Patent Application
20100035945
Pathogenic bacteria cause a variety of disease in humans, which manifest in a range of symptoms from mild to severe, and can lead to death. Worldwide infectious diseases are a leading cause of death. Pathogenic bacteria are of particular concern given the development of increased multi-drug resistance and horizontal transfer of resistance genes. This development of bacterial resistance to antibiotics is an ongoing and increasing problem. There is a continued need for new classes of antibiotics and, in particular, antibiotics that are less likely to lose efficacy due to resistance development by bacteria. The present invention provides a new class of antibiotics that interfere with DNA methylation by inhibiting DNA adenine methylase (“Dam”). Because Dam is required for virulence in a variety of bacteria, inhibiting Dam reduces virulence. Inhibitors of Dam are particularly beneficial as antibiotics because they do not affect mammalian cell DNA-MTases and, accordingly, have minimal toxicity for the host organism. In addition, because only bacterial virulence is reduced, the opportunity for bacteria to develop resistance to Dam inhibitors is also reduced.
DNA methylation is a process whereby methyl groups are added to DNA and provides a mechanism to control gene expression. Accordingly, DNA methylation plays an important role in a large number and variety of biological processes. DNA from most prokaryotes and eukaryotes contains the methylated bases 4-methylcyosine (N4mC), 5 methylcytosine (5mC) and 6-methyladenine (N6mA). Modifications by methylation are introduced after DNA replication by DNA methyltransferases (“MTases”). DNA MTases catalyze methyl group transfer from donor S-adenosyl-L-methionine (“AdoMet”) to produce S-adenosyl-L-homocysteine (AdoHcy) and methylated DNA (
While most prokaryote DNA MTases are components of restriction-modification systems and function as part of a phage defense mechanism, some MTases are not associated with cognate restriction enzymes; e.g. the E. coli DNA adenine MTase (Dam), which methylates an exocyclic amino nitrogen (N6) of the Adenosine in GATC sequence (
Dam methylation is important in prokaryotic DNA replication. For example, there is a cluster of GATC sites near the origin of replication of E. coli and Salmonella typhimurium, all of which are conserved between the two species. It is the hemimethylated GATC sites, produced immediately following DNA replication, that regulate the timing and targeting of a number of cellular functions (Messer & Noyer-Weidner, 1988). For example, SeqA specifically binds these hemimethylated GATC sites, causes delay of their full methylation (Guarne et al. 2002; Kang et al. 1999; Lu et al. 1994) and, in part, controls DNA replication.
DNA-adenine methylation at specific GATC sites plays a central role in bacterial gene expression, DNA replication, mismatch repair, and is essential for bacterial virulence for many Gram-negative bacteria. Dam methylation regulates the expression of certain genes in E. coli (Oshima et al. 2002; Lobner-Olesen et al. 2003), and the expression and secretion of Yop virulence proteins under non-permissive conditions in Yersinia pseudotuberculosis (Julio et al. 2002). The expression of pyelonephritis-associated pili (Pap) in uropathogenic E. coli is epigenetically controlled by the binding of the global regulator Lrp to a hemimethylated GATC site (Hernday et al. 2003). In addition, Dam methylation is important in the E. coli mismatch repair system formed by MutSI and MutH (Modrich, 1989; Yang, 2000). In contrast, DNA-adenine methylation has not been observed in humans or other higher eukaryotes.
The mechanism of DNA methylation and base flipping by the EcoDam enzyme has been extensively studied. EcoDam methylates DNA in a processive reaction, in which EcoDam transfers up to 55 methyl groups without dissociation from the DNA molecule (Urig et al., 2002). In such a mode of action, EcoDam exchanges AdoHcy for AdoMet while staying bound to the DNA duplex leading to a processive methylation of the DNA, a mechanism that also holds for other solitary MTases (i.e., no cognate restriction enzyme) (Berdis et al. 1998; Renbaum and Razin, 1992). In contrast, MTases belonging to a restriction-modification system often exhibit a distributive mechanism (as processive methylation of DNA interferes with the biological function of restriction-modification systems) (Jeltsch, 2002). The high processivity is essential to rapidly restore full methylation after replication.
DNA adenine methylation plays an essential role in bacterial virulence (Heithoff et al. 1999; Garcia-Del Portillo et al. 1999). The present invention, therefore, inhibits virulence by inhibiting Dam methylation. The involvement of Dam as a virulence factor was first described for Salmonella enterica serovar Typhimurium, where the dam mutant was out-competed by wildtype in establishing fatal infections in mice and where mice previously infected with the dam mutant were less susceptible to superinfection by the wildtype (Low et al. 2001). Salmonella is one of the most common enteric (intestinal) infections in the U.S. In some states (e.g. Georgia, Maryland) it is the most common, and overall it is the second most common, foodborne illness (usually slightly less frequent than a Campylobacter infection). According to the CDC, approximately 500 to 1,000 persons, or 31% of all food-related deaths are caused by Salmonella infections in the U.S. every year. Salmonella is a type of bacteria that causes typhoid fever and many other infections of intestinal origin. Typhoid fever, rare in the U.S., is caused by a particular strain designated Salmonella typhi. But illness due to other Salmonella strains, called “salmonellosis,” is common in the U.S. Today, the number of known strains (technically termed “serotypes” or “serovars”) of this bacterium total over 2,300 (from CDC web site). It was first shown in Salmonella typhimurium that Dam methylation regulates a bacterium's use of its armament of molecular tools to dodge the immune defenses of mammals. A dam− mutant was avirulent to mice at 10,000 times the LD50 of dam+ bacteria, although the mutant bacteria appeared to grow normally. Moreover, infecting mice with dam− mutant cells offered protection against further infection by wild type dam−. Accordingly, Dam is an appealing target for drug design (Heithoff et al. 1999; Low et al. 2001).
Yersinia Dam: Yersinia pestis is a species of bacteria that causes plague, an infection that leads to death quickly and that has caused several major epidemics in Europe and Asia over the last 2,000 years. One of the best known was called the Black Death because it turned the skin black. This plague epidemic in the 14th century killed more than one-third of the population of Europe within a few years. In some cities, up to 75 percent of the population died within days, with fever and ulcerated swellings on their skin. The last urban plague epidemic in the United States occurred in Los Angeles in 1925. Since then, an average of 13 cases of plague have been diagnosed each year, primarily in the Southwest, with about 80 percent occurring in the desert areas of New Mexico, Arizona or Colorado and about 9 percent in California. Worldwide, up to 3,000 cases of plague are reported to the World Health Organization each year. Plague is considered one of the most dangerous agents of biological warfare and could be utilized by terrorists in pneumonic form (identified as potential bioterrorism agents by the CDC).
