;
• Methyl 2-O-ethyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside;
• Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside;
• Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside;
• methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-6-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside;
• Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-6(S)-phenyl-α-D-mannohexopyranoside;
• Methyl 2,3-anhydro-4,6-O-p-methoxybenzylidene-α-D-mannopyranoside;
• Methyl 3-azido-4,6-O-p-methoxybenzylidene-α-D-altropyranoside;
• Methyl 3-azido-2-O-ethyl-4,6-O-p-methoxybenzylidene-α-D-altropyranoside;
• Methyl 3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altro-pyranosid;
• Methyl 3-azido-6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside;
• Methyl 6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranoside sodium salt;
• Methyl 6-O-benzoyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranoside ammonium salt;
• Methyl 3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside;
• Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranoside sodium salt;
• Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altro-pyranoside ammonium salt;
• Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-tbutyloxamido-α-D-altropyranoside;
• Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-oxamido-α-D-altropyranoside ammonium salt;
• Methyl 3-azido-6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside;
• and
• Methyl 6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranoside ammonium salt.

A method for identifying a binding site in a periplasmic molecular chaperone or an analogue thereof, comprising

• co-crystallizing the periplasmic molecular chaperone or an analogue thereof with a ligand binding to the periplasmic molecular chaperone or the analogue thereof,
• resolving the three-dimensional structure of the chaperone/ligand interaction, thereby resolving the three-dimensional structure of the periplasmic molecular chaperone or the analogue thereof when binding to the ligand,
• determining the site-point(s) involved in the intermolecular interaction between the periplasmic molecular chaperone or the analogue thereof and the ligand, and
• identifying the thus determined site-point(s) of the periplasmic molecular chaperone or the analogue thereof as a binding site in the periplasmic molecular chaperone or the analogue thereof.