# Patent

## Title
Compounds binding to the bacterial beta ring

## Abstract

The present invention relates to compounds which bind to the hydrophobic pocket of the β clamp, i.e., to the surface of the β ring with which said protein interacts with other proteins of the bacterial replication complex during DNA replication. These compounds are derived from the acetylated peptide AcQLDLF (P6) to improve their affinity to their target.

## Background
SEQUENCE LISTING SUBMISSION VIA EFS-WEB
A computer readable text file, entitled “045636-5242-SubstituteSequenceListing.txt” created on or about 14 Apr. 2015, with a file size of about 13 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
The present invention relates to bacterial replication. More precisely, the present invention concerns compounds which bind to the hydrophobic pocket of the β clamp, i.e., to the surface of the β ring with which said protein interacts with other proteins involved in DNA metabolism.
In all three domains of life, multicomponents complexes, the so-called replisomes, have evolved to ensure the faithful replication of chromosomal DNA. One central protein of these complexes forms a ring that encircles and slides along the double stranded DNA1, 2. A physical interaction between the clamp and the chromosomal replicase confers a high processivity to the enzyme3. In bacteria, the processivity factor, also referred to as the β ring, is a homodimer which results from the head-to-tail association of two monomers, each of them being shaped in three globular sub-domains1. In eukaryotes and archae, the β homolog factor, PCNA (for Proliferating Cell Nuclear Antigen), is a homotrimer with each monomer organized in two sub-domains2, 4.
Beside their role as processivity factors for chromosomal replicases, β and PCNA clamps also participate in various protein-protein interactions. They notably act as landing platforms for factors involved in DNA metabolism and cell cycle regulation5, particularly DNA polymerases involved in translesion synthesis6, 7, and factors promoting DNA repair8, 9, 10. All these factors possess a small conserved peptide sequence, which binds into a hydrophobic pocket located on one side of the ring. Noteworthy, these pockets differ significantly between bacterial rings and PCNA. A bioinformatics analysis performed on putative β ring partners led to define the bacterial consensus binding peptide QL[S/D]LF10. The absolute requirement of the interacting peptide for β ring partners binding has been further demonstrated biochemically and physiologically11, 12, 13, 14. Finally, the interaction between the ring and the interacting peptide of different β binding proteins have been structurally characterized15, 16, 17, 18 The peptide binding site is formed by a deep leucine-rich hydrophobic pocket (subsite 1) located between sub-domains two and three of the β monomer and connected via a groove to a second sub-site (subsite 2) located in sub-domain three17 (FIG. 1C). An additional interaction has also been observed in the case of the polymerase Pol IV, between the little finger domain of the enzyme and the edge of the β ring16.
The major contribution of the peptide-mediated interaction to a successful DNA replication and ultimately to cell survival, both in prokaryotes and eukaryotes, makes the ring interacting pockets potential targets for the development of new antibacterial or anticancer drugs, respectively. In a recent report, a chemical compound was identified from a library and shown to bind into the leucine rich sub-domain of the E. coli β ring interacting pocket with an affinity of 10−5 M18.
In the experimental work described below, a different, structure-based strategy was used to design short peptides with improved affinities for the β interacting pocket. The first step of this approach was to decipher the molecular basis of the interaction of the natural ligand in the binding pocket. Then, using these data, a first peptide (SEQ ID No: 6, P6) was designed, which was then further modified to improve its affinity. Several biophysical and biochemical methods were used to measure the strength of the interaction and to characterize the structure of the most efficient complexes formed. As a result, the binding efficiency of the modified ligand was improved by two orders of magnitude, reaching 10−8 M range.
Due to their very good affinity for the β interacting pocket, the compounds described in the present text are very promising leads for new antibiotic compounds.
According to a first aspect, the present invention pertains to a compound of formula (I)




wherein

    
    
        Gln is glutamine;
        R is selected in the group consisting of a C1-12-alkyl group optionally substituted by a C6-10-aryl group, a C2-12-alkenyl group optionally substituted by a C6-10-aryl group, a C3-6-cycloalkyl group, a C6-10-aryl group optionally substituted by a C1-4-alkyl, and a C1-5-alkyl-(O—CH2—CH2)t— group with t being an integer from 0 to 20 inclusive;
        R1 is the side chain of arginine or lysine (n.b.: when n>1, each R1 is, independently from each other, the side chain of arginine or lysine);
        R2 is a —(CH2)—C3-6-cycloalkyl group optionally substituted by a halogen and/or by a group selected amongst —NH2, —NH—CO—Ra, —CO2H, —NHRa and —NRaRb, wherein Ra and Rb are independently a C1-4-alkyl group;
        R3 is selected in the group consisting of a C1-8-alkyl group, the side chain of arginine or lysine, —(CH2)q—CO2R7a, —(CH2)q—CO—NHR7b, —CH2OR8 and — (CH2)qNHR9, wherein
        
            q is 1, 2, 3 or 4,
            R7a is a hydrogen atom, a C1-8-alkyl group, a C4-12-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-12-alkenylene, forming together with R6 a lactone or a polyether ring,
            R7b is a hydrogen atom, a C1-8-alkyl group, or —(CH2)q′—NH— with q′ being an integer between 2 and 8 inclusive and forming together with R6 a lactam,
            R8 is a hydrogen atom, a C1-8-alkyl group, a C4-12-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-12-alkenylene, forming together with R6 a lactone or a polyether ring,
            R9 is a hydrogen atom, or R9 together with R6 form a lactam;
        
        
        R4 is a C1-8-alkyl group optionally substituted by a C3-6-cycloalkyl group, or a halogen-C1-4-alkyl group;
        R5 is selected in the group consisting of a —(CH2)—C3-6-cycloalkyl group; —(CH2—CH2)—C3-6-cycloalkyl group; a —(CH2)—C6-10-aryl group optionally substituted by a halogen, a C1-2 alkyl group and/or a C1-2 alkoxy group; a —(CH2—CH2)—C6-10-aryl group optionally substituted by a halogen, a C1-2 alkyl group and/or a C1-2 alkoxy group; a —(CH2)—C5-10-heteroaryl group optionally substituted by a halogen and/or a C1-2 alkyl group; a —(CH2—CH2)—C5-10-heteroaryl group optionally substituted by a halogen and/or a C1-2 alkyl group;
        R6 is —CO2H, —CO2R10, —CO—NH2, —CO—NHR10, —OR10 when r is 1 or 2, —NH—CO—NHR10 when r is 1 or 2, or R6 is —CO—, —CO—O— or —O— and forms a lactam, a lactone, or a polyether ring with R7a, R7b, R8 or R9; wherein
        
            R10 is a C1-8-alkyl group optionally substituted by a C6-10-aryl group; a C3-6-cycloalkyl group; a C6-10-aryl group optionally substituted by a halogen, a C1-2-alkyl group and/or a C1-2-alkoxy group;
        
        
        m is 0 or 1;
        n is an integer from 0 to 9 inclusive;
        p is an integer from 0 to 10 inclusive;
        r is 0, 1 or 2.
    
    


In the above formula (I), the peptide linkages (—CO—NH—) can be replaced or modified to obtain synthetic pseudopeptides or peptidomimetics in which the peptide bond is modified, especially to become more resistant to proteolysis, provided the immunogenicity of and the toxicity of the molecule is not increased by this modification, and providing the pseudopeptide retains its affinity for the β interacting pocket.
The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein. The term “C1-12-alkyl” refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon group of 1 to 12 (inclusive) carbon atoms. Similarly, the terms: “C1-8-alkyl”, “C1-5-alkyl”, “C1-4-alkyl”, “C1-2-alkyl” and the like refer to branched or straight-chain monovalent saturated aliphatic hydrocarbon groups of, respectively, 1 to 8 (inclusive), 1 to 5 (inclusive), 1 to 4 (inclusive), 1 to 2 carbon atoms. This term is further exemplified by groups as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecanyl and their branched isomers. The “alkyl” group can optionally be mono-, di-, tri- or multiply-substituted by a halogen and/or a C6-10 aryl group, as defined below.
The term “C1-8-alkyl-(O—CH2—CH2)t-” refers to a —(O—CH2—CH2)t— substituted C1-8-alkyl group wherein the alkyl group is as defined above and t is an integer from 0 to 20 (inclusive), preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferred —(O—CH2—CH2)t— substituted alkyl group is a C1-5-alkyl-(O—CH2—CH2)t— group with t and alkyl as defined above.
The term “C2-12-alkenyl” refers to a branched or straight-chain monovalent unsaturated aliphatic hydrocarbon group having one or more carbon double bonds, of 2 to 12 (inclusive) carbon atoms, preferably 2 to 8 (inclusive) carbon atoms, more preferably 2 to 4 (inclusive) carbon atoms. This term is further exemplified by groups as vinyl, propylenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl and their straight-chain and branched and stereo isomers. The “alkenyl” group can optionally be mono-, di-, tri- or multiply-substituted by a halogen and/or a C6-10-aryl group, as defined below.
The term “C1-12-alkylene” refers to a divalent C1-12-alkyle with alkyl as defined above. Similarly, terms such as “C4-12-alkylene” or “C4-8-alkylene” and the like, refer to divalent C4-12-alkyl or divalent C4-8-alkyle group where alkyl is defined above. Examples of alkylene groups are —(CH2)—, —(CH2)2—, —(CH2)3—, —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, —(CH2)8—, —(CH2)9—, —(CH2)10—, —(CH2)11—, —(CH2)12—.
The term “C4-12-alkenylene” refers to a divalent C4-12-alkenyl of formula —(CH2)x—(CH═CH)y—(CH2)z— wherein x and z are, independently, 0, 1, 2, 3, 4, 5, 6, 7 or 8 and y is 1, 2, 3 or 4. Similarly, the term “C4-8-alkenylene”, refers to a divalent C4-8-alkenyl. Examples of alkenylene groups include butenyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, heptadienyl, octenyl, octadienyl, nonenyl, nonadienyl, decenyl, decadienyl, undecenyl, undecadienyl, undodecenyl, undodecadienyl, and their straight-chain and branched and stereo-isomers.
The term “C3-6-cycloalkyl” refers to a saturated or partially unsaturated cyclic hydrocarbon group having 3 to 6 (inclusive) carbon atoms. This term is further exemplified by groups as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The “C3-6-cycloalkyl” group can optionally be mono-, di-, tri- or multiply-substituted by a halogen as defined below, a C1-4-alkyl group as defined above, a —NH2, a —NH—CO2H, a —NH—CO—Ra, —CO2H, —NHRa and/or —NRaRb wherein Ra and Rb are independently a C1-4-alkyl group as defined above.
The term —(CH2)—C3-6-cycloalkyl group refers to a —CH2— substituted C3-6-cycloalkyl group wherein the cycloalkyl group is as defined above.
The term “C6-10 aryl” refers to a monocyclic or bicyclic aromatic ring system of 6 to 10 (inclusive) carbon atoms, preferably 6 carbon atoms. This term is further exemplified by groups as phenyl and naphtyl. The C6-10-aryl group can optionally be mono-, di-, tri- or multiply-substituted by a halogen as defined below and/or a C1-4-alkyl group as defined above.
The terms “halo” or “halogen” refers to fluorine, chlorine, bromine and iodine.
The term “halogen-C1-4-alkyl”, refers to a halogen substituted C1-4-alkyl group wherein both halogen and alkyl groups have the meaning as above. Preferred “halogen-C1-4-alkyl” groups are fluorinated “halogen-C1-4-alkyl” groups such as —CF3, —CH2—CF3, —CH(CF3)2, —CH(CH3)(CF3), —C4F9.
The term “C1-12-alkoxy” refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon group of 1 to 12 (inclusive) carbon atoms attached to an oxygen atom. Similarly, the terms “C1-8-alkoxy”, “C1-5-alkoxy”, “C1-4-alkoxy”, “C1-2-alkoxy” refer to branched or straight-chain monovalent saturated aliphatic hydrocarbon groups of, respectively, 1 to 8 (inclusive), 1 to 5 (inclusive), 1 to 4 (inclusive), 1 to 2 carbon atoms. Examples of “alkoxy” groups are methoxy, ethoxy, propoxy, butoxy, pentoxy, hexyloxy, heptyloxy, octyloxy, and their branched isomers.
The term “C5-10-heteroaryl” refers to a heterocyclic aryl group containing 1 to 3 heteroatoms in the ring with the remainder being carbon atoms. In the said heterocyclic aryl group, suitable heteroatoms include, without limitation, sulfur and nitrogen. Exemplary heteroaryl groups include indolyl, azaindolyl, thiophenyl, benzothiophenyl, thioazolyl, benzothiazolyl. The heteroaryl group can optionally be mono-, di-, tri- or multiply-substituted by a halogen and/or a C1-4-alkyl group, as defined above. When the heteroaryl group is mono-, di-, tri- or multiply-substituted by a C1-4-alkyl group, said alkyl group is preferably a methyl group.
The term “polyether ring”, refers ring containing 1, 2, or 3 ether groups, an ether group being an oxygen atom connected to two alkyl groups as defined above
The term “lactone” refers to a closed ring containing an oxygen atom adjacent to a carbonyl group (—CO—O—). It can be considered as the condensation product of an —OH group with a —CO2H group.
The term “lactam” refers to a closed ring containing an nitrogen atom adjacent to a carbonyl group (—CO—NH— or —CO—NR— with R being for example an alkyl group as defined above).
The terms “substituted” and “substitution and the like”, refer to the replacement of one, two, three or more atoms in a given group by one, two, three or more suitable substituents, including, without limitation, a halogen, a C6-10 aryl group, a C1-4-alkyl group, a C1-2-alkyl group, a C1-2-alkoxy group, a —NH2, a —NH—CO—Ra, —CO2H, —NHRa and/or —NRaRb wherein Ra and Rb are independently a C1-4-alkyl group, or a mixture of those substituents.
In some embodiments of the invention, the compounds of the invention can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereoisomeric mixtures. All such isomeric forms of these compounds are included in the present invention, unless expressly provided otherwise.
In some embodiments, the compounds of the invention can contain one or more double bonds and thus occur as individual or mixtures of Z and/or E isomers. All such isomeric forms of these compounds are included in the present invention, unless expressly provided otherwise.
In the embodiments where the compounds of the invention can contain multiple tautomeric forms, the present invention also includes all tautomeric forms of said compounds unless expressly provided otherwise.
In the embodiment where R7a together with R6 form a lactone or a polyether ring,

    
    
        R7a is C4-12-alkylene, preferably C4-8-alkylene group, and is linked to a —CO—O— or to a —O— functional group in R6, or
        R7a is C4-12-alkenylene, preferably C4-8-alkenylene group, and is linked to a —CO—O— or to a —O— functional group in R6.
    
    


In the embodiment where R7b together with R6 form a lactam, R3 is a —(CH2)q—CO—NHR7b and R7b is —(CH2)q′—NH— with q′ being 2, 3, 4, 5, 6, 7 or 8.
In the embodiment where R9 together with R6 form a lactam, R3 is a —(CH2)qNHR9 and R9 is a direct link between —(CH2)qNH— and a —CO— functional group in R6.
In the embodiment where R8 together with R6 form a lactone or a polyether ring:

    
    
        R8 is C4-12-alkylene, preferably C4-8-alkylene group, and is linked to a —CO—O— or to a —O— functional group in R6, or
        R8 is C4-12-alkenylene, preferably C4-8-alkenylene group, and is linked to a —CO—O— or to a —O— functional group in R6.
    
    


The terms “β ring”, “β protein” or “β clamp” herein designate the β subunit of a eubacterial DNA polymerase III, such as that of E. coli. The β subunit of DNA polymerase III of E. coli is in particular described in Kong et al. (1992)1.
Further definitions are added in the text, when necessary.
Particular embodiments of the compounds according to the invention are described in the following more detailed specification.
According to a particular embodiment of the compounds according to the invention, the R group indicated in the above formula (I) is selected amongst a C1-8-alkyl group optionally substituted by a C6-10-aryl group, a C2-8-alkenyl group optionally substituted by a C6-10-aryl group or a C1-5-alkyl-(O—CH2—CH2)t— group with t being 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, R is a C1-4-alkyl group optionally substituted by a C6-10-aryl group or a C2-4-alkenyl group optionally substituted by a C6-10-aryl group. Indeed, as described in the experimental part below, the inventors have observed that a N-terminal acetylation of the peptide P5 (QLDLF, SEQ ID No: 5) leads to a 10-fold increase of the affinity of the peptide for the β interacting pocket.
When m is not null and p is >1 in the above formula (I), for example when m=1 and p=2, 3, 4, 5, 6, 7, 8, 9 or 10, the above-described compounds are pegylated. The pegylation contributes to the stability of the whole molecule and can also have a positive effect for the entry of said compound into bacterial cells.
Particular compounds according to the invention have one or several arginines and/or lysines at the N-terminal extremity of the peptide part of the compound. For example, n is 1, 2, 3, 4 or 5. Indeed, these positively charged residues are known to favor membrane crossing. This feature is however not compulsory, and other compounds according to the invention do not have such residues (n=0 in formula I).
The inventors have also observed that substitution of the second amino acid of Ac-QLDLF (SEQ ID No: 6, P6) by a beta-cyclohexyl-L-alanyl (hereafter designated as “Cha”) leads to a further 6-fold increase of the affinity of said peptide with the β hydrophobic pocket. Other substitutions at the same position (see Table 4 in the experimental part) led to the above definition of R2 in formula (I). According to an advantageous embodiment, R2 is a —(CH2)—C3-6-cycloalkyl group.
By substituting the leucine of the acetylated peptide Ac-Gln-Cha-Asp-Leu-Phe 7 (SEQ ID No: 7, P7) by a number of different residues (see Table 5 of the experimental part below), the inventors could determine preferable embodiments for R4 group, in order to optimize the binding to the interacting pocket of the β ring. Accordingly, R4 is preferably selected amongst C1-5-alkyl groups, especially branched ones (such as valine, leucine or homoleucine lateral chains, for example), or amongst C1-3—, preferably C1-2-alkyl groups optionally substituted by a C3-6-cycloalkyl group (such as Cha and homoCha, for example).
As shown in Table 6 below, modifications introduced on the C-terminal phenylalanine benzyl ring led to an increase of the affinity of the molecule with the interacting pocket of the β ring. The binding affinity was found to increase with the size of the ring substituent (p-methyl<p-chloro<p-bromo<3,4-dichloro). The same table shows that other cyclic molecules can be used in this position. Contrarily, replacement of the phenylalanine lateral chain by a 2-amino-tetradecanoic acid led to a significant loss in affinity, thereby indicating an upper limit for the size of the group to be used at this position. Accordingly, in the compounds of the present invention, R5 is preferably a —(CH2)—C6-10-aryl group optionally substituted by a halogen, a C1-2 alkyl group and/or a C1-2 alkoxy group.
As exemplified in the experimental part below, excellent affinities are obtained with linear molecules having a peptidic skeleton. Such molecules excellently mimic the binding part of the proteins which naturally interact with the β ring. When the compounds according to the invention are linear, R3 and R6 are as follows:

    
    
        R3 is selected in the group consisting of a C1-8-alkyl group, the side chain of arginine or lysine, —(CH2)q—CO2R7a, —(CH2)q—CO—NHR7b, —CH2OR8, —(CH2)qNHR9, wherein
        
            q is 1, 2, 3, 4,
            R7a is a hydrogen atom, or a C1-8-alkyl group,
            R7b is a hydrogen atom, or a C1-8-alkyl group,
            R8 is a hydrogen atom, a C1-8-alkyl group,
            R9 is a hydrogen atom;
        
        
    
    


and

    
    
        R6 is —CO2H, —CO2R10, —CO—NH2, —CO—NHR10, —OR10 when r is 1 or 2, —NH—CO—NHR10 when r is 1 or 2; wherein
        
            R10 is a C1-8-alkyl group optionally substituted by a C6-10-aryl group; a C3-6-cycloalkyl group; a C6-10-aryl group optionally substituted by a halogen, a C1-2-alkyl group and/or a C1-2-alkoxy group.
        