E. coli Dam: Even though, E. coli is a major facultative inhabitant of the large intestine, it is one of the most frequent causes of some of the many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection, and traveler's diarrhea, and other clinical infections such as neonatal meningitis and pneumonia. There are hundreds of strains of this bacterium. One strain, Escherichia coli O157:H7, is an emerging cause of foodborne illness. It produces large quantities of one or more related, potent toxins that cause severe damage to the lining of the intestine. These toxins (verotoxin (VT), shiga-like toxin) are closely related or identical to the toxin produced by Shigella dysenteriae. Escherichia coli O157:H7 infection often leads to bloody diarrhea, and occasionally to kidney failure.
Klebsiella Dam: Although the role of Dam methylation in growth and virulence of Klebsiella has not been established in the art, we examine it because Klebsiella pneumoniae infections are common in hospitals where they cause pneumonia (characterized by emission of bloody sputum) and urinary tract infections in catheterized patients. Klebsiella infections tend to occur in people with a weakened immune system. In fact, K. pneumoniae is second only to E. coli as a urinary tract pathogen. Klebsiella infections are encountered far more often now than in the past especially in neonatal intensive care units. This is probably due to the bacterium's antibiotic resistance properties. Klebsiella species may contain resistance plasmids (R-plasmids) which confer resistance to such antibiotics as ampicillin, carbenicillin, and penicillin. Often, two or more powerful antibiotics are used to help eliminate a Klebsiella infection. To make matters worse, the R-plasmids can be transferred to other enteric bacteria not necessarily of the same species. Accordingly, there is a need for a new class of compounds to inhibit Klebsiella Dam, and thereby effectively treat these opportunistic hospital infections.
In addition, inactivation of Dam MTase attenuates Haemophilus influenzae virulence (Watson et al. 2004). Dam is associated with virulence factors for a growing list of bacterial pathogens including Neisseria meningitides, Yersinia pseudotuberculosis, Vibrio cholerae, Pasteurella multocida, Haemophilus influenzae and Yersinia enterecolitica. (see Low et al. 2001 and Table 1). Although Dam methylation is not essential for viability in many organisms, dam is an essential gene in Vibrio cholerae and Yersinia pseudotuberculosis, under tested growth conditions (Julio et al. 2001). Overproduction of Dam in Yersinia pseudotuberculosis attenuates virulence, secretion of several outer proteins (Yops) and heightened immunity (Julio et al. 2002), although the effect may be indirect through the inhibition of SeqA binding to hemimethylated GATC sites (Lobner-Olesen et al. 2005). A similar rationale may apply to dam plasmid attenuation of virulence in Pasteurella multocida which causes bovine respiratory disease (Chen et al. 2003). Among the Dam molecules examined to date, the Shigella flexnerii dam mutant shows the least effect on virulence (Honma et al. 2004).
Dam inhibitors are useful in reducing and/or preventing virulence associated with a number of pathogenic bacteria. For example, enteropathogenic E. coli (EPEC) is a significant public health concern, especially in developing countries, where it contaminates water supply and causes infant diarrhea (Gill and Hamer, 2001; Goosney et al. 2000; Knutton et al. 19989; Levine and Edelman, 1984), resulting in two million infant deaths per year. EPEC is closely related to enterohemorrhagic E. coli O157:H7 (EHEC), which causes diarrhea and hemorrhagic colitis that can lead to hemolytic uremic syndrome (Riley et al. 1983) and death. In Western nations EHEC is endemic in cattle (Mead et al. 1999), and has been a major source of contamination of ground beef (USDA, 2002). EHEC kills about 60 people per year and infects about 74,000 people in the United States alone (Mead et al. 1999). Currently, antibiotics are contraindicated for EHEC infections because they cause lysis and release of Shiga toxin, which causes renal failure and death. Development of drugs which inhibit expression of virulence factors offers a means to treat EHEC infections.
The present invention provides a method for rational design of, and screening to identify, specific inhibitors of Dam to reduce virulence of pathogenic bacteria. These specific inhibitors can be used to treat humans, as well as other higher eukaryotes that do not have detectable DNA-adenine methylation (Jeltsch, 2002). Accordingly, specific GATC methylation inhibitors can have broad anti-microbial action without affecting host function. There are a number of advantages for targeting factors that influence virulence over, for example, essential enzymes, and include: (1) selection of pathogenic over non-pathogenic bacteria without being toxic to non-pathogenic bacteria; (2) lack of immediate toxicity reduces the risk of rapid development of drug resistance; and (3) continued initial propagation of the pathogen allows the host to mount a stable immune response. Dam deletion mutants of Salmonella can be used as a live attenuated vaccine conferring cross-protective immunity (Dueger et al. 2001, 2003; Heithoff et al. 1999). However, dam mutants would have deficient mismatch DNA repair and consequently an increased rate of spontaneous mutation, which would not be a desirable trait for a live vaccine strain. Compounds having the capacity to affect virulence without affecting growth are less likely to elicit resistance compared to conventional antibiotics. Antibiotic resistance is one of the single greatest public health challenges facing humanity and developing compounds to affect virulence in a range of pathogens can significantly and positively impact treatment of infectious diseases. Because Dam inhibitors can affect the viability of many human bacterial pathogens, they may have widespread applicability in an era of bioterrorism concern. Inhibition of Dam by small molecule inhibitors provides a basis for identifying and developing a new class of antibiotics with broad anti-microbial action. We have determined the three-dimensional structures of two Dam MTases in complexes with DNA: the bacteriophage T4 Dam MTase and the E. coli Dam MTase. These high-resolution structures are used to identify, as well as rationally design, specific Dam MTase inhibitors. These inhibitors are useful in treating a host infected with pathogenic bacteria.
The present invention is for compounds and method of treating pathogenic organisms in a host. In particular, the invention provides a method to identify compounds capable of modifying activity of a DNA methyltransferase, including modifying activities of AdoMet-dependent MTases from pathogenic bacteria. AdoMet-dependent MTases and related proteins include: HhaI DNA MTase, HhaI MTase-DNA complex, PvuII endonuclease-DNA complex, PvuII DNA MTase, protein arginine MTases PRMT3 and PRMT1, small molecule histamine MTase and its complex with inhibitor, Dnmt3b PWWP domain, MBD4 glycosylase domain, histone H3 Lys9 MTase DIM-5 and its complex with substrate H3 peptide, phage T4 Dam and its complexes with DNA specifically and nonspecifically, protein glutamine-N5 MTase HemK, a nucleosomal dependent histone H3 lysine 79 MTase Dot1p, and HinP1I endonuclease, E. coli Dam and Dam from other pathogenic bacteria. In an embodiment Dam or Dim-5 enzyme activity is modified. In an embodiment Dam enzyme activity is modified. The Dam enzyme activity can be increased or it can be decreased. In a preferred embodiment the Dam enzyme activity is inhibited.