        
    
    


It is to be noted that R6 is directed towards the solvent. Hence, R6 can be replaced by virtually any kind of molecule. For example, it can advantageously be replaced by or attached to a molecule which helps the crossing of membranes and/or the internalization by the bacteria. Non-limitative examples of such molecules are cell-penetrating peptides (CPP) (Classes and prediction of cell-penetrating peptides, Lindgren M, Langel U., Methods Mol Biol. 2011, 683, p. 3-19). In case a CPP or another molecule is covalently bound to the compound via R6, a linker, made of one to 10, preferably 1 to 4 amino acids, can be added between the compound of the invention and said CPP. Such a linker can be, for example, a mere arginine or lysine, or a sequence of 2 to 4 amino-acids corresponding to the amino-acids immediately following the binding site of a natural ligand of the β ring, such as, for example, ASRQ (SEQ ID No: 31), which is the sequence following the binding site of the delta protein from the gamma complex. Indeed, as shown by Jeruzalmi et al (2001)15, 15a, this protein exhibits a bend towards the outside of the pocket. Hence, a CPP bound via a ASRQ linker to a compound according to the invention would not hinder the interaction of said compound with the β ring.
According to a particular embodiment of the linear compounds of the present invention, R3 and/or R6 are as follows:

    
    
        R3 is selected in the group consisting of the side chain of arginine, the side chain of lysine, —(CH2)q—CO2R7a and —(CH2)q—CO—NHR7b, wherein
        
            q is 1, 2, 3 or 4,
            R7a is a hydrogen atom, or a C1-8-alkyl group, and
            R7b is a hydrogen atom, or a C1-8-alkyl group,
        
        
    
    


and/or

    
    
        R6 is —CO2H or —CO—NH2.
    
    


In an alternative embodiment, the compounds according to the present invention are cyclic, a cycle being made between the R3 and R6 groups. This bridge between R3 and R6 groups eliminates carboxylates, thereby improving the capacity of the compounds to enter bacterial cells, without impacting R5, which is necessary for anchoring the compound in the pocket, and for the subsequent conformational modification of said pocket. According to this embodiment, R3 and R6 are as follows:

    
    
        R3 is selected in the group consisting of —(CH2)q—CO2R7a, —(CH2)q—CO—NHR7b, —CH2OR8, —(CH2)qNHR9, wherein
        
            q is 1, 2, 3 or 4,
            R7a is a C4-8-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-8-alkenylene, forming together with R6 a lactone or a polyether ring,
            R7b is —(CH2)q′—NH— with q′ being an integer from 2 to 8 inclusive and forming together with R6 a lactam,
            R8 is a C4-8-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-8-alkenylene, forming together with R6 a lactone or a polyether ring,
            R9 together with R6 form a lactam;
        
        
        R6 is —CO—, —CO—O— or —O— and forms a lactam, a lactone, or a polyether ring with R7a, R7b, R8 or R9.
    
    


Particular compounds according to the present invention are described in the experimental part which follows. Particular compounds having a very good to excellent affinity for the β ring are: P7 (SEQ ID No: 7), P11 (SEQ ID No: 11), P12 (SEQ ID No: 12), P13 (SEQ ID No: 13), P14 (SEQ ID No: 14), P16 (SEQ ID No: 16), P17 (SEQ ID No: 17), P23 (SEQ ID No: 23), P24 (SEQ ID No: 24), P25 (SEQ ID No: 25), P26 (SEQ ID No: 26), P27 (SEQ ID No: 27).
As described in the experimental part below and as perfectly known by skilled artisans, several techniques exist to measure the affinity of two interacting proteins. These techniques may give slightly different results. However, the relative affinity of two compounds for the β ring is not dependent from the technique used for measuring said affinities (FIG. 4B). In a preferred embodiment of the compounds according to the invention, the affinity of said compounds for the interacting pocket of the bacterial β ring is at least twice the affinity of the acetylated peptide of sequence AcQLDLF (SEQ ID No: 6, P6) with said interacting pocket.
The compounds described above can advantageously be used as antibacterial agents, since they inhibit, at least partially, the interaction between the β protein and proteins that interact therewith by binding to its hydrophobic pocket.
A pharmaceutical composition comprising, as an active agent, a compound as above-described, is also part of the present invention.



FIGURES LEGENDS
FIG. 1: Representations of the ligand binding pocket of the β ring of E. coli, from the co-crystal structure of the β ring with the C-terminal peptide of the E. coli DNA polymerase IV (R1Q2L3V4L5G6L7, SEQ ID No: 32) (PDB code 1OK7). A: unbound pocket: the M362 (Δ) residue is located close to the H175 β residue (*) and obstructs the path between subsite 1 (black dots area) and subsite 2 (white dots area). Water molecules are represented as medium grey balls. B: bound pocket. The peptide has been removed. The movement of residue M362 opens a cleft (dark arrow) which connects subsite 1 and subsite 2 and where the V4 peptide residue interacts (see C). Water molecules are displaced, as compared to A, so that the peptide can fit into subsite 1. Note the opening of the platform (white star) between M362 and R365 where the L3 peptide residue will be located. C: Same as B but with the peptide P1 bound into the pocket.
FIG. 2: A: Energetic contributions (Kcal/mol) of each peptide residue (R1Q2L3V4L5G6L7, SEQ ID No: 32) for the interaction within the binding pocket of the β ring (PDB 1OK7). Black: electrostatic contribution, dark grey: solvent accessible surface contribution, light grey: Van der Waals contribution, white: total contribution. B: Single residue contribution (kcal/mol) to the peptide binding. Native peptide P1 of E. coli DNA polymerase IV, from the structure 1OK7, is in black. The pentapeptide P6 is in grey (PDB 3Q4J).
FIG. 3: Detailed connectivities between β residues N320 and M362 in subsite 2 of the binding pocket, in absence (A) or presence (B) of the peptide. Balls represent water molecules. From PDB structure 1OK7, incorporated herein by reference.
FIG. 4: Polymerase competition assay. A: the β dependant activity of PolIV DNA polymerase is challenged by increasing concentrations of various peptides B: the table displays the IC50 determined for various peptides by the Pol IV based biochemical assay and the SPR assay. The histogram indicates that the same general trend is observed with both techniques despite a difference in sensitivity. Grey: biochemical assay, black: SPR assay. P15 sequence is Ac-RQLVLF, (SEQ ID No: 15), Scr: scrambled peptide: Ac-ChaFQLD, (SEQ ID No: 33).
FIG. 5: Superimposition of peptide-β complexes. A: A P6-β complex (pale colors) is superimposed on P12-β complex (dark colors) (rmsd: 0.95 Å). The first (Gln) and last (Phe) peptide residues are indicated. The Cha group of P12 (SEQ ID No: 12) peptide occupies the same position as the Leu2 residue of P6 (SEQ ID No: 6). The chloro-modified Phe residue of P12 is tilted toward the bottom of subsite 1 as compared to the cognate residue of P6. B: P14-β complex (pale colors) is superimposed on P12-β complex (dark colors) (rmsd: 0.56 Å). The chlorine atom in meta position forms an halogen bond with T172 residue.
FIG. 6: Superposition of the peptide free (dark) and peptide bound (pale) interacting pockets of 1OK7 structure. In the absence of peptide, the M362 side chain (dark) is located close to the H175 residue (closed conformation), and separates subsite 1 and subsite 2. When the peptide is bound, the M362 side chain (pale) is displaced away from the H175 (open conformation) allowing the opening of a cleft in which the peptide can bind. Residue R365 is also shifted upon peptide binding, triggering the opening of a small platform where the peptide L3 residue locates.
FIG. 7: Graphical representation of the quantitative analysis of polymerase competition assays performed with several peptides. The percentage of inhibition of β dependent E. coli DNA polymerase IV activity is plotted as a function of peptide concentration (μM). P15 sequence is Ac-RQLVLF (SEQ ID No: 15). Scr: scramble peptide: Ac-ChaFQLD (SEQ ID No: 33); (related to FIG. 4).
FIG. 8: Isothermal titration calorimetry (ITC).
A. Binding isotherms for the titration of the β ring with peptide P12 (SEQ ID No: 12) and P14 (SEQ ID No: 14). N: number of sites per β monomer.
B. Enthalpy-entropy compensation for selected natural and non-natural β binding peptides. The thermodynamics parameters are determined by ITC. Each value is the mean of two independent experiments monitoring the binding of each peptide (400 μM) to the β ring (20 or 30 μM) at 25° C. Each correlation point is labeled according to the corresponding peptide, and the respective AG values are plotted below. 1 cal=4.18J; (related to Table 11).
FIG. 9: Examples of compounds according to the invention are represented in FIG. 9 (A-H). Those include compounds wherein R=acetyl, cynamoyle, octanoyle; R1=Cl and R2=H, or R1=R2=Cl, or R1=R2=H, or R1=Me and R2=H, or R1=Br and R2=H; and R3=OH or NH2. Specific compounds P23 (SEQ ID No: 23), P24 (SEQ ID No: 24), P25 (SEQ ID No: 25), P26 (SEQ ID No: 26), P27 (SEQ ID No: 27), P28 (SEQ ID No: 28), P29 (SEQ ID No: 29) and P30 (SEQ ID No: 30) are disclosed in FIG. 9H.



EXAMPLES
Example 1
Structure-Based Design of Short Peptide Ligands Binding onto the E. coli Processivity Ring
1.1. Material and Methods
1.1.1. Protein Production, Purification and Characterization
The E. coli dnaN gene was cloned into pET15b plasmid (Invitrogen) using standard protocols. The resulting N-tagged protein was expressed in BL21 E. coli cells after IPTG induction (0.1 mM) at 28° C. The β protein fraction was first enriched on a Ni-NTA column, eluted with an histidine step (300 mM) and further purified on a MonoQ column in buffer containing 20 mM Tris HCl pH 7.5, 0.5 mM EDTA and 10% glycerol, using a gradient from 0 to 0.5 M NaCl. The quality of the protein was assessed by mass spectrometry in denaturing and native conditions
1.1.2. Peptide Synthesis
Peptides P1-P14 (SEQ ID Nos: 1 to 14) were synthesized in Fmoc chemistry by the stepwise solid-phase methodology28 on a home-made semi-automatic peptide synthesizer29. N—N-Fmoc protected amino acids (natural and non natural) are commercially available from Polypeptide Labs (Strasbourg, France). Resins for solid-phase peptide synthesis are commercially available from Polypeptide Labs (Strasbourg, France) and CBL Patras (Patras, Greece). Assembly of the protected peptide chains was carried out on a 100-μmol scale starting from either Fmoc-Leu-Wang resin (Peptides P1, P2, P4), Fmoc-Phe-Wang (Peptides P3, P5-P10) resin or o-chlorotrityl chloride resin (peptide P11-P14). For each coupling step, the reactants were introduced manually as a solution in dry DMF (2.0 mL). Nα-Fmoc amino acids (5.0 equivalent) with standard side-chain protecting groups were coupled 2 times by using BOP (5.0 equivalent), HOBt (5.0 equivalent) and DIEA (10.0 equiv) in dry DMF for 20 min. The washing of the resin as well as Fmoc deprotection (by using a freshly prepared solution of 20% piperidine in DMF) were performed automatically. The coupling and deprotection steps were monitored by the Kaiser test30. At the end of the elongation of the peptidic chain, the resin was washed with CH2Cl2 and dried with Et2O. A mixture of TFA/H2O/TIPS/DTT (8.8/0.5/0.2/0.5; 10.0 mL) was then added to the resin. The mixture was gently shaken for 2.5 h and the resulting solution was flushed through a frit in cold Et2O. The precipitate was recovered by centrifugation, dissolved in a mixture of AcOH and H2O and freeze-dried. The crude peptides were finally purified by HPLC (linear gradient, 5-65% B, 30 min) and freeze-dried. All peptides were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and their homogeneity was assessed by C18 RP-HPLC (purity of all peptides determined to be >90%).
Analytical data are reported in Table 8.
1.1.3. Molecular Dynamics
In the present work, the inventors used a protocol31 based on the MM/PBSA method32, 33, where conformations extracted from molecular dynamics simulations are processed using a simplified description for the solvent to yield an estimate of binding free energy. Individual contributions of each amino acid to the complex formation are estimated and important energetic amino acid “hot spots” are identified.
Structures
The initial structure for the apo protein was chain A from the PDB file 1OK717, while for the protein and native peptide it was chains B and C from the same PDB (1OK7). All crystallographic water molecules were retained.
MD Simulations
The CHARMM program34, version 32, with the CHARMM 22 all atom protein-nucleic acid force field35 was used for the molecular dynamics simulations. Hydrogen atoms were added using the HBUILD facility in the CHARMM program. A sphere of 37 Å containing 6840 water molecules (TIP3) was used to solvate the system. Stochastic boundary conditions were imposed and the calculation was limited to residues 7 Å around the peptide. The SHAKE algorithm was used to constrain hydrogen-heavy atom bond distances, and the simulations were done using Langevin algorithm. A 1-fs time step was used for the molecular dynamics simulation and the simulation time. A 12 Å cutoff was used; the van der Waals non bonded terms were treated with a SWITCH potential function whereas the electrostatic terms was evaluated with the SHIFT function.
Free Energy Decomposition of Interactions Between the E. coli β Clamp and the Different Peptides.
To obtain a semi-quantitative estimate of the contributions of all amino acids to the binding free energy for the formation of the β clamp-peptide complex, a molecular free energy decomposition scheme based on the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) analysis was performed, following the approach presented by Lafont et al.31. From this analysis, an estimation of the free energy of binding for molecular complexes can be obtained. Briefly, in the MM/PBSA approach, the free energy is estimated using a standard thermodynamic cycle of the form




where the binding free energy is calculated according to the equation:

ΔGassocsolution=ΔEMMgas−TΔS+MM+ΔGsolvation 

where ΔMMgas is the difference in the gas phase energy; ΔSMM is the change in entropy upon complex formation and ΔGsolvation is the change in solvation free energy. The gas phase energy differences contain terms for the intermolecular electrostatic and van der Waals energies, as well as the equivalent internal energy terms. These terms are based on the CHARMM force field in the present approach. The solvation free energy is divided into two contributions: an electrostatic and a nonpolar contribution. This latter term is approximated by an empirical relationship based on solvent accessible surface area and the electrostatic contribution is calculated here using the Poisson-Boltzmann equation.
Several approximations are introduced in the MM/PBSA method. The first was the neglect of conformational change upon complex formation, which is dictated by the absence of experimental structures for the unbound protein and peptides. To account for the unbound species in the calculations, their respective structures were obtained from the complex generated during the molecular dynamics simulations. With this approximation, there are no changes to the internal energy terms. The second approximation is the neglect of changes in configuration entropy due to binding. Although these simplifications preclude calculations of absolute values of the binding free energies, they have been shown in previous work to be satisfactory in the context of identifying interaction energy “hot spots” in protein-protein and protein-ligand complexes. Similar simplifications have been employed in other studies36 31 37. Via this approach, the total binding free energy can be decomposed into individual energetic contributions per residue. Decomposition of the binding free energy to individual amino acid contributions leads to the identification of amino acids that play a dominant role in binding and can contribute to reliable predictions of the role of particular amino acids in stabilizing complexes.
1.1.4. Structure-Based Design of Peptides
From the initial structural and energetic analysis of the RQLVLGL (SEQ ID No: 1, P1 in Table 1) peptide binding to the β clamp, modification of the sequence appeared potentially interesting in three positions (cf. FIG. 1): Q2; L3 and the hydrophobic L5-G6-L7 segment. In order to identify interesting modifications, the programs MCSS38 and SEED39 were used to dock small librairies of hydrophobic and polar small ligands (fragments) onto the surface of the β-clamp encompassing the peptide binding site. The protocol incorporated improved scoring functions with solvation corrections.40, 41. From this initial step, it appeared difficult to find replacements for the Q2 side-chain of the peptide that would correctly maintain the intricate hydrogen-bond network at this position (see FIG. 3) and therefore no modification of Q2 were attempted. For the other positions, improving interactions with optimized hydrophobic contacts appeared promising. Based on these initial data, a selection of peptides with modified side chains were constructed, docked into the structure and their interactions with the β clamp evaluated using the MM-PBSA protocol described above. The choice of side-chain replacements was based on the docking data, focusing on commercially available protected amino-acids. A force field adapted from CHARMM 2235 was used for non-natural amino acids. The most promising candidates were selected for synthesis.
1.1.5. β/Peptide Interaction in Solution: In Vitro Competition Assays.
5′ end radiolabelling, purification and annealing of synthetic primers were performed as previously described (Wagner et al., 1999). The 30/90mer synthetic construct was obtained by annealing the 30 mer primer (5′GTAAAACGACGGCCAGTGCCAAGCTTAGTC3′, SEQ ID No: 34) with the 90 mer template (5 ‘CCATGATTACGAATTCAGTCATCACCGGCGCCACAGACTAAGCTTGGCACTG GCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGG3’, SEQ ID No:35) to form a double stranded structure with 5′ and 3′ ssDNA overhangs of 25 and 35 nucleotides, respectively. All replication experiments (10 μl final volume) were carried out in buffer E (40 mM HEPES pH 7.5, 80 mM potassium glutamate, 160 μg/ml BSA, 16% glycerol, 0.016% NP40, 8 mM DTT). The 30/90 mer duplex (1 nM final concentration) was first incubated with single strand binding protein (SSB; Sigma; 90 nM final concentration) in the presence of ATP (200 μM) and MgCl2 (7.5 mM) at 37° C. for 10 min. γ complex (1 nM final concentration) (obtained as described by Dallmann et al, 1995) and β clamp (5 nM as dimer final concentration) were added at that stage and incubation was carried out at 37° C. for 10 min. Then, 7 μl of the mixture was added to 1 μl of either DMSO or 1 μl of peptide solution (as specified), incubated 5 min. at room temperature and further 2 hours at 4° C. 1 μl of PolIV was then added (1.5 nM final concentration), incubated 5 min. at room temperature and finally, the whole reaction was mixed with 1 μl of a dNTPs solution (200 μM each dNTP final concentration) and let react for 1 min. at room temperature. Reactions were quenched by the addition of 20 μl of 95% formamide/dyes solution containing 7.5 mM EDTA, heat-denatured and analysed by chromatography on 12% denaturing polyacrylamide gels. Radiolabelled products were visualised and quantified using a Personal Molecular Imager Fx and the Quantity One software (Bio-Rad).
1.1.6. SPR Assays.
SPR experiments were performed on a Biacore® 3000. The association constant (KA) of β with the natural Cter heptamer (P1, Table 7) of the DNA polymerase IV of E. coli were determined as follow: the β protein (0.125 μM to 2 μM) was injected on the immobilized P1 peptide at a flow rate of 500 μL·min−1. After subtraction of the background response, the data were fit to the 1:1 Langmuir model using BIAevaluation (BiacoreTM). The inhibition of P1-β interaction by peptides P2 to P14 (Table 7) was used to measure their affinity for β and was assessed according to the following procedure: complexes of β ring (0.25 μM) with various concentrations of challenging peptides (1.5 nM to 100 μM) were formed and injected on a chip loaded with the P1 peptide. IC50 values for each challenging peptide were determined by plotting the concentration of peptide against the percentage of binding inhibition. The IC50 value of each peptide was used to calculate Ki (Ki=(1+KA[β])/IC50) which measures the affinity of the challenging peptide for β in the competition assay, and AG was derived from Ki (ΔG=RT ln Ki).
1.1.7. Isothermal Titration Calorimetry.
ITC was performed by using a ITC200 microcalorimeter from MicroCal. Peptides (400 μM) were titrated in sequential injections (2 μl each) into a β ring solution (300 μl, 20 or 30 μM) at 25° C. Data were corrected from control experiments where peptides were injected in buffer solution (Hepes 10 mM pH 7.4, NaCl 0.15M, EDTA 3 mM, P20 0.005%). Data analysis was performed with Origin 7.0 software.
1.1.8. Crystallogenesis, Data Collection and Processing.
Crystallization experiments were essentially conducted as described previously17. Crystals of P12-β complexes were grown in capillaries in presence of 0.2% of agarose42. Crystallisation buffer contained 100 mM CaCl2, 100 mM Mes pH 6.0 and 30% PEG 400. Cryoprotection was performed by soaking crystals in the same buffer supplemented with 20% glycerol. Cryoprotected crystals were frozen in liquid ethane and X-ray diffraction data were collected at 100 K at beamline X06SA at the Swiss Light Source (Villigen PSI, Switzerland) and beamlines ID29 and ID14-4 at ESRF (Grenoble, France). Diffraction images were processed with XDS, XSCALE and XDSCONV43. The structures were solved by molecular replacement with MOLREP44, using the known beta structure as a search model PDB ID 1OK712. Alternate rounds of rebuilding and refinement, including noncrystallographic symmetry restraints, were carried out with PHENIX45, COOT46 and CNS47. Model statistics were obtained with Molprobity48. Molecular visualizations and structures illustrations were performed using PyMOL49. Data processing and refinement statistics are summarized in Table 11.
1.2. Results
1.2.1. Structure and Energetics of the Binding Pocket
The inventors have previously solved the structure of a complex formed between the E. coli β clamp and the 16 residues long C-terminal peptide of the E. coli DNA polymerase IV (PDB code: 1OK7)17. A first part of the present work aimed at unraveling the molecular basis of the peptide-pocket interactions. Molecular modeling approaches were used to determine the contribution of each residue of the last seven amino-acids of the C-terminal part of Pol IV (R1Q2L3V4L5G6L7, SEQ ID No: 32) to the overall interaction (FIG. 2A), using free energy decomposition (see Material and Methods). For each amino acid, the van der Waals, electrostatic and hydrophobic solvation contribution to binding have been calculated. Stabilizing interactions between the β ring and the peptide are essentially Van der Waals contacts (see FIG. 2A). Electrostatics contributions are poor, due to compensation between the protein-peptide interaction and the peptide desolvation cost. Hydrophobic solvation contributions are favorable but of lesser magnitude. The net contributions of residues Q2, L3, L5 and L7 is predominant to the overall interaction (FIG. 2B). G6 has no contribution while V4 which is oriented toward the solvent poorly contributes to the interaction.
Due to the good resolution, the inventors could also analyze the position of water molecules in the free and bound pockets of the 1OK7 structure. In the absence of peptide, four water molecules are located in subsite 1. Upon peptide binding, one is eliminated and one is repositioned close to the T142 and Y154 residues, allowing the L5-G6-L7 tripeptide to bind into the hydrophobic subsite 1 (FIG. 1AB). The two water molecules located on the platform in the apo monomer are dislodged upon peptide binding, thus making room for the peptide L3 residue to bind (FIG. 1BC). Finally, two water molecules are deeply inserted into the empty subsite 2. One of these two molecules interconnects the NαH of N320 and the Cα=O of M362 (FIG. 3) and is not exchanged with the solvent upon peptide binding, underlining its structural function. The second water molecule is replaced by the Cδ=0 of peptide Q2 residue, while its δ-amino group establishes bounds with the Cα=O of M362 and the Cα=O of peptide residue L2 (FIG. 3B).
This initial analysis led the inventors to design a minimal peptide binding sequence that was used as a starting point for ligand optimization. Because of the complex network of hydrogen bonds formed by the highly conserved Q residue in subsite 2, one cannot substitute this side chain without dramatically altering the interaction of the whole peptide. Alternatively, several other positions in the peptide sequence may accept modifications that could increase its affinity for the β clamp. Following the structural and energetic analysis of the binding pocket (see Material and Methods), several peptides were synthesized (Table 7 and Table 11) and their binding efficiencies were analyzed by surface plasmon resonance (SPR). The dissociation constant of the P1 natural heptapeptide was measured to be 2.85 (±0.94) 10−7 M. As compared to the whole polymerase, this peptide binds 30 fold less efficiently to the ring (table 1, compare P1 and PolIV), pinpointing the contribution of alternate regions of the enzyme to the interaction16, 14, 19 Removing the G residue of the terminal tripeptide (-LGL) results in a two to three fold decrease in interaction (table 1, compare P1 and P2), while replacing the terminal tripeptide with the consensus LF dipeptide does not affect the affinity (table 1, compare P1 and P3). However, a FL dipeptide totally disrupts peptide binding (table 1, P4). Substituting F for other aromatic residues (W, Y) at the C-terminal position does not contribute to any increased interaction (data not shown). In order to design the shortest peptide, the first (R1) residue was also removed, which does not seem to contribute significantly to the binding (table 1, P5, FIGS. 1 and 2)17, and the V4 was replaced by a D residue, as observed in the consensus sequence, in order to increase the solubility of the resulting pentapeptide P5 (QLDLF). Although its affinity for the β ring is low, it was increased by 10 fold upon acetylation (table 1, compare P5 and P6), thus providing a good compromise between interaction efficiency and ligand size.







TABLE 1







Influence of the C-terminal tripeptide sequence 


and effect of N-terminal acetylation on the  


interaction of peptide with the E. coli β   


clamp, as measured by SPR experiments.













se-
IC50 
Ki 
ΔG (Kcal/
Seq Id 


#
quence
(μM)
(106 M−1)
mol)
No:















PolIV

0.29
4.7
−9.09



 


P1
RQLVLGL
8.85
0.15
−7.06
1


 


P2
RQLVLL
21.53
0.0063
−6.54
2


 


P3
RQLVLF
8.62
0.15
−7.04
3


 


P4
RQLVFL
256
Ø
Ø
4


 


P5
QLDLF
12.44
0.11
−6.87
5


 


P6
AcQLDLF
1.12
1.2
−8.22
6





Ø: not determined.


Ki = (1 + KA[β])/IC50.


ΔG = −RT ln Ki.


PolIV: E coli DNA polymerase IV.






1.2.2. Crystal Structure of the P6-β Ring Complex.
The P6 peptide (AcQLDLF, SEQ ID No: 6) co-crystallized with the β ring in conditions similar to those previously described17 but the cell parameters lead to a VM value of 7.8, which corresponds to the presence of 3 dimers per asymmetric unit (Table 2). This structure was solved by molecular replacement at 2.3 Å resolution, using our previously determined structure (PDB 1OK7). The superposition of main chain atoms of each ring to the model led to rmsd values ranging from 0.70 Å to 1.06 Å, underlining the close structural similarity of each dimer. Each monomer of the three rings binds a peptide, and all ligands adopt a similar conformation in all six hydrophobic pockets, as indicated by a rmsd value ranging between 0.25 Å to 0.51 Å.







TABLE 2







Statistics on Data Collection and Refinement (related to FIG. 5).











Beta-P14
Beta-P6
Beta-P12


Structure
(PDB 3Q4L)
(PDB 3Q4J)
(PDB 3Q4K)










Data Collection










Space Group
P1
P1
P1











Unit cell
a (Å)
34.84
35.09
36.25



b (Å)
79.57
132.87
80



c (Å)
81.64
137.27
82.18



α (°)
65.28
62.73
66.15



β (°)
75.26
88.51
74.94



γ (°)
82.22
89.77
82.03










Beamline
ID29/ESRF
X06SA/SLS
ID14-4/ESRF


Wavelength (Å)
0.97623
0.915694
0.9794


Resolution limits (Å)
39.2-1.95
29.5-2.3
19.9-2.6


high resolution shell
 2.0-1.95
2.35-2.3
2.65-2.6


Reflections:


measured
221062
296785
42708


unique
54138
96508
22982


Completeness (%)
96.3 (93.8)*
98.6 (97.9)*
91.9 (60.5)*


Rmerge
0.06 (0.72)*
0.05 (0.36)*
0.067 (0.11)* 


I/σ
 13 (1.9)*
22.6 (3.6)* 
8.7 (4.0)*







Refinement










Reflections Rcryst/Rfree
54134/2750
96493/7742
22979/1160


Rcryst (%)
20.1
21.6
25.9


Rfree (%)†
23.2
25.0
30.6


Protein atoms
5579
17085
5471


Ligand atoms
106
196
103


Water molecules
299
357
129


Average B factor (Å2)


Protein
33.3
52.1
30.4


Ligand
39.4
66.5
27.1


Water
40.1
44.5
27.6


R.m.s.d. bond length
0.01
0.009
0.008


(Å)


R.m.s.d. angles length
1.13
1.15
1.11


(°)





*Values in parentheses correspond to high resolution shell in data collections.


†5% of the reflections were set aside for an Rfree test before initiating any refinement






The atomic coordinates of the peptide and the peptide binding site of the β clamp (residues ≦5 Å from the ligand) are disclosed in the following Table 3. The other residues have the same positions as in the previously determined structure (PDB 1OK7) also described in U.S. Pat. No. 7,635,583.







TABLE 3





Atomic coordinates of P6 residues and of the residues involved in the binding of P6


to the β clamp, in the crystal of P6 peptide co-crystallized with the β ring.


