The identification method can be conducted by providing a three-dimensional structure of a Dam enzyme or a Dam enzyme complex. The Dam enzyme can be the entire protein. Alternatively, the Dam enzyme can be a portion of the entire protein, wherein the portion contains one or more of an AdoMet binding pocket, channel into and out of the pocket, a hinge region between the catalytic and DNA binding domains, a DNA binding surface, a unique surface pocket, or any other region that can affect Dam enzyme activity. The structure can be from a Dam enzyme complexed with one or more of the methyl donor (e.g. AdoMet) and DNA. A modifier candidate structure is provided and an interaction energy value calculated from a simulated docking interaction with the candidate structure and the Dam enzyme. A candidate structure is identified as capable of modifying Dam enzyme activity by assessing the interaction energy value. The assessment can be done relative to a reference or “cut-off” value.
In an embodiment the Dam enzyme structure is that obtained from a bacteriophage or a bacterium. In an embodiment the Dam enzyme structure is that obtained from E. coli. The structure can be from any source, so long as the structure has sufficient resolution so that a meaningful interaction energy value can be obtained from the simulated docking interaction. Preferred structures are obtained from X-ray crystallography, including those crystal structures deposited with the Protein Data Bank and summarized in Table 8.
The docking interaction preferably occurs at a docking site. Docking sites for Dam include an active site where the methyl donor donates a methyl group to the DNA base and/or a pocket formed between the catalytic and DNA binding domains and/or the methyl donor binding sites. Docking sites include AdoMet binding pocket, a channel into and out of the pocket, a hinge region between the catalytic and DNA binding domains, a DNA binding surface, a unique surface pocket, and other sites that can specifically affect DAM enzyme activity.
The methods of the present invention include computer-assisted drug design wherein, based on the enzyme's 3-dimensional structure, an inhibitor candidate structure is generated by calculating an interaction energy value between the generated structure and the enzyme structure. The enzyme can be a Dam enzyme, and the Dam enzyme structure can be obtained from any organism, including from a pathogenic bacteria. The Dam enzyme structure can be obtained from an E. coli Dam.
Compounds identified by any of these methods can be further assessed as capable of modifying Dam enzyme activity using known in vitro and/or in vivo assays, including by biochemical assays (e.g. non-cell based), cell-based, and whole-animal studies.
DNA methylation in a bacterium can be inhibited by providing the compound identified by the present invention and contacting the bacterium with the compound in an amount sufficient to inhibit DNA methylation in the bacterium. In an embodiment the bacterium contains a methylase, and preferably a Dam methylase and/or a cell-cycle regulated DNA adenine methylase. The bacterium can contain a methylase; in a particular embodiment, the methylase is capable of adenine methylation at GATC or GANTC sites.
Compounds have been identified by the screening methods and verified as inhibiting DNA methylation by biochemical and whole-cell assays. These compounds can be used to inhibit DNA methylation by Dam in an organism by contacting the organism with the compound. In an embodiment the compound is Dam-iZ1, wherein Dam-iZ1 has structural formula:
Wherein A is a non-aromatic 5 or 6 member ring and wherein one or more of the ring members of A can be C, N, O or S, and A can be optionally substituted. Examples of preferred structures for A (with the * indicating bond location to Y2) are:
Each of X1-X5 is independently selected from the group consisting of H, halide, OH, OCH3, alkyl and alkylhalide. Y1 is NH or CH2. Y2 is N or CH. The dashed double bond to Y2 indicated the bend can be single or double. In a specific embodiment Y2 binds to A at the site indicated.
Compound Dam-iZ1 can be NCI 659390:
Compound Dam-iZ1 can be NCI 658343:
Compound Dam-iZ1 can be NCI 657589:
The term “aryl” refers to a group containing an unsaturated aromatic carbocyclic group of from 6 to 22 carbon atoms having a single ring (e.g., phenyl), one or more rings (e.g., biphenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Aryls include phenyl, naphthyl and the like. Aryl groups may contain portions that are alkyl, alkenyl or akynyl in addition to the unsaturated aromatic ring(s). The term “alkaryl” refers to the aryl groups containing alkyl portions, i.e., -alkylene-aryl and -substituted alkylene-aryl). Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
Alkyl, alkenyl, alkynyl and aryl groups are optionally substituted as described herein and may contain 1-8 non-hydrogen substituents dependent upon the number of carbon atoms in the group and the degree of unsaturation of the group.
The term “heteroaryl” refers to an aromatic group of from 2 to 22 carbon atoms having 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring). Heteroaryl groups may be optionally substituted.
As to any of the above groups which contain one or more substituents, it is understood, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. The compounds of this invention include all novel stereochemical isomers arising from the substitution of disclosed compounds.
Any of Compound Dam-iZ1, NCI-DTP Diversity Set compound numbers 659390, 658343, 657589, and any compound identified by the methods of the present invention can be used to inhibit DNA methylation by Dam in an organism by contacting the organism with any one or more of these compounds. In an embodiment, the organism is a bacterium, including an E. coli bacterium. In an embodiment the DNA methylation is inhibited by inhibition of a Dam methylase. In an embodiment, the concentration of compound to inhibit Dam is between about 10 μM and 400 μM. In an embodiment the concentration to inhibit Dam is between about 20 μM and 200 μM. In an embodiment the concentration to inhibit Dam is about 20 μM.
The invention includes methods of treating a host suspected of infection with a pathogenic bacterium comprising administering to the host a compound identified by any of the methods of the present invention, including a compound selected from the group consisting of Dam-iZ1 and NCI-DTP Diversity Set compound numbers 659390, 658343, and 657589. In an embodiment, the method of treating the host suspected of infection with a pathogenic bacterium reduces a virulence parameter of the bacterium. As used herein, virulence parameter is used broadly to refer to, for example, replication, adherence to host, colonization, motility, gene expression, metabolism, heat shock response, and other measurable parameters that are associated with virulence.
In an embodiment, the invention provides a method of treating a host suspected of infection with a pathogenic bacterium comprising administering to the host a compound capable of modification of pathogenesis by inhibiting a methylase. In an embodiment, the methylase is a Dam methylase. In an embodiment, the modification of pathogenesis involves a modification of virulence. In an embodiment, the modification of virulence is without a substantial effect on bacterial cell division.