ATOM
1
NE
ARG
B
152
10.195
−25.903
12.978
1.00
60.02
N


ATOM
2
CZ
ARG
B
152
9.832
−25.010
13.891
1.00
70.75
C


ATOM
3
NH1
ARG
B
152
10.045
−23.710
13.686
1.00
51.13
N1+


ATOM
4
NH2
ARG
B
152
9.228
−25.402
15.004
1.00
66.21
N


TER
5

ARG
B
152


ATOM
6
CG
LEU
B
155
6.034
−25.353
10.551
1.00
37.71
C


ATOM
7
CD1
LEU
B
155
6.887
−24.861
11.676
1.00
37.67
C


ATOM
8
CD2
LEU
B
155
4.805
−25.976
11.115
1.00
31.46
C


TER
9

LEU
B
155


ATOM
10
CB
THR
B
172
1.710
−23.748
14.242
1.00
31.63
C


ATOM
11
CG2
THR
B
172
2.028
−25.084
13.527
1.00
27.98
C


ATOM
12
OG1
THR
B
172
2.665
−23.450
15.241
1.00
32.30
O


ATOM
13
C
ASP
B
173
6.157
−21.665
14.133
1.00
35.44
C


ATOM
14
N
GLY
B
174
5.672
−22.551
14.996
1.00
34.81
N


ATOM
15
CA
GLY
B
174
6.511
−23.182
16.011
1.00
35.62
C


ATOM
16
C
GLY
B
174
6.492
−22.492
17.359
1.00
39.39
C


ATOM
17
O
GLY
B
174
6.970
−23.064
18.344
1.00
39.92
O


ATOM
18
N
HIS
B
175
5.986
−21.242
17.411
1.00
34.18
N


ATOM
19
CA
HIS
B
175
5.900
−20.479
18.650
1.00
33.72
C


ATOM
20
C
HIS
B
175
4.476
−20.329
19.088
1.00
35.43
C


ATOM
21
O
HIS
B
175
4.175
−20.368
20.282
1.00
34.79
O


ATOM
22
CB
HIS
B
175
6.562
−19.119
18.513
1.00
36.12
C


ATOM
23
CG
HIS
B
175
7.984
−19.194
18.096
1.00
41.64
C


ATOM
24
CD2
HIS
B
175
9.032
−19.835
18.668
1.00
44.85
C


ATOM
25
ND1
HIS
B
175
8.394
−18.617
16.936
1.00
45.18
N


ATOM
26
CE1
HIS
B
175
9.678
−18.899
16.829
1.00
45.14
C


ATOM
27
NE2
HIS
B
175
10.115
−19.589
17.878
1.00
45.20
N


ATOM
28
N
ARG
B
176
3.593
−20.133
18.121
1.00
31.08
N


ATOM
29
CA
ARG
B
176
2.181
−19.986
18.371
1.00
29.68
C


ATOM
30
C
ARG
B
176
1.413
−20.789
17.353
1.00
32.45
C


ATOM
31
O
ARG
B
176
1.918
−21.053
16.262
1.00
32.86
O


ATOM
32
N
LEU
B
177
0.240
−21.239
17.733
1.00
27.89
N


ATOM
33
CA
LEU
B
177
−0.619
−22.029
16.875
1.00
27.83
C


ATOM
34
CB
LEU
B
177
−0.579
−23.523
17.307
1.00
27.38
C


ATOM
35
CG
LEU
B
177
−1.466
−24.512
16.510
1.00
31.18
C


ATOM
36
CD1
LEU
B
177
−0.745
−25.845
16.280
1.00
30.87
C


TER
37

LEU
B
177


ATOM
38
CB
PRO
B
242
3.262
−29.933
14.003
1.00
42.35
C


ATOM
39
CG
PRO
B
242
3.185
−28.774
13.112
1.00
45.82
C


ATOM
40
CD
PRO
B
242
3.325
−29.320
11.745
1.00
40.86
C


TER
41

PRO
B
242


ATOM
42
O
VAL
B
247
−0.058
−27.602
22.929
1.00
52.04
O


ATOM
43
CB
VAL
B
247
0.470
−28.149
19.728
1.00
50.73
C


ATOM
44
CG1
VAL
B
247
0.574
−26.655
20.015
1.00
50.50
C


ATOM
45
CG2
VAL
B
247
1.641
−28.626
18.875
1.00
51.23
C


TER
46

VAL
B
247


ATOM
47
O
GLY
B
318
5.474
−15.393
28.086
1.00
28.75
O


ATOM
48
N
PHE
B
319
5.344
−13.225
27.583
1.00
24.23
N


ATOM
49
CA
PHE
B
319
4.851
−13.489
26.241
1.00
24.99
C


ATOM
50
C
PHE
B
319
5.356
−12.468
25.290
1.00
30.49
C


ATOM
51
O
PHE
B
319
5.591
−11.324
25.669
1.00
32.74
O


ATOM
52
CB
PHE
B
319
3.310
−13.478
26.174
1.00
27.25
C


ATOM
53
CG
PHE
B
319
2.640
−14.732
26.670
1.00
28.63
C


ATOM
54
CD1
PHE
B
319
2.741
−15.919
25.957
1.00
30.47
C


ATOM
55
CE1
PHE
B
319
2.128
−17.094
26.424
1.00
31.60
C


ATOM
56
N
ASN
B
320
5.468
−12.865
24.025
1.00
26.27
N


ATOM
57
CA
ASN
B
320
5.720
−11.953
22.949
1.00
26.75
C


ATOM
58
C
ASN
B
320
4.315
−11.306
22.760
1.00
28.81
C


ATOM
59
O
ASN
B
320
3.351
−11.990
22.409
1.00
25.74
O


ATOM
60
CB
ASN
B
320
6.143
−12.740
21.690
1.00
31.09
C


ATOM
61
CG
ASN
B
320
6.252
−11.902
20.458
1.00
38.50
C


ATOM
62
ND2
ASN
B
320
7.226
−12.202
19.631
1.00
36.32
N


TER
63

ASN
B
320


ATOM
64
CB
TYR
B
323
2.398
−14.188
20.062
1.00
29.74
C


ATOM
65
CG
TYR
B
323
3.671
−14.541
19.312
1.00
34.89
C


ATOM
66
CD2
TYR
B
323
4.613
−15.405
19.867
1.00
36.98
C


ATOM
67
CE2
TYR
B
323
5.769
−15.758
19.176
1.00
38.52
C


ATOM
68
CZ
TYR
B
323
5.978
−15.280
17.899
1.00
45.00
C


ATOM
69
OH
TYR
B
323
7.102
−15.660
17.220
1.00
51.04
O


TER
70

TYR
B
323


ATOM
71
O
SER
B
343
6.499
−19.652
31.418
1.00
42.19
O


ATOM
72
CA
VAL
B
344
7.142
−22.358
31.029
1.00
30.95
C


ATOM
73
C
VAL
B
344
6.382
−23.225
30.039
1.00
37.16
C


ATOM
74
O
VAL
B
344
6.960
−23.833
29.135
1.00
38.77
O


ATOM
75
CB
VAL
B
344
8.406
−23.037
31.630
1.00
34.02
C


ATOM
76
CG1
VAL
B
344
9.318
−22.002
32.284
1.00
33.14
C


TER
77

VAL
B
344


ATOM
78
CB
SER
B
346
1.690
−23.500
25.230
1.00
34.25
C


ATOM
79
OG
SER
B
346
0.915
−24.661
25.493
1.00
39.01
O


TER
80

SER
B
346


ATOM
81
C
VAL
B
360
−0.613
−20.918
21.452
1.00
27.37
C


ATOM
82
O
VAL
B
360
−0.111
−20.800
20.340
1.00
24.84
O


ATOM
83
CB
VAL
B
360
−1.624
−23.300
21.499
1.00
27.65
C


ATOM
84
CG1
VAL
B
360
−0.575
−23.807
22.494
1.00
27.63
C


ATOM
85
C
VAL
B
361
1.982
−19.886
23.474
1.00
27.90
C


ATOM
86
CG1
VAL
B
361
1.873
−16.988
22.556
1.00
22.99
C


ATOM
87
N
MET
B
362
3.180
−20.112
23.023
1.00
28.61
N


ATOM
88
CA
MET
B
362
4.274
−20.561
23.871
1.00
28.56
C


ATOM
89
C
MET
B
362
4.839
−19.321
24.530
1.00
31.58
C


ATOM
90
O
MET
B
362
5.039
−18.292
23.870
1.00
29.49
O


ATOM
91
CB
MET
B
362
5.340
−21.302
23.049
1.00
31.41
C


ATOM
92
CG
MET
B
362
6.222
−22.193
23.888
1.00
35.60
C


ATOM
93
SD
MET
B
362
5.377
−23.603
24.664
1.00
38.96
S


ATOM
94
CE
MET
B
362
6.619
−24.060
25.847
1.00
34.70
C


ATOM
95
N
PRO
B
363
5.071
−19.362
25.842
1.00
29.24
N


ATOM
96
CA
PRO
B
363
5.609
−18.178
26.510
1.00
28.99
C


ATOM
97
C
PRO
B
363
7.074
−17.892
26.226
1.00
33.83
C


ATOM
98
O
PRO
B
363
7.743
−18.614
25.456
1.00
33.34
O


ATOM
99
CB
PRO
B
363
5.341
−18.479
27.991
1.00
30.94
C


ATOM
100
CG
PRO
B
363
5.412
−19.947
28.091
1.00
35.37
C


ATOM
101
CD
PRO
B
363
4.870
−20.473
26.798
1.00
31.25
C


ATOM
102
N
MET
B
364
7.545
−16.777
26.784
1.00
29.71
N


ATOM
103
CA
MET
B
364
8.945
−16.382
26.731
1.00
30.02
C


ATOM
104
C
MET
B
364
9.502
−16.624
28.124
1.00
40.88
C


ATOM
105
O
MET
B
364
8.772
−16.499
29.120
1.00
40.62
O


ATOM
106
CB
MET
B
364
9.118
−14.915
26.403
1.00
30.48
C


ATOM
107
CG
MET
B
364
8.757
−14.585
25.034
1.00
32.54
C


ATOM
108
SD
MET
B
364
8.724
−12.808
24.682
1.00
35.07
s


ATOM
109
CE
MET
B
364
10.528
−12.292
24.937
1.00
31.33
C


ATOM
110
N
ARG
B
365
10.767
−17.037
28.190
1.00
42.29
N


ATOM
111
CA
ARG
B
365
11.463
−17.315
29.447
1.00
44.24
C


ATOM
112
C
ARG
B
365
11.620
−15.996
30.209
1.00
47.68
C


ATOM
113
O
ARG
B
365
12.039
−14.991
29.621
1.00
46.53
O


ATOM
114
CB
ARG
B
365
12.812
−18.035
29.173
1.00
49.79
C


ATOM
115
CG
ARG
B
365
13.354
−18.871
30.335
1.00
61.58
C


ATOM
116
CD
ARG
B
365
12.589
−20.152
30.620
1.00
75.54
C


ATOM
117
NE
ARG
B
365
13.073
−21.279
29.817
1.00
90.27
N


ATOM
118
CZ
ARG
B
365
12.957
−22.563
30.161
1.00
100.27
C


ATOM
119
NH1
ARG
B
365
13.409
−23.516
29.355
1.00
79.16
N1+


ATOM
120
NH2
ARG
B
365
12.398
−22.901
31.318
1.00
86.12
N


TER
121

ARG
B
365


HETATM
122
O
HOH
B
384
8.833
−14.385
20.130
1.00
33.25
O


HETATM
123
O
HOH
B
407
10.652
−12.727
21.066
1.00
31.86
O


HETATM
124
O
HOH
B
465
12.648
−14.060
22.219
1.00
36.65
O


HETATM
125
O
HOH
B
466
13.941
−12.371
23.870
1.00
26.96
O


HETATM
126
C
ACE
H
69
12.190
−16.728
25.287
1.00
47.99
C


HETATM
127
O
ACE
H
69
11.809
−17.683
25.955
1.00
46.88
O


HETATM
128
CH3
ACE
H
69
13.141
−15.743
25.924
1.00
48.05
C


ATOM
129
N
GLN
H
70
11.778
−16.484
24.012
1.00
43.64
N


ATOM
130
CA
GLN
H
70
10.826
−17.283
23.246
1.00
42.45
C


ATOM
131
C
GLN
H
70
11.026
−18.818
23.243
1.00
48.79
C


ATOM
132
O
GLN
H
70
11.987
−19.340
22.644
1.00
49.83
O


ATOM
133
CB
GLN
H
70
10.668
−16.743
21.816
1.00
43.26
C


ATOM
134
CG
GLN
H
70
9.503
−17.399
21.019
1.00
48.43
C


ATOM
135
CD
GLN
H
70
8.133
−17.259
21.688
1.00
51.59
C


ATOM
136
NE2
GLN
H
70
7.769
−16.030
22.096
1.00
32.22
N


ATOM
137
OE1
GLN
H
70
7.418
−18.250
21.901
1.00
38.15
O


ATOM
138
N
LEU
H
71
10.077
−19.531
23.889
1.00
44.09
N


ATOM
139
CA
LEU
H
71
10.074
−20.993
23.930
1.00
42.82
C


ATOM
140
C
LEU
H
71
9.337
−21.541
22.690
1.00
47.21
C


ATOM
141
O
LEU
H
71
8.603
−20.812
22.008
1.00
46.65
O


ATOM
142
CB
LEU
H
71
9.507
−21.552
25.254
1.00
42.99
C


ATOM
143
CG
LEU
H
71
10.264
−21.176
26.550
1.00
48.52
C


ATOM
144
CD1
LEU
H
71
9.369
−21.316
27.773
1.00
48.02
C


ATOM
145
CD2
LEU
H
71
11.512
−22.045
26.736
1.00
53.09
C


ATOM
146
N
ASP
H
72
9.557
−22.813
22.379
1.00
45.35
N


ATOM
147
CA
ASP
H
72
8.966
−23.458
21.213
1.00
45.33
C


ATOM
148
C
ASP
H
72
7.805
−24.369
21.558
1.00
46.72
C


ATOM
149
O
ASP
H
72
7.847
−25.031
22.589
1.00
44.98
O


ATOM
150
CB
ASP
H
72
10.057
−24.179
20.397
1.00
47.71
C


ATOM
151
CG
ASP
H
72
10.805
−23.219
19.472
1.00
73.93
C


ATOM
152
OD1
ASP
H
72
11.558
−22.358
19.986
1.00
75.90
O


ATOM
153
OD2
ASP
H
72
10.576
−23.278
18.231
1.00
87.47
O1−


ATOM
154
N
LEU
H
73
6.766
−24.394
20.694
1.00
44.17
N


ATOM
155
CA
LEU
H
73
5.598
−25.260
20.839
1.00
44.82
C


ATOM
156
C
LEU
H
73
5.949
−26.725
20.585
1.00
49.95
C


ATOM
157
O
LEU
H
73
5.343
−27.628
21.189
1.00
49.17
O


ATOM
158
CB
LEU
H
73
4.487
−24.829
19.868
1.00
44.08
C


ATOM
159
CG
LEU
H
73
3.484
−23.817
20.345
1.00
45.47
C


ATOM
160
CD1
LEU
H
73
2.433
−23.649
19.295
1.00
45.34
C


ATOM
161
CD2
LEU
H
73
2.807
−24.251
21.651
1.00
38.54
C


ATOM
162
N
PHE
H
74
6.897
−26.941
19.644
1.00
47.70
N


ATOM
163
CA
PHE
H
74
7.378
−28.265
19.211
1.00
48.65
C


ATOM
164
C
PHE
H
74
8.752
−28.089
18.510
1.00
75.49
C


ATOM
165
O
PHE
H
74
9.100
−26.937
18.126
1.00
77.08
O


ATOM
166
CB
PHE
H
74
6.340
−28.932
18.271
1.00
49.99
C


ATOM
167
CG
PHE
H
74
5.819
−28.030
17.171
1.00
50.87
C


ATOM
168
CD1
PHE
H
74
6.502
−27.909
15.963
1.00
52.73
C


ATOM
169
CD2
PHE
H
74
4.661
−27.281
17.352
1.00
52.30
C


ATOM
170
CE1
PHE
H
74
6.047
−27.044
14.972
1.00
52.86
C


ATOM
171
CE2
PHE
H
74
4.230
−26.379
16.375
1.00
53.68
C


ATOM
172
CZ
PHE
H
74
4.918
−26.281
15.186
1.00
51.51
C


ATOM
173
OXT
PHE
H
74
9.469
−29.102
18.345
1.00
100.17
O1−


TER
174

PHE
H
74


HETATM
175
O
HOH
H
86
5.592
−15.725
23.553
1.00
33.16
O


END









A free energy decomposition analysis (see Material and Methods for details) of this complex was performed (FIG. 2B) and the most important interactions are similar to the initial complex 1OK7, as expected. The canonical sequence LF advantageously replaces the LGL sequence in C-ter of the peptide (FIG. 2B). The P6 peptide acetyl group also forms two hydrogen bonds with the Nα of residues R365 and L366 of the β monomer which probably account for the 10 fold increase in stability of the P6 peptide as compared to P5 (Table 1). Despite its reduced size, the P6 peptide therefore has an increased affinity for the β-clamp with respect to the original peptide P1.
1.2.3. Design of Non-Natural Peptides Ligands with Increased Binding Affinity.
P6 was further used as a lead to introduce modifications aimed at increasing the affinity of the ligand for the β clamp. Because the natural ligand binds to the pocket essentially through hydrophobic interactions, the aim was to extend the network of such interactions. A first set of modifications concerned position 2, where the leucine residue was replaced by a cyclohexyl-L-alanyl group (Cha) (P7, table 4 and table 7). An initial modeling analysis, using programs MCSS and SEED, indicated that this modification provides a Van der Waals energy contribution two-fold higher than that with the natural L3 residue, and is the most efficient group tested (Table 4). It also results in a 6 fold increase in the interaction, as measured by SPR (Table 4 and Table 7, compare P6 and P7). Attempts to increase the side chain length resulted in a drastic reduction of the affinity (Table 4 and Table 7, P8) while other modifications like homoleucyl (hLeu) or neopentylglycyl (NptGly, also called tertiomethylbutylglycine, tBMG) did not yield any gain in affinity (Table 4 and Table 7, P9 and P10), probably indicating that the area available for an efficient interaction is limited.







TABLE 4







Effect of site-specific modifications of


the second residue on the interaction


 of peptide with the E. coli β clamp.












#
sequence
IC50 (μM)
SEQ ID No:
















P6
AcQLDLF
1.12
6



 



P7
Ac Q ChaDLF
0.17
7



 



P8
Ac Q hCha DLF
82.8
8



 



P9
Ac Q hLeu DLF
0.74
9



 



P10
Ac Q NptGly DLF
0.99
10







Cha: cyclohexylalanine,



hCha: homocyclohexylalanine,



hLeu: homoleucine,



NptGly: neopentylglycyl.






A second set of similar modifications was introduced at position L4, but no increase in affinity was observed as compared to P7 (Table 5).







TABLE 5







Effect of site-specific modifications  


of the L4, residue on the interaction 


of peptide with the E. coli β clamp.












#
sequence
IC50 (μM)
SEQ ID No:







P6
AcQLDLF
1.12
 6



 



P7
Ac Q Cha DLF
0.17
 7



 



P16
Ac Q Cha D hLeu F
0.23
16



 



P17
Ac Q Cha D Cha F
0.31
17



 



P18
Ac Q Cha D hCha F
0.72
18



 



P19
Ac Q ChaD NptGly F
5.63
19










Finally, several modifications were introduced on the terminal phenylalanine benzyl ring (Table 6). The binding affinity was found to increase significantly with the size of the ring substituent (p-methyl<p-chloro<p-bromo<3,4-dichloro)(see P11, P12, P13, and P14, respectively). An IC50 value of 70 nM was measured for the 3,4-dichlorophenylalanine containing peptide (P14), which represent a 15, 115 and 4 fold increase as compared to P6, P1 and the full PolIV enzyme, respectively (Table 6 and Table 7). To the contrary, replacement of the terminal phenylalanine by a cyclohexyl-L-alanyl group (Cha), a tryptophan or a 2-amino-tetradecanoic acid (Atda) (P20, P21 and P22, Table 6) led to a decreased affinity.







TABLE 6







Effect of modifications on the 


terminal phenylalanine benzyl ring   


on the interaction of peptide with  


the E. coli β clamp.












#
sequence
IC50 (μM)
SEQ ID No:
















P6
AcQLDLF
1.12
6



 



P11
Ac-Q Cha DL pMeF
0.26
11



 



P12
Ac-Q Cha DL pClF
0.16
12



 



P13
Ac-Q Cha DL pBrF
0.10
13



 



P14
Ac-Q Cha DL diClF
0.072
14



 



P20
Ac-Q Cha DL Cha
0.41
20



 



P21
Ac-Q Cha DLW
0.64
21



 



P22
Ac-Q Cha DL Atda
3.72
22










The interactions of the various peptides with the β ring were also evaluated using a polymerase competition assay where primer elongation performed by the polIV DNA polymerase is challenged by various concentrations of the peptides to be tested17. While the β independent activity of the polymerase is insensitive to the peptides, indicating that they have no direct effect on the enzyme activity, the β dependent elongation activity is differentially inhibited depending on the affinity of the peptide for its target (FIG. 4). A quantitative analysis of the biochemical assays is shown in FIG. 7. Although this approach is less sensitive than SPR, a good correlation is observed between the two methods (FIG. 4).







TABLE 7







Influence of the C-terminal tripeptide sequence


and effect of site specific modifications on 


the interaction of peptide with the E. coli β


clamp, as measured by SPR experiments.















Ki
ΔG 
Seq



se-
IC50
(106 
(Kcal/
Id


#
quence
(μM)
M−1)
mol)
No:















PolIV

0.29
4.7
−9.09



 


P1
RQLVLGL
8.85
0.15
−7.06
 1


 


P2
RQLVLL
21.53
0.0063
−6.54
 2


 


P3
RQLVLF
8.62
0.15
−7.04
 3


 


P4
RQLVFL
256
Ø
Ø
 4


 


P5
QLDLF
12.44
0.11
−6.87
 5


 


P6
AcQLDLF
1.12
1.2
−8.22
 6


 


P7
Ac Q ChaDLF
0.17
8.01
−9.42
 7


 


P8
Ac Q hCha DLF
82.8
16.4
−5.74
 8


 


P9
Ac Q Hol DLF
0.74
1.84
−8.54
 9


 


P10
Ac Q NptGly DLF
0.99
1.36
−8.36
10


 


P11
Ac-Q Cha DL pMeF
0.26
8.43
−9.44
11


 


P12
Ac-Q Cha DL pClF
0.16
13.7
−9.73
12


 


P13
Ac-Q Cha DL pBrF
0.096
13.49
−9.71
13


 


P14
Ac-Q Cha DL diClF
0.077
17
−9.85
14





Ø: not determined.


Ki = (1 + KA[β])/IC50.


ΔG = −RT ln Ki.


PolIV: E coli DNA polymerase IV.






The following table 8 presents the analytical data concerning the most relevant peptides described in this study.








TABLE 8







Sequence and analytical data of C-terminal


peptides of the E. coli DNA polymerase IV


and analogues (related to FIG. 1 and 


table 7).
















PU-

MS 





HPLC
RITY

FOUND
SEQ


Pep-

tR
[%]
CALC.
[M + 
ID


tide
compound
(min)
HPLC
MASS
H+]
No:
















P1
H-Arg-Gln-Leu-Val-
11.97
98.2
799.0
799.9
 1



Leu-Gly-Leu-OH—







 


P2
H-Arg-Gln-Leu-Val-
11.50
93.0
740.5
741.9
 2



Leu-Leu-OH







 


P3
H-Arg-Gln-Leu-Val-
12.13
92.1
774.5
775.8
 3



Leu-Phe-OH







 


P4
H-Arg-Gln-Leu-Val-
12.02
92.5
774.5
775.5
 4



Phe-Leu-OH







 


P5
H-Gln-Leu-Asp-Leu-
12.55
90
634.3
634.8
 5



Phe-OH







 


P6
Ac-Gln-Leu-Asp-Leu-
13.25
92.5
676.3
677.3
 6



Phe-OH







 


P7
Ac-Gln-Cha-Asp-Leu-
15.12
95
716.8
717.7
 7



Phe-OH







 


P8
Ac-Gln-hCha-Asp-Leu-
16.23
50.93
730.8
731.4
 8



Phe-OH







 


P9
Ac-Gln-Hol-Asp-Leu-
14.23
90
690.8
691.0
 9



Phe-OH







 


P10
Ac-Gln-NptGly-Asp-
13.99
94
702.8
691.7
10



Leu-Phe-OH







 


P11
Ac-Gln-Cha-Asp-Leu-
15.88
97
716.4
732.7
11



pMePhe-OH







 


P12
Ac-Gln-Cha-Asp-Leu-
16.27
93
736.3
752.7
12



pClPhe-OH







 


P13
Ac-Gln-Cha-Asp-Leu-
16.45
95
794.3
799.8
13



pBrPhe-OH







 


P14
Ac-Gln-Cha-Asp-Leu-
17.18
91
784.3
786.9
14



(3,4-di-Cl)Phe-OH





Cha: beta-cyclohexyl-L-alanyl;


hCha: L-homoCha;


Hol: L-homoleucyl;


NptGly: neopentylglycyl;


pMePhe: 4-methyl-L-phenylalanyl;


pClPhe: 4-chloro-L-phenylalanyl;


pBrPhe: 4-bromo-L-phenylalanyl;


(3,4-di-Cl)Phe: 3,4-dichloro-L-phenylalanyl






1.2.4. Crystal Structure of the Modified Peptides β Ring Complexes.
Peptides P12 and P14 were co-crystallized with the β ring. The structures were solved by molecular replacement at 2.6 Å and 1.95 Å resolution respectively, using the 1OK7 structure as a search model. As for the previous P6-β complex, both complexes crystallized in space group P1 but with one ring per asymmetric unit (Table 3). Each monomer of the rings binds a peptide, and both ligands adopt a similar configuration (rmsd=0.70 Å and 0.78 Å for the peptides atoms of P12 and P14, respectively), indicating that the modified peptides essentially adopt the same conformation and location in the binding pocket. The Cha moiety is located at the same position as the L3 residue of P6 but extends its interaction further within a hydrophobic pocket shaped by the P363, V344, M362 and R365 β residues (FIG. 5A) and interacts with the last three residues. These extra interactions probably account for the increased affinity of P7 as compared to P6 (table 1). Additionally, the Nα of this modified residue interacts with the Cα=O of P363. The p-chloro and 3,4-dichloro F residues of P12 and P14 are almost superimposed (FIG. 5B) and interact with β residues T172, L177 and V247. The chlorine atom in meta position in P14 establishes an halogen bond with the hydroxyl oxygen of T172, with contact distance and angle in good agreement with previously published data (d=3.17 Å and θ=148.71°)20. In contrast, the para chlorine atoms of both P12 and P14 do not establish such type of interaction because the distances with adjacent oxygen atoms of β residues are too large.
The atomic coordinates P12 and P14, co-crystalized with the β clamp, are disclosed in the following Tables 9 and 10. These tables indicate the atomic coordinates of the peptides and of the binding site of the β clamp (residues≦5 Å from the ligand). The other residues have the same positions as in the previously determined structure (PDB 1OK7) also described in U.S. Pat. No. 7,635,583.







TABLE 9





Atomic coordinates of P12 residues and of the residues involved in the binding of P12


to the β clamp, in the crystal of P12 peptide co-crystallized with the β ring.


























ATOM
1
NH1
ARG
A
152
−13.878
13.827
24.977
1.00
41.13
N1+


TER
2

ARG
A
152


ATOM
3
CD2
LEU
A
155
−9.376
16.306
22.697
1.00
33.78
C


TER
4

LEU
A
155


ATOM
5
CB
THR
A
172
−5.579
13.660
24.332
1.00
10.80
C


ATOM
6
CG2
THR
A
172
−6.162
15.062
24.379
1.00
3.00
C


ATOM
7
OG1
THR
A
172
−6.396
12.784
25.111
1.00
11.87
O


TER
8

THR
A
172


ATOM
9
N
GLY
A
174
−9.375
11.662
24.270
1.00
22.35
N


ATOM
10
CA
GLY
A
174
−10.197
11.573
25.471
1.00
21.94
C


ATOM
11
C
GLY
A
174
−9.904
10.341
26.321
1.00
26.11
C


ATOM
12
O
GLY
A
174
−10.187
10.331
27.522
1.00
24.70
O


ATOM
13
N
HIS
A
175
−9.341
9.286
25.699
1.00
23.67
N


ATOM
14
CA
HIS
A
175
−9.001
8.008
26.347
1.00
23.91
C


ATOM
15
C
HIS
A
175
−7.479
7.869
26.584
1.00
24.57
C


ATOM
16
O
HIS
A
175
−7.046
7.395
27.635
1.00
24.56
O


ATOM
17
CB
HIS
A
175
−9.546
6.822
25.519
1.00
25.86
C


ATOM
18
CG
HIS
A
175
−10.971
7.005
25.080
1.00
30.11
C


ATOM
19
CD2
HIS
A
175
−11.486
7.140
23.838
1.00
32.30
C


ATOM
20
ND1
HIS
A
175
−12.005
7.082
25.997
1.00
32.23
N


ATOM
21
CE1
HIS
A
175
−13.107
7.245
25.288
1.00
31.59
C


ATOM
22
NE2
HIS
A
175
−12.845
7.286
23.985
1.00
32.09
N


ATOM
23
N
ARG
A
176
−6.685
8.290
25.610
1.00
18.69
N


ATOM
24
CA
ARG
A
176
−5.234
8.264
25.702
1.00
18.59
C


ATOM
25
C
ARG
A
176
−4.604
9.609
25.304
1.00
24.08
C


ATOM
26
O
ARG
A
176
−5.276
10.463
24.713
1.00
23.83
O


ATOM
27
N
LEU
A
177
−3.347
9.832
25.715
1.00
19.54
N


ATOM
28
CA
LEU
A
177
−2.653
11.088
25.503
1.00
18.04
C


ATOM
29
CB
LEU
A
177
−2.873
11.987
26.745
1.00
17.92
C


ATOM
30
CG
LEU
A
177
−1.963
13.211
26.971
1.00
21.84
C


ATOM
31
CD1
LEU
A
177
−2.328
14.347
26.066
1.00
21.30
C


ATOM
32
CD2
LEU
A
177
−2.004
13.663
28.416
1.00
22.36
C


TER
33

LEU
A
177


ATOM
34
CA
PRO
A
242
−8.284
20.266
27.211
1.00
10.30
C


ATOM
35
C
PRO
A
242
−7.012
20.416
28.076
1.00
16.74
C


ATOM
36
CB
PRO
A
242
−8.798
18.838
27.188
1.00
11.44
C


ATOM
37
CG
PRO
A
242
−8.164
18.255
25.944
1.00
16.16
C


ATOM
38
CD
PRO
A
242
−8.332
19.361
24.955
1.00
11.62
C


ATOM
39
N
ASP
A
243
−7.224
20.463
29.391
1.00
14.24
N


ATOM
40
CA
ASP
A
243
−6.222
20.608
30.438
1.00
14.52
C


ATOM
41
C
ASP
A
243
−5.454
19.282
30.618
1.00
21.86
C


ATOM
42
O
ASP
A
243
−5.842
18.424
31.416
1.00
25.37
O


ATOM
43
N
TYR
A
244
−4.471
19.055
29.748
1.00
15.95
N


ATOM
44
CA
TYR
A
244
−3.688
17.827
29.764
1.00
15.04
C


ATOM
45
CB
TYR
A
244
−2.900
17.645
28.437
1.00
14.90
C


TER
46

TYR
A
244


ATOM
47
NH1
ARG
A
246
−9.401
15.913
36.324
1.00
21.44
N1+


ATOM
48
CB
VAL
A
247
−4.905
13.902
32.710
1.00
29.59
C


ATOM
49
CG1
VAL
A
247
−5.480
12.551
33.104
1.00
29.67
C


ATOM
50
CG2
VAL
A
247
−6.024
14.870
32.343
1.00
29.06
C


TER
51

VAL
A
247


ATOM
52
O
PHE
A
278
−11.396
−2.943
23.858
1.00
27.53
O


TER
53

PHE
A
278


ATOM
54
N
ASN
A
320
−7.095
−1.216
26.969
1.00
26.00
N


ATOM
55
CB
ASN
A
320
−8.050
−0.275
24.887
1.00
19.44
C


ATOM
56
CG
ASN
A
320
−8.114
−0.289
23.378
1.00
25.87
C


ATOM
57
ND2
ASN
A
320
−9.197
0.247
22.826
1.00
19.30
N


ATOM
58
OD1
ASN
A
320
−7.179
−0.699
22.685
1.00
16.42
O


TER
59

ASN
A
320


ATOM
60
CD2
TYR
A
323
−6.890
3.229
24.491
1.00
20.56
C


ATOM
61
CE2
TYR
A
323
−8.089
3.767
24.028
1.00
21.88
C


ATOM
62
cz
TYR
A
323
−8.254
4.015
22.675
1.00
30.51
C


ATOM
63
OH
TYR
A
323
−9.422
4.548
22.199
1.00
34.44
O


TER
64

TYR
A
323


ATOM
65
O
SER
A
343
−8.420
0.538
36.986
1.00
43.35
O


ATOM
66
CA
VAL
A
344
−9.412
2.967
38.022
1.00
37.68
C


ATOM
67
C
VAL
A
344
−8.749
4.286
37.541
1.00
39.37
C


ATOM
68
O
VAL
A
344
−9.430
5.162
37.004
1.00
37.60
O


ATOM
69
CB
VAL
A
344
−10.716
3.216
38.843
1.00
41.47
C


ATOM
70
CG1
VAL
A
344
−11.660
2.025
38.749
1.00
41.11
C


TER
71

VAL
A
344


ATOM
72
CB
SER
A
346
−4.248
7.581
33.672
1.00
36.35
C


TER
73

SER
A
346


ATOM
74
O
VAL
A
360
−2.861
8.288
27.992
1.00
21.29
O


ATOM
75
CG1
VAL
A
360
−2.051
9.515
31.551
1.00
22.57
C


TER
76

VAL
A
360


ATOM
77
N
MET
A
362
−5.771
5.847
29.897
1.00
24.01
N


ATOM
78
CA
MET
A
362
−6.824
5.610
30.866
1.00
25.90
C


ATOM
79
C
MET
A
362
−7.277
4.123
30.792
1.00
30.54
C


ATOM
80
O
MET
A
362
−7.461
3.593
29.689
1.00
29.30
O


ATOM
81
CB
MET
A
362
−7.994
6.560
30.587
1.00
29.26
C


ATOM
82
CG
MET
A
362
−8.871
6.790
31.773
1.00
34.65
C


ATOM
83
SD
MET
A
362
−8.107
7.741
33.104
1.00
40.06
s


ATOM
84
CE
MET
A
362
−9.291
7.371
34.427
1.00
36.64
C


ATOM
85
N
PRO
A
363
−7.464
3.423
31.935
1.00
28.36
N


ATOM
86
CA
PRO
A
363
−7.885
2.016
31.872
1.00
27.57
C


ATOM
87
C
PRO
A
363
−9.367
1.784
31.553
1.00
32.79
C


ATOM
88
O
PRO
A
363
−10.161
2.723
31.445
1.00
30.76
O


ATOM
89
CB
PRO
A
363
−7.534
1.508
33.276
1.00
29.22
C


ATOM
90
CG
PRO
A
363
−7.733
2.646
34.141
1.00
33.99
C


ATOM
91
CD
PRO
A
363
−7.288
3.852
33.338
1.00
30.19
C


ATOM
92
N
MET
A
364
−9.735
0.505
31.409
1.00
33.12
N


ATOM
93
CA
MET
A
364
−11.116
0.063
31.233
1.00
34.28
C


ATOM
94
C
MET
A
364
−11.510
−0.892
32.363
1.00
38.91
C


ATOM
95
CB
MET
A
364
−11.392
−0.494
29.827
1.00
37.35
C


ATOM
96
CG
MET
A
364
−10.591
−1.704
29.437
1.00
42.38
C


ATOM
97
SD
MET
A
364
−10.644
−1.982
27.634
1.00
48.21
s


ATOM
98
CE
MET
A
364
−12.355
−2.535
27.415
1.00
45.40
C


ATOM
99
N
ARG
A
365
−12.802
−0.893
32.770
1.00
36.16
N


ATOM
100
CA
ARG
A
365
−13.345
−1.727
33.857
1.00
54.18
C


ATOM
101
O
ARG
A
365
−13.715
−3.627
32.448
1.00
15.77
O


ATOM
102
CB
ARG
A
365
−14.777
−1.280
34.190
1.00
54.40
C


ATOM
103
CG
ARG
A
365
−14.987
−0.808
35.628
1.00
61.81
C


ATOM
104
CD
ARG
A
365
−16.323
−0.093
35.795
1.00
67.50
C


ATOM
105
NE
ARG
A
365
−16.385
0.668
37.050
1.00
70.43
N


ATOM
106
CZ
ARG
A
365
−17.177
1.718
37.255
1.00
69.73
C


ATOM
107
NH1
ARG
A
365
−17.980
2.151
36.291
1.00
47.59
N1+


ATOM
108
NH2
ARG
A
365
−17.162
2.350
38.424
1.00
45.96
N


TER
109

ARG
A
365


HETATM
110
O
HOH
A
397
−12.948
−0.572
24.569
1.00
24.99
O


HETATM
111
O
HOH
A
422
−15.206
5.096
38.996
1.00
47.81
O


HETATM
112
O
HOH
A
433
−8.020
1.912
27.730
1.00
9.25
O


HETATM
113
O
HOH
C
24
−11.224
1.716
24.238
1.00
30.48
O


HETATM
114
C
ACE
C
367
−14.628
0.704
29.591
1.00
35.56
C


HETATM
115
O
ACE
C
367
−14.221
1.136
30.667
1.00
30.52
O


HETATM
116
CH3
ACE
C
367
−15.612
−0.454
29.524
1.00
29.87
C


ATOM
117
N
GLN
C
368
−14.292
1.401
28.359
1.00
30.56
N


ATOM
118
CA
GLN
C
368
−13.424
2.699
28.412
1.00
25.45
C


ATOM
119
C
GLN
C
368
−13.778
3.790
29.559
1.00
32.24
C


ATOM
120
O
GLN
C
368
−14.830
4.442
29.488
1.00
32.94
O


ATOM
121
CB
GLN
C
368
−13.142
3.398
26.993
1.00
29.55
C


ATOM
122
CG
GLN
C
368
−11.862
4.252
26.986
1.00
29.78
C


ATOM
123
CD
GLN
C
368
−10.609
3.491
27.350
1.00
23.65
C


ATOM
124
NE2
GLN
C
368
−9.792
3.893
28.463
1.00
24.50
N


ATOM
125
OE1
GLN
C
368
−10.426
2.411
26.809
1.00
27.24
O


TER
126

GLN
C
368


HETATM
127
N
ALC
C
369
−12.687
4.147
30.488
1.00
28.64
N


HETATM
128
CA
ALC
C
369
−12.514
5.508
30.964
1.00
29.77
C


HETATM
129
C
ALC
C
369
−11.636
6.300
29.995
1.00
30.06
C


HETATM
130
O
ALC
C
369
−10.844
5.719
29.263
1.00
32.57
O


HETATM
131
CB
ALC
C
369
−11.826
5.516
32.374
1.00
29.41
C


HETATM
132
CG
ALC
C
369
−12.462
4.800
33.632
1.00
27.38
C


HETATM
133
CD1
ALC
C
369
−11.611
3.659
34.076
1.00
26.63
C


HETATM
134
CD2
ALC
C
369
−13.892
4.431
33.398
1.00
29.06
C


HETATM
135
CE1
ALC
C
369
−12.176
2.854
35.208
1.00
26.40
C


HETATM
136
CE2
ALC
C
369
−14.487
3.799
34.618
1.00
36.93
C


HETATM
137
cz
ALC
C
369
−13.665
2.730
35.276
1.00
30.08
C


ATOM
138
N
ASP
C
370
−11.984
7.629
29.488
1.00
34.94
N


ATOM
139
CA
ASP
C
370
−12.550
8.830
30.301
1.00
34.00
C


ATOM
140
C
ASP
C
370
−11.474
9.729
31.120
1.00
30.52
C


ATOM
141
O
ASP
C
370
−11.377
9.697
32.347
1.00
25.64
O


ATOM
142
CB
ASP
C
370
−14.029
8.546
30.909
1.00
38.94
C


ATOM
143
CG
ASP
C
370
−15.012
8.727
29.752
1.00
40.34
C


ATOM
144
OD1
ASP
C
370
−16.182
8.114
29.765
1.00
59.35
O


ATOM
145
OD2
ASP
C
370
−14.418
9.287
28.668
1.00
30.05
O1−


ATOM
146
N
LEU
C
371
−10.560
10.539
30.269
1.00
32.14
N


ATOM
147
CA
LEU
C
371
−9.624
11.571
30.769
1.00
33.35
C


ATOM
148
C
LEU
C
371
−10.269
12.914
31.093
1.00
36.56
C


ATOM
149
O
LEU
C
371
−9.703
13.671
31.882
1.00
38.88
O


ATOM
150
CB
LEU
C
371
−8.465
11.790
29.754
1.00
29.71
C


ATOM
151
CG
LEU
C
371
−7.489
10.663
29.542
1.00
32.92
C


ATOM
152
CD1
LEU
C
371
−6.748
10.753
28.242
1.00
21.26
C


ATOM
153
CD2
LEU
C
371
−6.637
10.343
30.752
1.00
23.07
C


TER
154

LEU
C
371


HETATM
155
N
200
C
372
−11.514
13.352
30.399
1.00
31.07
N


HETATM
156
CA
200
C
372
−12.085
14.735
30.622
1.00
32.30
C


HETATM
157
C
200
C
372
−13.594
14.731
30.756
1.00
37.62
C


HETATM
158
O
200
C
372
−14.200
15.440
31.810
1.00
41.91
O


HETATM
159
CB
200
C
372
−11.640
15.817
29.612
1.00
30.34
C


HETATM
160
CG
200
C
372
−10.185
15.711
29.135
1.00
25.13
C


HETATM
161
CD1
200
C
372
−9.909
15.210
27.863
1.00
20.80
C


HETATM
162
CD2
200
C
372
−9.161
16.082
29.995
1.00
22.71
C


HETATM
163
CE1
200
C
372
−8.592
15.088
27.455
1.00
22.75
C


HETATM
164
CE2
200
C
372
−7.842
15.960
29.593
1.00
22.47
C


HETATM
165
CZ
200
C
372
−7.572
15.459
28.324
1.00
26.57
C


HETATM
166
CL
200
C
372
−5.931
15.288
27.829
1.00
37.22
CL


HETATM
167
OXT
200
C
372
−14.362
13.982
29.828
1.00
44.19
O


END
















TABLE 10





Atomic coordinates of P14 residues and of the residues involved in the binding of P14


to the β clamp, in the crystal of P14 peptide co-crystallized with the β ring.


