In an embodiment, the invention provides a crystal of Escherichia coli Dam. In an embodiment, the invention provides a crystal of a Escherichia coli Dam complex. In an embodiment, the complex comprises E. coli Dam and cognate DNA. In an embodiment, the complex comprises E. coli Dam and noncognate DNA. In an embodiment, the complex further comprises a cofactor or cofactor analog. In an embodiment, the cofactor or cofactor analog is selected from the group consisting of AdoMet, AdoHcy, and sinefungin. In a particular embodiment, the crystal has a set of atomic structure coordinates of
The invention may be further understood by the following non-limiting examples. All references cited herein are hereby incorporated by reference to the extent not inconsistent with the disclosure herewith. Although the description herein contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of the invention. For example, thus the scope of the invention should be determined by the appended claims and their equivalents, rather than by the examples given. In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the invention.
List of abbreviations: A/E (attaching and effacing); ATCC (American Type Culture Collection); Dam (DNA-adenine MTase); AdoHcy (S-adenosyl-L-homocysteine); AdoMet (S-adenosyl-L-methionine); EcoDam (E. coli Dam); EHEC (enterohemorrhagic E. coli O157:H7); EPEC (enteropathogenic E. coli); HTA (high throughput assay); ISS (in silico screening); MTases (methyltransferases); NCI (National Cancer Institute); PDB (Protein Data Bank).
General Crystallization and Structure Determination Techniques. Dam enzymology and assay development have been examined. See, for example, Roth & Jeltsh (2000); Urig et al. (2002); Humeny et al. (2003); Liebert et al. (2004); Horton et al. (2004). Utilizing standard procedures for protein purification, crystallization and structure determination, we have solved de novo structures of many AdoMet-dependent MTases and related proteins: HhaI DNA MTase, HhaI MTase-DNA complex, PvuII endonuclease-DNA complex, PvuII DNA MTase, protein arginine MTases PRMT3 and PRMT1, small molecule histamine MTase and its complex with inhibitor, Dnmt3b PWWP domain, MBD4 glycosylase domain, histone H3 Lys9 MTase DIM-5 and its complex with substrate H3 peptide, phage T4 Dam and its complexes with DNA specifically and nonspecifically, protein glutamine-N5 MTase HemK, a nucleosomal dependent histone H3 lysine 79 MTase Dot1p, and HinP1I endonuclease.
Once the enzymes are purified and concentrated to approximately 10 mg/ml, the crystallization conditions are searched using three screens (300 conditions) currently available in the lab. If further screens are necessary, we can use other commercially available screens of thousands of conditions, including different precipitants, buffers, etc. We screen the crystallization in three parallel lines: First, for the apo-enzyme, second, for the binary complex of MTase-AdoMet or AdoHcy complex, the cofactor is added during the last column of purification (usually a gel filtration column) and during the concentration step, and third, for the ternary complex of MTase-AdoHcy-DNA, the protein/DNA ratio as well as the DNA length and sequence is varied. In addition, we use hemimethylated GATC for crystallization (N6-methyl-Ade in one of the strands) because it is the nature substrate present immediately following DNA replication.
We use three approaches to solve the structure of EcoDam and other Dam molecules when X-ray diffraction quality crystals are obtained: (1) Molecular replacement; (2) Multi- or singly-wavelength anomalous diffraction (MAD or SAD) of Seleno-Met; and (3) Multiple isomorphous replacement (MIR) of heavy atom derivatives.
Molecular replacement: For the EcoDam structure determination (see
We use the same approach to solve the Dam structures of Salmonella and H. influenzae when the crystals become available. In addition, the molecular replacement solution can also be used to locate the Se or mercury sites via an anomalous difference Fourier map in the MAD or MIR data (below). A combination of the molecular replacement and experimental phases can greatly improve the quality of the electron density map and make it suitable for interpretation of the structure. We used a similar approach to solve the structure of HemK (Yang et al., 2004).
Multi- or single-wavelength anomalous diffraction (MAD or SAD) of Seleno-Met: EcoDam contains three methionines, and we have replaced the methionines in the protein with Se-Met by overexpressing the protein in the medium that supplies Se-Met. Preliminary X-ray data have been collected at APS SERCAT beamline for two wavelengths near the Se-absorption edge at ˜2.3 Å resolution; however these data were not needed because we solved the structure by molecular replacement (above).
Multiple isomorphous replacement (MIR) of heavy atom derivatives: If needed, isomorphous heavy atom derivatives are obtained by soaking the crystals in a variety of reagents containing heavy atoms. We initially focus on mercurial compounds. The mercury atom reacts with the sulfur atom of cysteine and EcoDam contains five cysteine residues. The first T4Dam structure was solved via mercury derivatives (Yang et al., 2003).
Any one of a variety of Dam molecules from pathogenic bacteria can be crystallized to obtain a high-resolution three-dimensional structure via X-ray crystallography. For example, Dam molecules can be obtained from Salmonella enterica serovar typhimurium, Yersinia pestis, and Klebsiella pneumoniae. The three enzymes (278, 271 and 275 residues, respectively) are similar in size to E. coli Dam (278 residues). Kpn Dam has been expressed in E. coli and purified. In addition, Shigella flexnerii and Salmonella pseudotuberculosis dam genes have been cloned and the proteins have been expressed in E. coli and are catalytically active (data not shown). The purified Dam protein is used to obtain a crystal structure by crystallographic methods known in the art.
The Kpn genomic DNA was obtained from ATCC (Manassas, Va.); the Dam gene was acquired from the genomic DNA using PCR (Dam sequence are available from publicly accessible databases, e.g. see e.g.
T4Dam structure has been solved by X-ray crystallography. See Yang et al. “Structure of the bacteriophage T4 DNA adenine methyltransferase Nature Struct. Biol. 10: 849-55 (2003) and Horton et al. “Transition from nonspecific to specific DNA interactions along the substrate recognition pathway of Dam methyltransferase” Cell 121. 349 61 (2005), both incorporated by reference, and specifically incorporated by reference for crystallographic methods, data and solution structure of T4Dam. The coordinates of the binary and ternary structures of T4Dam are deposited in the Protein Data Bank (see Table 8 for a summary of structures deposited with the PDB and PDB ID Nos).
Bacteriophage T4Dam contains two domains: (i) a seven-stranded catalytic domain harboring the binding site for AdoHcy and (ii) a DNA binding domain consisting of a five-helix bundle and a beta-hairpin loop. (
Structure of non-specific T4Dam-DNA-AdoHcy complex: We crystallized a ternary complex of T4Dam with both AdoHcy and a synthetic 12 base pair DNA (ACAGGATCCTGT)—the minimum substrate for T4Dam (Hattman and Malygin, 2004). In the crystal, the DNA duplexes are stacked head-to-end, forming a pseudo-continuous DNA duplex.
An explanation for the non-specific binding between T4Dam and DNA is that T4Dam methylates DNA with multiple GATC sites in a processive manner; i.e., more than one methyl group may be transferred per bound Dam monomer. In the ternary crystal structure, the T4Dam-AdoHcy complex may be on the duplex in a fashion that corresponds to the stage following methyl transfer. That is, it is not in contact with the GATC target site; rather it contacts the phosphodiester backbone and is primed for diffusion and/or exchange of AdoHcy with AdoMet. This ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.
Structure of a semi-specific complex: In addition to the blunt-end GATC-containing 12mer DNA, we also use a 13mer specific DNA with a 5′ overhang Ade in one strand (ACCATGATCTGAC) and a 5′-overhang Thy in the other strand (TGTCAGATCATGG), so that the Ade and Thy are base paired at the joint. As with the non-specific binding, two Dam molecules bind one DNA duplex, except the helical axis of the two DNA molecules are shifted relative to on another by about 12 Å (
Structure of a ternary complex containing both semi-specific and specific contacts: In addition to the 12mer and 13mer, we constructed a 15mer oligo (TCACAGGATCCTGTG) with the end sequence mimicking part of the recognition sequence. In addition, we also reduced the ratio of protein to DNA. We observed: (1) all of the joints between neighboring DNA molecules are occupied by a Dam molecule (molecules B, C, D, and E in
These observations indicate the Dam enzyme preferentially binds at the joint of two DNA molecules, which mimics damaged DNA or altered recognition sites. This is surprising but consistent with biochemical data, which suggest that binding specificity for DNA MTases is determined by the nucleotides flanking the target nucleotide and DNA MTases bind more tightly to substrates containing mismatches at the target base (Cheng and Roberts, 2001). In other words, DNA MTases do not depend on the flappable target base for their binding specificity. For Dam, having only one-half of the recognition site on one strand appears to be sufficient for stable complex formation provided that the 5′ G:C base pairs are present at both ends of the palindrome (Hattman and Malygin, 2004). This is what we observe for the joint formed by the 15mer duplex DNA.
Full-Site Recognition involves a Protein Side Chain Intercalation. Only one T4Dam (molecule E) occupies a GATC site (orange DNA) (
The phenyl ring of F111 intercalates into the DNA helix and stacks between the adjacent A:T base pair and the Thy:S112 “base-amino acid” pair, resulting in a local doubling in helical rise (
Interaction with a Noncanonical Site. F111 intercalation by molecule E into the central AT stacking effectively causes a one-base-pair lengthening of the DNA molecule depicted in orange (FIGS. 7A and B). The expansion is propagated toward one end of the DNA molecule, resulting in two disordered nucleotides of the neighboring duplex (magenta). The 5′-overhanging Thy of the magenta DNA is pushed out and apparently becomes disordered, resulting in the Cyt of the next base pair stacking with F111 of Dam molecule D. The side chain of S112 approaches the Cyt base with the side chain hydroxyl oxygen and the exocyclic amino nitrogen N4 of the Cyt at a van der Waals distance, partly because of repulsion force between the N4 amino nitrogen (NH2) and the main chain amide nitrogen (NH) (
Stabilization of the Flipped Adenine in the Presence of Sinefungin. Thus far we had prepared ternary complexes using the methylation reaction product AdoHcy. The protein-AdoHcy interactions for each protamer are nearly identical to those described in the T4Dam-AdoHcy binary complex (Yang et al., 2003). In the full-site recognition complex between Dam molecule E and the orange DNA (
The new crystal contains two T4Dam molecules (not shown), one bound in the joint of two DNA duplexes, similar to the Dam C molecules in
Biochemical Analysis of EcoDam Variants. EcoDam has considerable sequence similarity (25% identity) to T4Dam (Hattman et al., 1985) but has significantly higher sequence conservation with Dam enzymes from pathogenic bacteria. For example, the E. coli and S. typhimurium Dam proteins are 92% identical (differing at only 22 of 278 residues) and have no gaps in their alignment. Because of the biological importance of the Dam family, we investigated whether the T4Dam structures contribute to understanding the function of these orthologs. To this end we studied the effects of substituting Ala for residues in EcoDam (
The R124A and Y119A variants were the most strongly affected by the Ala substitution; their catalytic activity was reduced more than 100-fold (
To further investigate the process of DNA recognition, the rate of DNA methylation by the wild-type and variant enzymes was determined using duplexes containing a single hemimethylated target (N6-methyl-Ade in the bottom strand, third base pair in
In contrast to the first position, the third and fourth bases of GATC are recognized more accurately. At both positions, transitions (Thy3 to Cyt or Cyt4 to Thy) are less deleterious than transversions, indicating that conservative exchanges are more tolerable. The contact between T4Dam R116 and Gua4 (
We found a similar base pair-specific loss of specificity associated with T4Dam residues P126 and M114, which recognize the T:A base pair at the third position of GATC (see
The activity of the L122A variant of EcoDam (M114 in T4Dam;
In addition to residues making base-specific contacts, we studied the aromatic residue that intercalates into the DNA (Y119 in EcoDam, F111 in T4Dam;
DNA recognition by proteins is essential for specific expression of genes in any living organism. Although the principle of proteins recognizing DNA sequences by contacts in the major groove has been known for decades (Seeman et al., 1976), there is no general code allowing one to deduce amino acid motifs from their target DNA sequences. Notable exceptions are the C2H2-type zinc fingers, where the DNA recognition process is sufficiently understood to define a DNA recognition code of this family of proteins (Pabo et al., 2001). Consequently, the rational design not only of DNA-interacting enzymes but also of even noncatalytic proteins is still in its infancy.