ATOM
1
NH1
ARG
A
152
−13.000
14.676
25.384
1.00
41.87
N1+


TER
2

ARG
A
152


ATOM
3
CD2
LEU
A
155
−8.771
17.218
22.511
1.00
32.76
C


TER
4

LEU
A
155


ATOM
5
CB
THR
A
172
−5.062
14.269
24.018
1.00
29.73
C


ATOM
6
CG2
THR
A
172
−5.795
15.620
24.122
1.00
26.88
C


ATOM
7
OG1
THR
A
172
−5.728
13.271
24.786
1.00
28.48
O


TER
8

THR
A
172


ATOM
9
N
GLY
A
174
−8.679
12.290
24.030
1.00
26.79
N


ATOM
10
CA
GLY
A
174
−9.451
12.319
25.270
1.00
28.12
C


ATOM
11
C
GLY
A
174
−9.367
11.041
26.069
1.00
32.47
C


ATOM
12
O
GLY
A
174
−9.848
10.979
27.199
1.00
31.98
O


ATOM
13
N
HIS
A
175
−8.715
10.024
25.494
1.00
30.75
N


ATOM
14
CA
HIS
A
175
−8.481
8.734
26.139
1.00
30.80
C


ATOM
15
C
HIS
A
175
−6.976
8.530
26.320
1.00
30.70
C


ATOM
16
O
HIS
A
175
−6.557
8.002
27.335
1.00
29.87
O


ATOM
17
CB
HIS
A
175
−9.131
7.602
25.325
1.00
32.00
C


ATOM
18
CG
HIS
A
175
−10.595
7.832
25.079
1.00
35.24
C


ATOM
19
CD2
HIS
A
175
−11.259
7.998
23.916
1.00
37.80
C


ATOM
20
ND1
HIS
A
175
−11.496
7.948
26.130
1.00
37.36
N


ATOM
21
CE1
HIS
A
175
−12.677
8.158
25.576
1.00
37.24
C


ATOM
22
NE2
HIS
A
175
−12.587
8.212
24.247
1.00
38.20
N


ATOM
23
N
ARG
A
176
−6.162
8.980
25.337
1.00
25.93
N


ATOM
24
CA
ARG
A
176
−4.709
8.899
25.425
1.00
23.72
C


ATOM
25
C
ARG
A
176
−4.078
10.213
25.009
1.00
27.35
C


ATOM
26
O
ARG
A
176
−4.648
10.950
24.212
1.00
24.69
O


ATOM
27
N
LEU
A
177
−2.885
10.489
25.534
1.00
25.59
N


ATOM
28
CA
LEU
A
177
−2.204
11.738
25.266
1.00
25.33
C


ATOM
29
CB
LEU
A
177
−2.431
12.671
26.488
1.00
25.95
C


ATOM
30
CG
LEU
A
177
−1.853
14.080
26.442
1.00
29.85
C


ATOM
31
CD1
LEU
A
177
−2.845
15.053
27.040
1.00
31.56
C


TER
32

LEU
A
177


ATOM
33
CA
PRO
A
242
−7.701
20.996
27.178
1.00
30.57
C


ATOM
34
C
PRO
A
242
−6.353
21.180
27.870
1.00
35.62
C


ATOM
35
O
PRO
A
242
−5.277
21.201
27.239
1.00
31.98
O


ATOM
36
CB
PRO
A
242
−8.234
19.577
27.285
1.00
32.60
C


ATOM
37
CG
PRO
A
242
−7.827
18.915
26.040
1.00
37.83
C


ATOM
38
N
ASP
A
243
−6.480
21.417
29.176
1.00
34.91
N


ATOM
39
CA
ASP
A
243
−5.451
21.642
30.163
1.00
35.21
C


ATOM
40
C
ASP
A
243
−4.812
20.307
30.500
1.00
38.35
C


ATOM
41
O
ASP
A
243
−5.193
19.613
31.455
1.00
37.41
O


ATOM
42
N
TYR
A
244
−3.865
19.930
29.652
1.00
34.76
N


ATOM
43
CA
TYR
A
244
−3.152
18.662
29.719
1.00
34.71
C


ATOM
44
C
TYR
A
244
−2.245
18.565
30.939
1.00
39.89
C


ATOM
45
CB
TYR
A
244
−2.366
18.444
28.424
1.00
34.64
C


TER
46

TYR
A
244


ATOM
47
NH1
ARG
A
246
−9.362
18.335
34.959
1.00
56.46
N1+


ATOM
48
CB
VAL
A
247
−4.800
14.658
32.377
1.00
45.61
C


ATOM
49
CG1
VAL
A
247
−6.175
15.272
32.614
1.00
44.77
C


ATOM
50
CG2
VAL
A
247
−4.260
15.077
31.013
1.00
45.81
C


TER
51

VAL
A
247


ATOM
52
O
GLY
A
318
−7.101
−0.481
31.341
1.00
28.54
O


ATOM
53
N
PHE
A
319
−6.439
−2.005
29.819
1.00
25.76
N


ATOM
54
CA
PHE
A
319
−6.062
−1.039
28.794
1.00
25.29
C


ATOM
55
C
PHE
A
319
−6.489
−1.419
27.399
1.00
26.95
C


ATOM
56
O
PHE
A
319
−6.510
−2.591
27.065
1.00
25.62
O


ATOM
57
CB
PHE
A
319
−4.532
−0.862
28.741
1.00
26.12
C


ATOM
58
CG
PHE
A
319
−3.977
0.004
29.823
1.00
26.52
C


ATOM
59
CD1
PHE
A
319
−4.229
1.368
29.839
1.00
30.64
C


ATOM
60
CE1
PHE
A
319
−3.731
2.174
30.867
1.00
31.78
C


ATOM
61
N
ASN
A
320
−6.726
−0.410
26.564
1.00
23.99
N


ATOM
62
CA
ASN
A
320
−6.956
−0.616
25.155
1.00
24.11
C


ATOM
63
C
ASN
A
320
−5.554
−0.926
24.635
1.00
27.35
C


ATOM
64
O
ASN
A
320
−4.654
−0.066
24.695
1.00
24.65
O


ATOM
65
CB
ASN
A
320
−7.569
0.636
24.508
1.00
23.58
C


ATOM
66
CG
ASN
A
320
−7.625
0.595
23.004
1.00
34.43
C


ATOM
67
ND2
ASN
A
320
−8.716
1.031
22.426
1.00
24.70
N


TER
68

ASN
A
320


ATOM
69
CB
TYR
A
323
−3.987
3.146
23.786
1.00
24.01
C


ATOM
70
CG
TYR
A
323
−5.324
3.669
23.316
1.00
27.27
C


ATOM
71
CD2
TYR
A
323
−6.338
3.953
24.224
1.00
27.85
C


ATOM
72
CE2
TYR
A
323
−7.551
4.485
23.803
1.00
28.48
C


ATOM
73
cz
TYR
A
323
−7.760
4.733
22.459
1.00
39.89
C


TER
74

TYR
A
323


ATOM
75
O
SER
A
343
−8.221
1.305
36.401
1.00
35.47
O


ATOM
76
CA
VAL
A
344
−9.112
3.668
37.518
1.00
31.45
C


ATOM
77
C
VAL
A
344
−8.465
5.023
37.143
1.00
35.04
C


ATOM
78
O
VAL
A
344
−9.164
5.988
36.815
1.00
35.77
O


ATOM
79
CB
VAL
A
344
−10.398
3.839
38.375
1.00
35.78
C


ATOM
80
CG1
VAL
A
344
−11.101
2.498
38.569
1.00
35.22
C


TER
81

VAL
A
344


ATOM
82
CB
SER
A
346
−3.897
8.687
33.543
1.00
33.22
C


ATOM
83
OG
SER
A
346
−3.189
9.493
34.475
1.00
39.59
O


TER
84

SER
A
346


ATOM
85
C
VAL
A
360
−1.708
8.441
28.606
1.00
31.20
C


ATOM
86
O
VAL
A
360
−2.317
8.921
27.649
1.00
30.76
O


ATOM
87
CB
VAL
A
360
−0.897
10.498
29.926
1.00
31.93
C


ATOM
88
CG1
VAL
A
360
−2.031
10.304
30.938
1.00
32.54
C


ATOM
89
C
VAL
A
361
−4.106
6.199
29.791
1.00
28.46
C


ATOM
90
CG1
VAL
A
361
−3.751
4.254
27.465
1.00
28.23
C


ATOM
91
N
MET
A
362
−5.362
6.501
29.534
1.00
27.43
N


ATOM
92
CA
MET
A
362
−6.410
6.303
30.527
1.00
28.06
C


ATOM
93
C
MET
A
362
−6.834
4.822
30.434
1.00
30.78
C


ATOM
94
O
MET
A
362
−7.011
4.331
29.325
1.00
29.96
O


ATOM
95
CB
MET
A
362
−7.591
7.238
30.220
1.00
30.87
C


ATOM
96
CG
MET
A
362
−8.518
7.477
31.376
1.00
34.33
C


ATOM
97
SD
MET
A
362
−7.736
8.227
32.828
1.00
37.58
s


ATOM
98
CE
MET
A
362
−9.105
8.087
34.005
1.00
34.59
C


ATOM
99
N
PRO
A
363
−6.981
4.091
31.569
1.00
26.83
N


ATOM
100
CA
PRO
A
363
−7.383
2.677
31.502
1.00
25.64
C


ATOM
101
C
PRO
A
363
−8.853
2.456
31.064
1.00
31.65
C


ATOM
102
O
PRO
A
363
−9.600
3.409
30.801
1.00
29.62
O


ATOM
103
CB
PRO
A
363
−7.148
2.203
32.941
1.00
26.32
C


ATOM
104
CG
PRO
A
363
−7.447
3.380
33.754
1.00
30.73
C


ATOM
105
CD
PRO
A
363
−6.797
4.505
32.971
1.00
26.98
C


ATOM
106
N
MET
A
364
−9.251
1.179
30.970
1.00
28.59
N


ATOM
107
CA
MET
A
364
−10.606
0.744
30.632
1.00
28.63
C


ATOM
108
C
MET
A
364
−11.156
0.010
31.848
1.00
33.94
C


ATOM
109
O
MET
A
364
−10.376
−0.602
32.572
1.00
30.49
O


ATOM
110
CB
MET
A
364
−10.559
−0.264
29.473
1.00
30.86
C


ATOM
111
CG
MET
A
364
−10.480
0.379
28.110
1.00
34.27
C


ATOM
112
SD
MET
A
364
−10.177
−0.880
26.856
1.00
37.20
S


ATOM
113
CE
MET
A
364
−11.844
−1.575
26.686
1.00
34.67
C


ATOM
114
N
ARG
A
365
−12.490
0.042
32.084
1.00
36.79
N


ATOM
115
CA
ARG
A
365
−13.039
−0.701
33.225
1.00
38.26
C


ATOM
116
C
ARG
A
365
−13.003
−2.213
32.924
1.00
41.53
C


ATOM
117
O
ARG
A
365
−13.457
−2.647
31.869
1.00
41.37
O


ATOM
118
CB
ARG
A
365
−14.441
−0.209
33.655
1.00
40.76
C


ATOM
119
CG
ARG
A
365
−14.900
−0.881
34.954
1.00
49.70
C


ATOM
120
CD
ARG
A
365
−16.194
−0.321
35.483
1.00
57.33
C


ATOM
121
NE
ARG
A
365
−15.967
0.754
36.451
1.00
62.68
N


ATOM
122
CZ
ARG
A
365
−16.403
1.998
36.291
1.00
83.24
C


ATOM
123
NH1
ARG
A
365
−17.085
2.335
35.202
1.00
79.01
N1+


ATOM
124
NH2
ARG
A
365
−16.159
2.916
37.217
1.00
72.47
N


TER
125

ARG
A
365


HETATM
126
O
HOH
A
393
−12.258
0.479
23.904
1.00
30.49
O


HETATM
127
O
HOH
A
434
−11.324
4.559
22.768
1.00
39.46
O


HETATM
128
O
HOH
A
453
−10.378
2.373
23.903
1.00
29.67
O


HETATM
129
O
HOH
A
463
−8.977
20.736
30.414
1.00
61.87
O


HETATM
130
O
HOH
A
490
−11.833
19.233
26.362
1.00
59.30
O


HETATM
131
O
HOH
A
494
−9.709
19.924
32.759
1.00
46.54
O


HETATM
132
O
HOH
A
516
−16.291
−3.291
31.343
1.00
50.13
O


HETATM
133
O
HOH
C
45
−6.988
2.274
27.402
1.00
24.52
O


HETATM
134
O
HOH
C
219
−14.432
8.577
32.316
1.00
42.19
O


HETATM
135
O
HOH
C
236
−12.880
18.057
33.132
1.00
45.93
O


HETATM
136
C
ACE
C
367
−14.148
1.368
29.378
1.00
30.45
C


HETATM
137
O
ACE
C
367
−13.835
1.713
30.502
1.00
36.32
O


HETATM
138
CH3
ACE
C
367
−15.035
0.134
29.141
1.00
23.39
C


ATOM
139
N
GLN
C
368
−13.746
1.957
28.290
1.00
33.32
N


ATOM
140
CA
GLN
C
368
−12.873
3.100
28.115
1.00
32.69
C


ATOM
141
C
GLN
C
368
−13.283
4.349
28.853
1.00
33.34
C


ATOM
142
O
GLN
C
368
−14.330
4.821
28.622
1.00
32.62
O


ATOM
143
CB
GLN
C
368
−12.617
3.443
26.659
1.00
30.16
C


ATOM
144
CG
GLN
C
368
−11.470
4.502
26.458
1.00
29.48
C


ATOM
145
CD
GLN
C
368
−10.087
4.218
27.096
1.00
29.80
C


ATOM
146
NE2
GLN
C
368
−9.514
3.231
26.666
1.00
25.11
N


ATOM
147
OE1
GLN
C
368
−9.495
4.916
28.005
1.00
31.46
O


TER
148

GLN
C
368


HETATM
149
N
ALC
C
369
−12.348
4.783
29.710
1.00
32.73
N


HETATM
150
CA
ALC
C
369
−12.500
6.004
30.548
1.00
34.14
C


HETATM
151
C
ALC
C
369
−11.878
7.192
29.752
1.00
36.64
C


HETATM
152
O
ALC
C
369
−10.984
7.069
28.869
1.00
32.46
O


HETATM
153
CB
ALC
C
369
−11.739
5.830
31.889
1.00
34.55
C


HETATM
154
CG
ALC
C
369
−12.265
4.559
32.650
1.00
35.64
C


HETATM
155
CD1
ALC
C
369
−13.808
4.654
32.962
1.00
39.49
C


HETATM
156
CD2
ALC
C
369
−11.323
4.206
33.835
1.00
33.24
C


HETATM
157
CE1
ALC
C
369
−14.214
3.964
34.302
1.00
41.77
C


HETATM
158
CE2
ALC
C
369
−11.911
3.020
34.514
1.00
36.86
C


HETATM
159
CZ
ALC
C
369
−13.067
3.588
35.295
1.00
40.10
C


ATOM
160
N
ASP
C
370
−12.406
8.334
30.145
1.00
40.84
N


ATOM
161
CA
ASP
C
370
−12.054
9.670
29.629
1.00
43.94
C


ATOM
162
C
ASP
C
370
−10.905
10.238
30.494
1.00
41.92
C


ATOM
163
O
ASP
C
370
−10.900
10.094
31.706
1.00
39.99
O


ATOM
164
CB
ASP
C
370
−13.194
10.609
29.791
1.00
48.03
C


ATOM
165
CG
ASP
C
370
−14.398
10.233
29.003
1.00
55.27
C


ATOM
166
OD1
ASP
C
370
−14.373
9.336
28.084
1.00
56.63
O


ATOM
167
OD2
ASP
C
370
−15.398
10.873
29.360
1.00
59.41
O1−


ATOM
168
N
LEU
C
371
−9.929
10.917
29.882
1.00
43.04
N


ATOM
169
CA
LEU
C
371
−8.785
11.787
30.606
1.00
42.42
C


ATOM
170
C
LEU
C
371
−9.335
12.911
31.330
1.00
47.23
C


ATOM
171
O
LEU
C
371
−8.808
13.271
32.377
1.00
47.55
O


ATOM
172
CB
LEU
C
371
−7.621
12.265
29.726
1.00
37.30
C


ATOM
173
CG
LEU
C
371
−6.599
11.206
29.251
1.00
32.23
C


ATOM
174
CD1
LEU
C
371
−5.841
11.622
27.996
1.00
27.72
C


ATOM
175
CD2
LEU
C
371
−5.712
10.688
30.370
1.00
32.57
C


TER
176

LEU
C
371


HETATM
177
N
ZCL
C
372
−10.331
13.589
30.796
1.00
51.48
N


HETATM
178
CA
ZCL
C
372
−11.007
14.833
31.271
1.00
56.21
C


HETATM
179
C
ZCL
C
372
−12.356
14.343
31.982
1.00
59.74
C


HETATM
180
O
ZCL
C
372
−13.391
14.104
31.274
1.00
58.69
O


HETATM
181
CB
ZCL
C
372
−11.091
16.085
30.288
1.00
55.89
C


HETATM
182
CG
ZCL
C
372
−9.672
16.291
29.799
1.00
57.05
C


HETATM
183
CD1
ZCL
C
372
−9.347
15.682
28.574
1.00
57.40
C


HETATM
184
CD2
ZCL
C
372
−8.663
17.020
30.494
1.00
57.44
C


HETATM
185
CE1
ZCL
C
372
−8.088
15.803
28.089
1.00
59.91
C


HETATM
186
CE2
ZCL
C
372
−7.347
17.172
29.984
1.00
57.91
C


HETATM
187
CZ
ZCL
C
372
−7.057
16.515
28.746
1.00
57.04
C


HETATM
188
CLE1
ZCL
C
372
−7.803
15.045
26.635
1.00
66.69
CL


HETATM
189
CLZ
ZCL
C
372
−5.507
16.525
27.966
1.00
50.27
CL


HETATM
190
OXT
ZCL
C
372
−12.339
14.160
33.254
1.00
61.54
O


END









1.2.5. Thermodynamic Analysis of the Pseudo Peptides Interaction with the Ring.
ITC experiments were conducted on selected peptides in order to determine the thermodynamics parameters of their interaction with the ring (Table 11 and FIG. 8). Although the Kd values determined in these experimental conditions were slightly different from those obtained by SPR, the same tendency was observed for all peptides (Table 11). The largest differences are observed for low affinity peptides (P1, P3, P6) while, for higher affinity peptides (P7, P11, P12, P13 and P14), both techniques yielded similar values. The introduction of modifications, Cha group in position 2 and p-methyl, p-chloro and p-bromo groups on F5, increases the affinity of the ligand, reaching respectively about 380, 100, 65 and 150 nM range, as compared to the 1-2 μM affinity of the reference natural peptide P1 (Table 11). A strong correlation is observed between ΔH and ΔS values (FIG. 8), which reflects an enthalpy-entropy compensatory effect, already observed in other systems21, 22. This correlation accounts for the small ΔG variation among the various peptides (FIG. 8). As reported earlier23, this correlation suggests that the observed desolvation of the pocket upon ligand interaction plays a major role in the binding process.







TABLE 11







ITC experiments on selected β binding peptides.















Beta
Pep


Kd
IC50
ΔH
ΔS (cal/
ΔG


(μM)
(μM)
Name
N
(nM)
SPR (nM)
(cal/Mol)
mol/deg)
(Kcal/mol)


















30
400
P1 (SEQ ID
1.33
1579
8850
−4087
12.8
−7.9




No: 1)










RQLVLGL
1.31
2012

−5500
7.6
−7.7


 


30
400
P3 (SEQ ID 
1.54
2816
8620
−5769
6.05
−7.5




No: 3)










RQLVLF
1.45
2320

−5699
6.6
−7.6


 


30
400
P6 (SEQ ID 
1.11
820
1120
−1.13 104
−10.4
−8.1




No: 6)










AcQLDLF
1.14
613

−1.11 104
−9.08
−8.3


 


30
400
P7 (SEQ ID
0.74
246
 170
−2.13 104
−41.2
−8.9




No: 7)










AcQChaDLF
0.76
222

−2.05 104
−38.5
−8.9


 


20
400
P11 (SEQ ID
0.95
401
 260
−1.48 104
−20.4
−8.6




No: 11)










AcQChaDLpMeF
0.95
362

−1.44 104
−19.0
−8.6


 


20
400
P12 (SEQ ID
1.02
89
 160
−1.5  104
−18.1
−9.6




No: 12)










AcQChaDLpClF
1.06
122

−1.43 104
−16.3
−9.4


 


20
400
P13 (SEQ ID
0.85
136
  96
−1.81 104
−29.3
−9.3




No: 13










AcQChaDLpBrF
0.83
167

−1.52 104
−20.2
−9.1


 


20
400
P14 (SEQ ID
0.91
73
  77
−1.89 104
−30.8
−9.6




No: 14)










AcQChaDLdiClF
0.90
55

−1.81 104
−27.6
−9.8





ΔG = ΔH-TΔS.


All experiments were performed at 25° C. Results from SPR experiments (IC50) are


added for comparison purposes.


N: number of binding sites per β monomer.