Here we describe six unique T4Dam-DNA interactions along the substrate-recognition pathway (
Interestingly, the phosphate-interacting residues R95 and N118, which hydrogen bond with the first and second phosphates 5′ to the Gua4 in the specific complex (or in any complex involving R116-Gua4 interaction;
Our data suggest a temporal order for the formation of specific contacts during the one-dimensional sliding of T4Dam along the DNA. The contact of R116 to the fourth base pair of the GATC site is observed in the 1/4- and 3/4-site recognition complexes. Next, the contacts of P126 and M114 to the third base pair are formed. All of these residues are strictly conserved within the Dam MTase family. The contact of R130 to Gua1 that is specific to T4Dam is formed later. This result agrees with a similar conclusion drawn from rapid kinetics experiments with M.EcoRV variants (Beck and Jeltsch, 2002). In this enzyme, substitutions of amino acids conserved in the enzyme family (such as N136A) interfered with specific complex formation at an early state, while substitutions of amino acids characteristic of EcoRV (such as R145A) interfered with complex formation at later stages. This finding might illustrate a general pathway for changes of DNA specificity of proteins and enzymes during molecular evolution. The recent study on human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) suggested that the recruitment of multiple AGT molecules to the same region of DNA might aid the search for DNA damage through a process of directional bias (Daniels et al., 2004). However, such directional bias was only observed for the repair of single-stranded DNA by AGT but not for double-stranded DNA, and the system cannot be directly compared to the Dam MTases because T4Dam and EcoDam move along double-stranded DNA (
We analyzed the biochemical effects of altering the contacts described above in double-mutant cycles (Fersht et al., 1992). This involved shortening the respective amino acid side chains and using DNA substrates with near-cognate sites. We found that it was possible to predictably design MTase variants that no longer recognize one specific base pair within their recognition site. The EcoDam R124A variant displayed a change in specificity because it had a significantly higher catalytic activity toward a near-cognate site. In addition, the EcoDam P134A variant (the analog of the T4Damh MTase) methylated a near-cognate site at almost the same rate as wild-type EcoDam modified the canonical site, indicating a broadened specificity (
We have identified two types of protein DNA contacts, discriminatory and antidiscriminatory. A discriminatory contact is one that stabilizes the transition state of enzymatic catalysis and specifically accelerates the reaction with the cognate site. The contact between R116 of T4Dam (R124 of EcoDam) and the Gua4 is an example of a discriminatory contact. Disruption of the contact by removal of the amino acid side chain led to a strongly reduced activity of the enzyme variant. An antidiscriminatory contact, e.g., the contact between P126 of T4Dam (P134 of EcoDam) and the third base pair of the recognition site, is one that does not significantly accelerate the reaction with the cognate site but disfavors activity at near-cognate sites because steric clashes may occur if the wrong DNA sequence is bound. This would strongly interfere with methylation of most noncanonical DNA sequences and lead to an efficient counterselection against methylation of nontarget sites. This is illustrated by the high activity and broadened specificity of EcoDam variants P134A and P134G.
Comparison with restriction-modification MTase M.DpnII: T4Dam and MTase M.Dpnii (Tran et la. 1998) both recognize and methylate the same GATC sequence, and have quite similar structure, but differ substantially in their processivity. It has been suggested that processive enzymes, like T4Dam, tend to more fully enclose their substrates. Breyer and Matthews (2001). However, since the M.DpnII-DNA complex structure is currently not available, a direct structural comparison with T4Dam cannot be made.
The cofactor (e.g. AcoHcy) binding site in all structures of T4Dam examined thus far is in a closed acidic pocket. In contrast, an open conformation was observed in the binary M.DpnII-AdoMet structure, where the AdoMet is largely visible and the pocket is opened up (Tran et al 1998). This difference suggests that the exchange of AdoHcy with AdoMet, the rate limiting step in the overall T4Dam methylation process (Malygin et al. 2000), requires a conformational change in the protein. This conformational change in T4Dam, which includes Trp185, was demonstrated by quenching of intrinsic tryptophan fluorescence that results from T4Dam binding either AdoMet of AdoHcy (Tuzikov et al. 1997)
Transition from Nonspecific to Specific DNA Interactions along the Substrate-Recognition Pathway of Dam Methyltransferase. DNA methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and antidiscriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.
Structural snapshots along the substrate recognition pathway. In the study of T4Dam (
Information on various structure of the enzyme in complex with DNA and mechanistic insights into the DNA methylation process impacts the ISS process and thus, the identification of possible leads, because one can design inhibitors that are selective for certain conformational states. Determining the areas of protein surface responsible for non-specific and specific DNA interactions assists in targeting these areas individually in ISS. Inhibitors interfering with specific DNA binding prevent the transition of non-specific to specific DNA interaction, or they can interfere with sliding of the enzyme along the DNA. Inhibitors that block AdoMet exchange can affect processivity.
Adenine methylation in hemimethylated GATC sites produced by DNA replication regulates bacterial cell functions including gene expression, mismatch repair, and virulence in many Gram-negative bacteria. The widespread and conserved enzyme DNA adenine methyltransferase (Dam) in γ-proteobacteria methylates GATC sites by scanning the genome. Structures have been solved for Escherichia coli Dam (EcoDam), interacting with a cognate and a non-cognate site in the presence of cofactor analogs. The non-cognate complex allowed identification of a potential DNA binding element, TA(G/A)AC, immediately flanking GATC sites in many Dam-regulated promoters. Accompanied by biochemical studies, the structures reveal a chronological order of formation of specific enzyme-DNA interactions. Contacts to the non-target strand in the second (3′) half of the GATC site are established early in the recognition pathway, initially to the fourth, and then to the third base pair. Then, intercalation of specific protein side chains into the DNA helix between the second and third base pairs occurs in concert with flipping of the target Ade. Contact to the first Gua in GATC is established later. The flipped target Ade bound to an alternative base-binding site suggests a possible late intermediate in the base-flipping pathway. The orphan Thy can adopt an intrahelical or extrahelical position.
We report two crystal structures of EcoDam, bound to cognate or non-cognate DNA, in the presence of a cofactor analog. The non-cognate DNA complex allowed us to identify a potential DNA binding element. TA(G/A)AC. immediately flanking GATC sites in many Dam-regulated promoters, which suggests a mechanism of regulation of dam methylation in bacterial DNA. Together with parallel biochemical studies, we verified structural predictions and reconciled the effect of site-directed mutations on DNA binding, target-sequence specificity and base flipping. By combining structural and kinetic data we also determined the sequential order for formation of specific contacts between the enzyme and the DNA and base flipping. The flipped target Ade bound to an alternative base-binding site suggests a possible late intermediate in the base-flipping pathway. The ‘orphan’ Thy can adopt an intrahelical or extrahelical position.
His Tag-EcoDam is expressed in HMS174(DE3) cells and purified using Ni2+-affinity, UnoS, and S75 Sepharose sizing columns. A 0.5-liter induced culture yields approximately 7 mg purified His Tag-EcoDam. In the last purification step and during concentration, cofactor analog AdoHcy or sinefungin is added is added to the protein at approximately 2:1 molar ratio. Concentrated binary complexes are mixed with oligonucleotide duplex (synthesized by New England Biolabs, Inc) at a protein to DNA ratio of about 2:1 and allowed to stand on ice for at least two hours before crystallization. Final protein concentration for crystallization is about 15 mg/mL. In hanging drop crystallization trials with AdoHcy, the ternary complex crystals appeared under low salt conditions of 100 mM KCl, 10 mM MgSO4, 5-15% PEG400, and 100 mM buffer (MES or HEPES) pH 6.6-7.4 (the cognate crystal form in Table 4). In crystallization trials with sinefungin, the ternary complex crystals grew under similar low salt conditions, but resulted in different cell dimensions (the non-cognate crystal form in Table 4).