The effect of the various modifications introduced in the peptides can be estimated from the ΔΔG values (see Table 12). The Cha moiety in position 2 contributes to the increased interaction by −0.66 kcal/mol as compared to the natural pentapeptide P6. While the introduction of a methyl substitution on the ring of the terminal phenylalanine residue is detrimental to the affinity (+0.25 kcal/mol), halogen modification results in a graduate effect on ligand affinity (p-bromo<p-chloro<3,4-dichloro). The chloro modification in para position contributes for the main part to the increased affinity (−0.6 kcal/mol). This contribution can result partly from an increased hydrophobic character of the halogen modified F residue24, as well as from dehydration of both peptide and sub site 125 and reorganization of water molecules as observed by comparing free and bound β interacting pockets (FIG. 1AB). In comparison, the second chlorine atom (meta position) in P14 only contributes for −0.2 kcal/mol and the para-bromo modification contribution to the binding energy is two times lower (−0.28 kcal/mol) than the corresponding para-chloro modification.







TABLE 12







effects of modifications introduced in the various pentapeptides


measured from the ΔΔG values extracted from ITC experiments data. The


P6 sequence (AcQLDLF) is chosen as a reference. ΔΔG = ΔGj − ΔGi.









ΔGi/ΔGj



















P6/
P7/
P7/
P7/
P7/
P11/
P11/
P11/
P12/
P13/
P13/



P7
P11
P12
P14
P13
P12
P14
P13
P14
P12
P14






















Compared
L/
F/
F/
F/
F:
pMeF/
pMeF/diClF
pMeF/
pClF/
pBrF/
pBrF/


residues
Cha
pMeF
pClF
diClF
pBrF
pClF

pBrF
diClF
pClF
diClF


ΔΔG
−0.66
+0.25
−0.6
−0.8
−0.28
−0.85
−1.05
−0.53
−0.2
−0.32
−0.52


(Kcal/mol)









1.3. Discussion.
1.3.1. The Fully Efficient β Binding Pocket is Formed Upon Ligand Binding.
Many cellular factors involved in replication and genome integrity survey processes require a peptide mediated interaction with the replisome sliding clamp in order to fulfill their function. This interaction has been structurally fully characterized in prokaryotes16, 17, 18 and eucaryotes2 26. In our previously published structure (1OK7), the β ring interacts with only one peptide, leaving one binding pocket free17. This gives the opportunity to compare the structure of a peptide-free versus a peptide-bound pocket and to get insights into the dynamic of the pocket upon peptide binding. Although the general structures of the free or bound pockets are similar, as estimated by the Cα chain conformation (rmsd=0.36 Å), the side chains of several residues undergo major movements (FIG. 6), notably residues M362 and S346 side chains which are displaced in a concerted way, and residue R365. The concerted shift of M362 and S346 side chains probably is a structural marker for the presence of a peptide ligand in the binding pocket. In absence of the ligand, these residues adopt a so-called close conformation where the M362 side chain is oriented toward residue H175 and separates subsite 1 and subsite 2 (PDB IDs 2POL, 1MMI and 1OK7 (monomer A)) (FIG. 6). Alternatively, in all β ring bound structures (1UNN, 1OK7 (monomer B), 3D1E, 3D1F), the residues are shifted by 180° in an open conformation, allowing the opening of a cleft joining the two subsites (FIG. 1BC). One exception is found in the structure of β co-crystallized with the Pol II peptide (3D1E) where no ligand is observed in monomer B, although the two residues adopt an open conformation18. This may result from a partial occupancy of the pocket, making difficult the detection of the peptide.
Residue R365 is also shifted by an angle of 46° toward residue L366, triggering the opening of a platform shaped by R365, P363, M362 and V344, where the L3 residue of the peptide locates (FIG. 1BC). The global dynamic of this structural modification has been modeled, showing that, as the ligand binds into the pocket, a groove forms that joins subsite 1 and subsite 2, in which the extended peptide can adapt.
The correlation between side chains orientations and the presence of a peptide in the binding pocket suggests that these two side chains might play a strategic function in the ligand binding process. The pocket could adopt two configurations: a closed configuration where the M362 side chains lies in the path between the two subsites of the pocket, thus impeding the formation of an efficient binding site (FIG. 1A). Alternatively, an open configuration where the M362 side chains shifts by about 180°, allowing ligand binding into the groove that joins the two subsites, as well as the opening of the platform so that the peptide establishes optimal interactions. At present, it is not known if the fully efficient binding pocket is readily available at the surface of the protein, or is structured by the binding of a specific ligand, according to a bona fide induced fit model. Previous observations for a ligand binding site on the cytokine IL-227 reveal that a portion of the binding site is adaptive and can form a hydrophobic channel upon ligand binding. A similar adaptive process could occur for the β binding pocket and would ensure the binding specificity of ligand proteins. Although the details of the dynamic process of this binding site formation are not known yet, this observation is likely to have major implications for the design of high affinity ligands.
1.3.2. Structure-Based Design of High Affinity Ligands.
The inventors have studied the interaction of various peptides with the E. coli processivity ring. Starting from the natural sequence of the Pol IV DNA polymerase interacting peptide (P1, RQLVLGL, SEQ ID No: 1), they have sequentially modified the peptide in order to increase its interaction strength and concomitantly to limit its size. The affinities were measured by biochemical assays, SPR and ITC, and despite a difference in sensitivity of these various techniques, the relative affinities of the peptides were similar. From the P1-β complex (PDB 1OK7), a minimal acetylated peptide (P6, AcQLDLF) was first delineated and found to bind about eight times tighter than P1. Then, the crystallographic structure of the P1-β and P6-β complexes were used together with modelling informations to guide modifications at specific positions (corresponding to residues L2 and F5 of P6, AcQLDLF), resulting in a dramatic increase of the modified peptides affinity for the β ring interacting pocket. This gain essentially results from an increase in hydrophobic interactions. A first improvement was achieved by the introduction of a cyclohexylalanyl residue at the P6-L2 position. Although position 2 does not correspond to a major binding pocket on the β-clamp, the simple modification to a cyclohexylalanyl residue (P7) improved the affinity by a factor 6 with respect to the P6 peptide (Table 1), indicating that shallow secondary sites can still prove useful in a global optimization scheme.
The other interesting gain was achieved by the addition of halogen atoms on the benzyl ring of the F residue. Interestingly, the chlorine atom when introduced at the meta position, forms a halogen bond that further strengthens the interaction. P14, which combines both the Cha and a 3,4-dichloro phenylalanine residue, displays the highest affinity, around 60 (±10) nM as measured by ITC, which represents a 10 to 15 fold increase in binding as compared to P6 and a 4 fold increased as compared to the whole PolIV DNA polymerase. The double F ring substitution contributes for −0.8 kcal/mol to the overall interaction. The para-chloro substitution provides most of the contribution to the binding while the meta-chloro, involved in a halogen bond, only contributes for one fourth of the total free energy.
Interestingly, halogen substituted aromatic ligands were also recently independently identified for the β-clamp using a chemical library screening strategy. A compound, named RU67, was identified as a β ligand inhibiting the E. coli pol III enzyme with a Ki of 10 μM18. It contains a di-bromo substituted aromatic ring which is deeply inserted in sub site 1 and almost superimposes with the peptide L4 residue of the Pol IV binding peptide. One bromine atom does not form any interaction while the other forms a halogen bond with β residue T172 (d=3.02 Å and θ=133.21°). This latter interaction is similar to that observed for the chlorine atom in meta position of F5 in P14.
This specific positioning of halogen substituted ligands in subsite 1 of the β-clamp contrasts with the wider range of positions occupied by natural amino acids in the same pocket. Indeed, the comparison of different structures of β in complex with various natural peptides (1OK7, 3D1E, 3D1F)17, 18 reveals that, while most of the peptide residues adopt the same overall conformation within the pocket, the position of the last C-terminal residues of the interacting sequence varies in subsite 1. This suggests that no specific interaction is established with specific β residue, but instead that this hydrophobic interaction, delocalized in all subsite 1, contributes mainly to paste the peptide onto the β surface.
In conclusion, the structure-based approach described herein allows the design of ligands that bind two orders of magnitude tighter than the natural peptide P1, reaching the 10−8 M range, and 4 fold more than the Pol IV enzyme. This increase in affinity relies both on chemical substitutions introduced on the peptide that increase the hydrophobic interactions and on the fact that the bidentate interaction of the ligand in the binding pocket is retained. This interaction mode promotes the modeling of an efficient binding site, possibly through an induced-fit process.
Further designs of high affinity ligands should also take into account the dynamic nature of the binding site formation. These observations are likely to have major implications for the development of new antibiotic compounds.
Example 2
Synthesis and Functional Characterization of Further Peptides Derived from P7 (SEQ ID No: 7), P12 (SEQ ID No: 12), P14 (SEQ ID No: 14), P23-P30 (SEQ ID Nos: 23-30)
Compounds P23 to P28 (SEQ ID No: 23 to SEQ ID No: 28), which are analogues of P7 and P14, have been prepared to (i) remove carboxylic side functions and monitor the influence of R and guanidino groups and conformational constraint. Compounds P23 to P28 have been prepared as previously reported in example 1 starting from Fmoc-Phe Wang resin or from o-chlorotrityl chloride resin. The linear precursor of P29 (Ac-RQChaKLF-OH, P27, SEQ ID No: 27) is prepared as described in example 1. In the case of compound P30 (SEQ ID No: 30), the linear precursor P28 (SEQ ID No: 28) requires the use of a beta amino acid (Fmoc-beta-HPhe-OH) which is commercially available from Sigma-Aldrich. In the case of P29 and P30, lactam formation between the amino group of the lysine side chain and the C-terminal carboxylic function was performed using standard methods as described in the literature (Robert C. Reid, Giovanni Abbenante, Stephen M. Taylor, and David P. Fairlie; J. Org. Chem., 2003, 68 (11), pp 4464-4471). The cyclization which requires the activation and coupling of a peptidyl-Arg residue (P27 or P28) was carried out in DMF at room temperature using BOP as a coupling agent and diisopropylethylamine as the base and monitored by analytical
RP-HPLC. Briefly, a solution of the fully deprotected peptide 27 or 28 (1 equivalent) in DMF (10-1M) and diisopropylethylamine (5.5 equivalent) was stirred at room temperature (about 20° C.) until homogeneous. Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate or BOP reagent (1.08 equivalent) was added, and the solution was stirred at room temperature (about 20° C.) for 2 h. A saturated Bicarbonate solution was added and the solid product was filtered off, washed on the filter with ether, and dried under high vacuum. The crude product was dissolved in 50% CH3CN/50% water (1 L) and purified by C18 RP-HPLC. The peak containing the cyclic peptide was collected and lyophilized.
Example 3
New Peptides Derived from P14, SPR Experiments
Based on the crystals analysis, the inventors have used peptide P14 (Bu29) as a scaffold to derive new variants with the aim of deciphering new properties to the ligand such as increased affinity, increased solubility, or increased cell penetration.







TABLE 13







sequences of the P14 derivative peptides.










Peptide





name
Sequence
Mw
SEQ ID No:













P14
Ac Q Cha D L diClF

14


 


P14-4
Ac R Q Cha N L diClF
940
36


 


P14-5
Cin R Q Cha N L diClF
1028
37


 


P14-6
Ac R Q Cha R L diClF
980.5
38


 


P14-7
Ac R Q Cha R L F
913
39


 


P14-8
Ac R Q Cha K L F
886
40





Ac: acetyl group;


Cin: cinnamoyl group.






The rationale directing the design of these peptides was 1) to extend the interaction of the N-terminal part of the peptide with the edge of the binding pocket, 2) to take advantage of the position of the D residue of peptide P14, that points towards the solvent, to introduce positively charged residues that will increase the solubility and the membrane penetrating capabilities of the peptide.
The affinity of these peptides for the binding pocket was measured by SPR, by defining the concentration of ligand that challenges 50% of the binding of the natural peptide P1 (RQLVLGL), thus defining a Ki. Results are given in table 14







TABLE 14







relative affinity of P14 derivative pep- 


tides for the beta ring binding pocket, 


as measured by SPR experiments.