Structural determination of the cognate ternary complex proceeded by molecular replacement with the program REPLACE (Tong and Rossmann, 1997) using protein coordinates of a DpnM monomer structure (PDB 2DPM) (Tran et al., 1998). The DpnM model was modified based on pair-wise sequence alignment of EcoDam with DpnM; differing amino acids in DpnM were changed to those in EcoDam and visually given the best rotamer using the program O (Jones et al., 1991), and some amino acids were deleted in the loop regions. DNA molecules were built manually into densities of difference maps. Refinement proceeded with the program CNS (Brunger et al., 1998). Structure of the non-cognate ternary complex was determined using a protein monomer from the refined cognate complex structure as a search model.
Site directed mutagenesis was performed as described (Jeltsch and Lanio, 2002). EcoDam wild type and its variants were purified as described (Horton et al., 2005). DNA binding was analyzed using surface plasmon resonance in a BiaCore X instrument as described (Horton et al., 2005). Methylation of oligonucleotide substrates (purchased from Thermo Electron, Dreieich, Germany in purified form) was carried out as described (Horton et al., 2005). Methylation experiments were performed in 50 mM Hepes (pH 7.5), 50 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 0.2 μg/μl BSA containing 0.76 μm [methyl-3H]AdoMet (NEN) at 37° C. as described (Roth and Jeltsch, 2000) using single-turnover-conditions with 0.5 μM oligonucleotide substrate and 0.6 μM enzyme for specificity analysis (
S1=(kGATG+kGATA+kGATT+kGAGC+kGAAC+kGACC)/(kAATC+kTATC+kCATC)
To measure equilibrium base flipping, the fluorescence change of oligonucleotides containing 2AP was detected in the absence and presence of EcoDam using 2 μM of enzyme and 0.5 μM DNA in 50 mM Hepes (pH 7.5), 50 mM NaCl containing 100 μM AdoHcy at ambient temperature (
We crystallized a ternary complex containing EcoDam, AdoHcy, and a 12-mer oligodeoxynucleotide duplex containing a single centrally located GATC target site (
It is difficult to produce E. coli Dam crystals, and is particularly difficult without DNA. Utilizing insight obtained from successful crystallization of T4Dam, we designed an oligonucleotide with the following properties: (1) optimized length to maximize the DNA-mediated protein-protein contacts in the crystal packing lattice; and (2) the two end-sequences of the oligonucleotide to mimic a GATC site if the two DNA duplexes are stacked head to tail. Accordingly, we use 12-mer DNA 5′-TCTAGATCTAGA-3′. In addition, protein to DNA ratio (>2:1) is varied so that all joints between neighboring DNA duplexes and the central GATC site are occupied by Dam molecules. With these properties, we successfully crystallized EcoDam in complex with DNA and AdoHcy. The crystals diffracted X-rays to higher resolution. In particular, orthorhombic crystal form (space group P212121) with unit cells of a=44.8 Å, b=70.2 Å, c=96.5 Å were grown with 5-15% PEG 400, 100 mM KCl, 10 mM MgSO4, and 100 mM buffer (MES or HEPES) pH 6.6-7.4. A data set diffracted to 1.89 Å resolution is shown in Table 5. The structure was solved by molecular replacement using T4Dam as an initial search model, and the model refined to an R-factor of 0.186 and R-free of 0.215 (
A different hexagonal crystal form (space group of P3(1,2)21) with the same 12 bp DNA has also been observed (unit cells a=b=159.5 Å, c= 93.7 Å) under the conditions of 1.5 M Li(SO4)2, 50 mM MgSO4, 100 mM buffer (MES or HEPES) pH 6.8-7.2.
Overall structure of EcoDam: Two EcoDam monomers (molecules A and B) and one DNA duplex are contained in the crystallographic asymmetric unit. EcoDam molecule A primarily binds to a single DNA duplex, while EcoDam molecule B binds the joint between the two DNA duplexes (
EcoDam-DNA phosphate interactions: The EcoDam molecule spans ten base pairs, four base pairs on 5′ side and five on 3′ side of the flipped-out target Ade (
EcoDam-DNA base interactions: The methylation target, the Ade of the second base pair in GATC (Ade2), flips out from the DNA helix (
Recognition of the first base pair by N-terminal K9: Their recognition of the first base pair is one of the most interesting deviations between T4Dam and EcoDam. In the T4Dam structure, the first Gua of the GATC site is contacted by R130 with bifurcated hydrogen bonds to the N7 and O6 atoms of Gua1 (Horton et al., 2005). R130 is located at the end of the β-hairpin, but it is not conserved among the Dam-related MTases (
To further investigate DNA recognition by EcoDam, we compared the rates of DNA methylation of the canonical versus variant duplexes, all containing a single hemimethylated target (
A specificity factor (S1) for the recognition of Gua1 was calculated for K9A, which is given by the average of the methylation rates of all near cognate substrates carrying an alteration at the first base pair divided by the average methylation rate of all other near cognate substrates. On the basis of S1, in comparison to wild type EcoDam, K9A has an at least 800-fold reduced recognition of the first base pair (
Interaction with the target Ade and base flipping: Incorporation of the nucleotide analog 2-aminopurine (2AP) into synthetic oligodeoxynucleotide duplexes has been used extensively to probe conformational changes, such as base flipping (Allan et al., 1998; Allan and Reich, 1996; Holz et al., 1998; Stivers, 1998), because 2AP fluorescence increases dramatically when it is removed from the stacking environment of double helical DNA (Ward et al., 1969). Fluorescence changes of a hemimethylated G-2AP-TC substrate, which carries 2AP at the position of the target Ade, was correlated with base flipping by EcoDam (Liebert et al., 2004). Base flipping by EcoDam comprises two steps: (i) flipping of the target base out of the DNA helix, and (ii) binding of the flipped base into the active site pocket of the enzyme (formed by the D181-P-P-Y184 motif). Target base flipping leads to a complete loss of the stacking interactions of the Ade with the neighbor bases which causes a strong increase in fluorescence. During binding of the flipped base into the active site pocket it stacks to aromatic residue(s), which leads to a reduction of 2AP fluorescence during this step of trapping (Liebert et al., 2004). Rapid kinetic measurements with a hemimethylated G-2AP-TC substrate demonstrated that, in the presence of AdoMet, base flipping by EcoDam was a biphasic process (
In agreement with these observations, in the current structure formed in the presence of AdoHcy, the flipped target Ade lies against the protein surface (side chains of Y184 and H222) outside the active-site pocket (
The existence of a conformation in which the flipped Ade is not bound to the active site suggests that the base flipping occurs through a series of intermediates (Banerjee et al., 2005; Horton et al., 2004; Liebert et al., 2004). The Ade-binding mode observed here in the EcoDam complex could mimic a late intermediate in the Ade-flipping pathway; viz., one just before insertion of the base into the active-site pocket. Alternatively, the conformation could be viewed as an intermediate formed immediately after release from the active-site pocket. Therefore, we reasoned that product AdoHcy might signal the enzyme to exchange for AdoMet prior to the binding of the flipped Ade into the active site pocket. This coupling could be mediated by the dynamic conformations of the loop adjacent to the active-site (see