Peptide

Ki
SEQ ID


name
Sequence
(nM)
No:













P7
Ac Q Cha D L F
170
 7


 


P14
Ac Q Cha D L diClF
85
14


 


P14-4
Ac R Q Cha N L diClF
insoluble
36


 


P14-5
Cin R Q Cha N L diClF
3250
37


 


P14-6
Ac R Q Cha R L diClF
>10 000
38


 


P14-7
Ac R Q Cha R L F
>10 000
39


 


P14-8
Ac R Q Cha K L F
904
40









CONCLUSIONS
None of the peptides is a better binder than P14. One of them, P14-8, still bind the target with a 1 μM affinity.
As compared to P14 (SEQ ID No: 14), the introduction of R and K residues in the sequence (P14-8, SEQ ID No: 40) results in a 10 fold decrease in affinity.
The diClF to F substitution results in a 2 fold decrease in affinity (compare P14 and P7). Thus the R and K subtitutions in peptide P14-8 may contribute to a 5 fold decrease (1000/200) of the affinity as compared to P14.
P14-7 and P14-8 differ from each other by the 5th residue (R for #7 and K for #8). Comparison of the relative affinity of these two peptides suggests that K residue is a better choice to maintain affinity. Moreover, comparing P7 and P14-7, which differ by R1 and R5 residues, indicates an affinity ratio of at least 50 (#10 000/200).
Altogether, this suggests that the K5 residue in P14-8 may contribute to decrease the affinity by a factor of 10 (P14-7/P7=5 and P14-8/P7=50).
REFERENCES


    1. Kong, X. P., Onrust, R., O'Donnell, M. & Kuriyan, J. (1992). Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69, 425-37.
    2. Gulbis, J. M., Kelman, Z., Hurwitz, J., O'Donnell, M. & Kuriyan, J. (1996). Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell 87, 297-306.
    3. Shamoo, Y. & Steitz, T. A. (1999). Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex. Cell 99, 155-66.
    4. Matsumiya, S., Ishino, Y. & Morikawa, K. (2001). Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus. Protein Sci 10, 17-23.
    5. Johnson, A. & O'Donnell, M. (2005). Cellular DNA replicases: components and dynamics at the replication fork. Annu Rev Biochem 74, 283-315.
    6. Fuchs, R. P. & Fujii, S. (2007). Translesion synthesis in Escherichia coli: lessons from the NarI mutation hot spot. DNA Repair (Amst) 6, 1032-41.
    7. Prakash, S., Johnson, R. E. & Prakash, L. (2005). Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu Rev Biochem 74, 317-53.
    8. Kleczkowska, H. E., Marra, G., Lettieri, T. & Jiricny, J. (2001). hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication foci. Genes Dev 15, 724-36.
    9. Lopez de Saro, F. J., Marinus, M. G., Modrich, P. & O'Donnell, M. (2006). The beta sliding clamp binds to multiple sites within MutL and MutS. J Biol Chem 281, 14340-9.
    10. Dalrymple, B. P., Kongsuwan, K., Wijffels, G., Dixon, N. E. & Jennings, P. A. (2001). A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems. Proc Natl Acad Sci USA 98, 11627-32.
    11. Wagner, J., Fujii, S., Gruz, P., Nohmi, T. & Fuchs, R. P. (2000). The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity. EMBO Rep 1, 484-8.
    12. Becherel, O. J., Fuchs, R. P. & Wagner, J. (2002). Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E. coli cells. DNA Repair (Amst) 1, 703-8.
    13. Lenne-Samuel, N., Wagner, J., Etienne, H. & Fuchs, R. P. (2002). The processivity factor beta controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo. EMBO Rep 3, 45-9.
    14. Heltzel, J. M., Maul, R. W., Scouten Ponticelli, S. K. & Sutton, M. D. (2009). A model for DNA polymerase switching involving a single cleft and the rim of the sliding clamp. Proc Natl Acad Sci USA 106, 12664-9.
    15. Jeruzalmi, D., Yurieva, O., Zhao, Y., Young, M., Stewart, J., Hingorani, M., O'Donnell, M. & Kuriyan, J. (2001). Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III. Cell 106, 417-28.
    15a. Jeruzalmi D, O'Donnell M, Kuriyan J. (2001) Crystal structure of the processivity clamp loader gamma (gamma) complex of E. coli DNA polymerase III. Cell 106, 429-41.
    16. Bunting, K. A., Roe, S. M. & Pearl, L. H. (2003). Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp. Embo J 22, 5883-92.
    17. Burnouf, D. Y., Olieric, V., Wagner, J., Fujii, S., Reinbolt, J., Fuchs, R. P. & Dumas, P. (2004). Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases. J Mol Biol 335, 1187-97.
    18. Georgescu, R. E., Yurieva, O., Kim, S. S., Kuriyan, J., Kong, X. P. & O'Donnell, M. (2008). Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp. Proc Natl Acad Sci USA 105, 11116-21.
    19. Wagner, J., Etienne, H., Fuchs, R. P., Cordonnier, A. & Burnouf, D. (2009). Distinct beta-clamp interactions govern the activities of the Y family PolIV DNA polymerase. Mol Microbiol 74, 1143-51.
    20. Auffinger, P., Hays, F. A., Westhof, E. & Ho, P. S. (2004). Halogen bonds in biological molecules. Proc Natl Acad Sci USA 101, 16789-94.
    21. Rinnenthal, J., Klinkert, B., Narberhaus, F. & Schwalbe, H. (2010). Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution. Nucleic Acids Res 38, 3834-47.
    22. Strazewski, P. (2002). Thermodynamic correlation analysis: hydration and perturbation sensitivity of RNA secondary structures. J Am Chem Soc 124, 3546-54.
    23. Ackroyd, P. C., Cleary, J. & Glick, G. D. (2001). Thermodynamic basis for sequence-specific recognition of ssDNA by an autoantibody. Biochemistry 40, 2911-22.
    24. Voth, A. R. & Ho, P. S. (2007). The role of halogen bonding in inhibitor recognition and binding by protein kinases. Curr Top Med Chem 7, 1336-48.
    25. Camacho, C. J., Kimura, S. R., DeLisi, C. & Vajda, S. (2000). Kinetics of desolvation-mediated protein-protein binding. Biophys J 78, 1094-105.
    26. Vijayakumar, S., Chapados, B. R., Schmidt, K. H., Kolodner, R. D., Tainer, J. A. & Tomkinson, A. E. (2007). The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase. Nucleic Acids Res 35, 1624-37.
    27. Arkin, M. R., Randal, M., DeLano, W. L., Hyde, J., Luong, T. N., Oslob, J. D., Raphael, D. R., Taylor, L., Wang, J., McDowell, R. S., Wells, J. A. & Braisted, A. C. (2003). Binding of small molecules to an adaptive protein-protein interface. Proc Natl Acad Sci USA 100, 1603-8.
    28. Goodman, M., Felix, A., Moroder, L. & Toniolo, C. H.-W. (2002). Synthesis of Peptides and Peptidomimetics. Methods of Organic Chemistry., Vol. E 22a-e: Thieme: Stuttgart, New York, 2002.
    29. Neimark, J. & Briand, J. P. (1993). Development of a fully automated multichannel peptide synthesizer with integrated TFA cleavage capability. Pept Res 6, 219-28.
    30. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. (1970). Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34, 595-8.
    31. Lafont, V., Schaefer, M., Stote, R., Altschuh, D. & Dejaegere, A. (2007). Protein-protein recognition and interaction hot spots in an antigen-antibody complex: free energy decomposition identifies “efficient amino acids. Proteins: Structure Function and Bioinformatics 67, 418-434.
    32. Kollman, P. A., Massova, I., Reyes, C., Kuhn, B., Huo, S., Chong, L., Lee, M., Lee, T., Duan, Y., Wang, W., Donini, O., Cieplak, P., Srinivasan, J. & Case, D. A. (2000). Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc. Chem. Res. 33, 889-97.
    33. Gohlke, H., Kiel, C. & Case, D. A. (2003). Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes. J Mol Biol 330, 891-913.
    34. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S. & M., K. (1983). CHARMM: A Program for Macromolecular Energy Minimization and Dynamics Calculations. J. Comp. Chem 4, 187-217.
    35. MacKerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D. & Karplus, M. (1998). All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102, 3586-3616.
    36. Hendsch, Z. S. & Tidor, B. (1999). Electrostatic interactions in the GCN4 leucine zipper: substantial contributions arise from intramolecular interactions enhanced on binding. Protein Sci 8, 1381-92.
    37. Gouda, H., Kuntz, I. D., Case, D. A. & Kollman, P. A. (2003). Free energy calculations for theophylline binding to an RNA aptamer: Comparison of MM-PBSA and thermodynamic integration methods. Biopolymers 68, 16-34.
    38. Miranker, A. & Karplus, M. (1991). Functionality maps of binding sites: a multiple copy simultaneous search method. Proteins 11, 29-34.
    39. Majeux, N., Scarsi, M., Apostolakis, J., Ehrhardt, C. & Caflisch, A. (1999). Exhaustive docking of molecular fragments with electrostatic solvation. Proteins 37, 88-105.
    40. Sirockin, F., Sich, C., Improta, S., Schaefer, M., Saudek, V., Froloff, N., Karplus, M. & Dejaegere, A. (2002). Structure activity relationship by NMR and by computer: a comparative study. J Am Chem Soc 124, 11073-84.
    41. Majeux, N., Scarsi, M. & Caflisch, A. (2001). Efficient electrostatic solvation model for protein-fragment docking. Proteins 42, 256-68.
    42. Biertumpfel, C., Basquin, J., Suck, D. & Sauter, C. (2002). Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr 58, 1657-9.
    43. Kabsch, W. (1993). Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Crystallogr 26, 795-800.
    44. Collaborative Computational Project, N. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr D50, 760-763.
    45. Adams, P. D., Grosse-Kunstleve, R. W., Hung, L. W., Loerger, T. R., McCoy, A. J. & al., e. (2002). PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr D50 58, 1948-1954.
    46. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr D50 60, 2126-2132.
    47. Brünger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kuntsleve, R. V. & al., e. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D 54, 905-921.
    48. Chen, V. B., Arendall, W. B. r., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W. & Richardson, J. S. R., D. C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D 66, 12-21.
    49. DeLano, W. L. (2008). The PyMOL Molecular Graphics System. DeLano Scientific LLC., Palo Alto, Calif., USA.
    50. Dallmann H G et al. (1995). DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Central role of tau in initiation complex assembly and in determining the functional asymmetry of holoenzyme. J Biol Chem. 270(49):29555-62.

## Claims

1. A compound of formula (I)




wherein
Gln is glutamine;
R is selected from the group consisting of a C1-12-alkyl group optionally substituted by a C6-10-aryl group, a C2-12-alkenyl group optionally substituted by a C6-10-aryl group, a C3-6-cycloalkyl group, a C6-10-aryl group optionally substituted by a C1-4-alkyl, and a C1-5-alkyl-(O—CH2—CH2)t— group with t being an integer from 0 to 20 inclusive;
R1 is the side chain of arginine or lysine;
R2 is a —(CH2)—C3-6-cycloalkyl group optionally substituted by a halogen and/or by a group selected from the group consisting of —NH2, —NH—CO—Ra, —CO2H, —NHRa and —NRaRb, wherein Ra and Rb are independently a C1-4-alkyl group;
R3 is selected from the group consisting of a C1-8-alkyl group, the side chain of arginine or lysine, —(CH2)q—CO2R7a, —(CH2)q—CO—NHR7b, —CH2OR8 and —(CH2)qNHR9, wherein
q is 1, 2, 3 or 4,
R7a is a hydrogen atom, a C1-8-alkyl group, a C4-12-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-12-alkenylene, forming together with R6 a lactone or a polyether ring,
R7b is a hydrogen atom, a C1-8-alkyl group, or —(CH2)q′—NH— with q′ being an integer between 2 and 8 inclusive and forming together with R6 a lactam,
R8 is a hydrogen atom, a C1-8-alkyl group, a C4-12-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-12-alkenylene, forming together with R6 a lactone or a polyether ring,
R9 is a hydrogen atom, or R9 together with R6 form a lactam;

R4 is a C1-8-alkyl group optionally substituted by a C3-6-cycloalkyl group, or a halogen-C1-4-alkyl group;
R5 is selected from the group consisting of a —(CH2)—C3-6-cycloalkyl group; —(CH2—CH2)—C3-6-cycloalkyl group; a —(CH2)—C6-10-aryl group optionally substituted by a halogen, a C1-2 alkyl group and/or a C1-2 alkoxy group; a —(CH2—CH2)—C6-10-aryl group optionally substituted by a halogen, a C1-2 alkyl group and/or a C1-2 alkoxy group; a —(CH2)—C6-10-heteroaryl group optionally substituted by a halogen and/or a C1-2 alkyl group; and a —(CH2—CH2)—C5-10-heteroaryl group optionally substituted by a halogen and/or a C1-2 alkyl group;
R6 is —CO2H, —CO2R10, —CO—NH2, —CO—NHR10, —OR10 when r is 1 or 2, —NH—CO—NHR10 when r is 1 or 2, or R6 is —CO—, —CO—O— or —O— and forms a lactam, a lactone, or a polyether ring with R7a, R7b, R8 or R9; wherein
R10 is a C1-8-alkyl group optionally substituted by a C6-10-aryl group; a C3-6-cycloalkyl group; a C6-10-aryl group optionally substituted by a halogen, a C1-2-alkyl group and/or a C1-2-alkoxy group;

m is 0 or 1;
n is an integer from 0 to 9 inclusive;
p is an integer from 0 to 10 inclusive;
r is 0, 1 or 2.

2. The compound of claim 1, wherein
R is a C1-8-alkyl group optionally substituted by a C6-10-aryl group, a C2-8-alkenyl group optionally substituted by a C6-10-aryl group or a C1-6-alkyl-(O—CH2—CH2)t— group with t being an integer from 0 to 10 inclusive.

3. The compound of claim 1, wherein
R is a C1-4-alkyl group optionally substituted by a C6-10-aryl group or a C2-4-alkenyl group optionally substituted by a C6-10-aryl group.

4. The compound of claim 1, wherein n is an integer comprised between 1 and 5 inclusive.

5. The compound of claim 1, wherein n is 0.

6. The compound of claim 1, wherein
R2 is a —(CH2)—C3-6-cycloalkyl group.

7. The compound of claim 1, wherein
R4 is a C1-5-alkyl group or a C1-2-alkyl group optionally substituted by a C3-6-cycloalkyl group.

8. The compound of claim 1, wherein
R5 is a —(CH2)—C6-10-aryl group optionally substituted by a halogen, a C1-2 alkyl group and/or a C1-2 alkoxy group.

9. The compound of claim 1, wherein
R3 is selected from the group consisting of a C1-8-alkyl group, the side chain of arginine or lysine, —(CH2)q—CO2R7a, —(CH2)q—CO—NHR7b, —CH2OR8, and —(CH2)qNHR9, wherein
q is 1, 2, 3, 4,
R7a is a hydrogen atom, or a C1-8-alkyl group,
R7b is a hydrogen atom, or a C1-8-alkyl group,
R8 is a hydrogen atom, a C1-8-alkyl group,
R9 is a hydrogen atom;


and
R6 is —CO2H, —CO2R10, —CO—NH2, —CO—NHR10, —OR10 when r is 1 or 2, —NH—CO—NHR10 when r is 1 or 2; wherein
R10 is a C1-8-alkyl group optionally substituted by a C6-10-aryl group; a C3-6-cycloalkyl group; a C6-10-aryl group optionally substituted by a halogen, a C1-2-alkyl group and/or a C1-2-alkoxy group.

10. The compound of claim 1, wherein
R3 is selected from the group consisting of the side chain of arginine, the side chain of lysine, —(CH2)q—CO2R7a and —(CH2)q—CO—NHR7b, wherein
q is 1, 2, 3 or 4,
R7a is a hydrogen atom, or a C1-8-alkyl group, and
R7b is a hydrogen atom, or a C1-8-alkyl group.

11. The compound of claim 1, wherein
R6 is —CO2H or —CO—NH2.

12. The compound of claim 1, wherein
R3 is selected from the group consisting of —(CH2)q—CO2R7a, —(CH2)q—CO—NHR7b, —CH2OR8, and —(CH2)qNHR9, wherein
q is 1, 2, 3 or 4,
R7a is a C4-8-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-8-alkenylene, forming together with R6 a lactone or a polyether ring,
R7b is —(CH2)q′—NH— with q′ being an integer from 2 to 8 inclusive and forming together with R6 a lactam,
R8 is a C4-8-alkylene group forming together with R6 a lactone or a polyether ring, or a C4-8-alkenylene, forming together with R6 a lactone or a polyether ring,
R9 together with R6 form a lactam;

R6 is —CO—, —CO—O— or —O— and forms a lactam, a lactone, or a polyether ring with R7a, R7b, R8 or R9.

13. The compound of claim 1, which is selected from the group consisting of Ac-Gln-Cha-Asp-Leu-Phe (SEQ ID NO:7), Ac-Gln-Cha-Asp-Leu-pMePhe (SEQ ID NO:11), Ac-Gln-Cha-Asp-Leu-pClPhe (SEQ ID NO:12), Ac-Gln-Cha-Asp-Leu-pBrPhe (SEQ ID NO:13), Ac-Gln-Cha-Asp-Leu-diClPhe (SEQ ID NO:14), Ac-Gln-Cha-Asp-hLeu-Phe (SEQ ID NO:16), and Ac-Gln-Cha-Asp-Cha-Phe (SEQ ID NO:17).

14. The compound of claim 1, wherein the affinity of said compound for the interacting pocket of the bacterial β ring is at least twice the affinity of the acetylated peptide of sequence AcQLDLF (SEQ ID NO:6) with said interacting pocket.

15. A pharmaceutical composition comprising, as an active agent, a compound according to claim 1.

16. A method of treating an infection by E. coli in a subject in need thereof comprising administering the compound of claim 1 to the subject.