Interaction with the orphan Thy: a double base flipping: The conformation of the orphan Thy (opposite the flipped Ade) represents a major difference between the EcoDam-DNA complexes formed by molecule A versus molecule B. Unexpectedly, in molecule A the orphan Thy in the center is also flipped out of the DNA helix, where it is stabilized by the n-stacking interactions with the guanidino group of R137 (
Y119 intercalation is necessary for base flipping: The Y119 aromatic ring intercalates into the DNA duplex and stacks between the third base pair of GATC and the Thy:N120 “base-amino acid” pair in the joint (
Recognition of the third base pair: discrimination of unmethylated and hemimethylated DNA. The third base pair of GATC makes van der Waals contacts with two hydrophobic side chains of L122 and P134 (
The mechanism of this pronounced change in the catalytic properties of L122A is not clear. Without wishing to be bound by any particular theory, we postulate that the Ala at position 122 interacts with the methyl group of methylated Ade3, to compensate for the loss of the contact between L122 and Thy3 (see
Recognition of the fourth base pair: the first step of specific DNA interactions: The Gua in the fourth base pair of GATC interacts via its O6 and N7 atoms with the guanidino group of R124 in a bifurcated hydrogen bonding pattern (
As shown in
These findings demonstrate that there is a coupling between DNA recognition and base flipping by EcoDam. The contacts of the β-hairpin loop with second half of the recognition sequence (the third and fourth base pairs) are required to position the enzyme on the target sequence. In particular, we hypothesize that the Gua4 base contact by R124, and its flanking phosphate contacts by conserved residues (see above), positions EcoDam on the DNA duplex such that other residues involved in base flipping (such as Y119) and DNA recognition (such as L122 and P134) can approach the DNA and induce base flipping. This notion is further supported by the next structure.
Interaction with a non-canonical site: implication in regulation of pap expression: A second crystal form was produced in the presence of the AdoMet analog sinefungin (adenosyl ornithine) (Table 4) and the same 12-mer blunt-end oligodeoxynucleotide duplex for crystallization. There were at least three unexpected observations. First, although an EcoDam molecule (designated as molecule C, to distinguish it from the A and B molecules shown in
Five base pairs in the joint are in contact with molecule C (
The Pap regulon contains two GATC sites (
Coupling of base flipping and DNA recognition: A central mystery of DNA methylation concerns the mechanism by which DNA MTases cause flipping of the target base within their recognition sequences. The present structures of the cognate and non-cognate complexes shed some light on this process. Comparison of the DNA conformation in the non-canonical site (bound with molecule C) with that in the canonical site (bound with molecule A or B) reveals the detailed conformational changes that take place in the earlier stages of DNA recognition and the final stage of base flipping. Shown in
To study the coupling of base flipping and DNA recognition, we investigated base flipping by EcoDam variants with an altered specificity by P134G and P134A (which show reduced discrimination at the third base pair) and K9A (which has relaxed recognition of the first base pair). In agreement with their high catalytic activities P134G (
On the basis of the structural comparison shown in
Compounds identified from the in silico screen (ISS) (see Example 3) can be further studied structurally by co-crystallization with Dam. The structural information obtained from these co-crystals can be used to identify site(s) of structural variability to generate derivatives around the same core chemical structure, via synthesis of a compound library, with more desirable properties.
Crystallization of mutant Dam or pap-associated GATC substrate to address processivity of DNA methylation: EcoDam methylates DNA in a highly processive reaction (Urig et al., 2002). After each methylation event the coenzyme product AdoHcy must be exchanged with AdoMet before next round of reaction. Processive methylation requires that this exchange occur while the enzyme stays bound to the DNA. An inhibitor that prevents the sliding of the enzyme along the DNA and/or blocks AdoHcy/AdoMet exchange can affect processivity, and thereby inhibit methylation.
In T4Dam structure, a channel connects the coenzyme binding site and the solvent (not shown). This channel is important for processive methylation by Dam, as it can allow the exchange of coenzyme without releasing the enzyme from DNA. In the context of this invention, the channel provides an additional docking site unique for Dam, with the potential of finding more specific inhibitors. These inhibitors can either prevent AdoHcy/AdoMet exchange or they can diffuse into the AdoMet binding pocket and sterically interfere with AdoMet binding. These possible modes of action can be distinguished by comparing the effects of the inhibitors on AdoMet binding and processivity of DNA methylation. Ile51 forms one wall of the channel in T4Dam. We have changed the corresponding residue (Ile55 in EcoDam) to Trp and Arg in an attempt to block the channel and interfere with processivity. Initial results show that blocking the channel by the I55W substitution strongly compromises activity. The I55R variant is as active as the wildtype enzyme on short oligonucleotide substrates. The processivity of I55R mutant is examined using the assay described in Urig et al. (2002), and measure the Kd of AdoMet binding to both mutants. Co-crystal structures of these mutants with coenzyme indicates whether/how these substitutions affects coenzyme interaction. If this channel indeed affects coenzyme binding/exchange, we can pursue ISS of this site to identify additional Dam inhibitors.
The Pap regulon contains two GATC sites separated by 103 bp (
In bacteria usually all MTase target sites are methylated. However, the ability of an MTase to regulate the expression of genes critically depends on the existence of a methylation pattern, which means that certain sites must be protected against the constitutive methylation. It is very likely that some signals in the sequence context of the pap-sites contribute to this protection and the loss of processivity of EcoDam in methylation of the pap site could be one effect. The identification of these signals that prevent processive methylation can help find other bacterial genes that are differentially methylated, and whose expression may be modulated by Dam methylation. This approach can help to understand how dam methylation regulates pathogenicity of bacteria.
Given the recent success in identifying the same inhibitor of the TGF-β receptor kinase by two different routes—one using ISS and one using high-throughput screening (HTS) (Sawyer et al., 2003; Singh et al., 2003), it is evident that the appropriately guided ISS approaches can be as successful as HTS (Liu et al., 2004) (Waszkowycz, 2002). In addition, molecular docking is less labor intensive. For example, it has a 6% hit rate, compared with <0.2% for HTS in the screen for tuberculosis target dihydrodipicolinate reductase against the Merck chemical collection (Paiva et al., 2001).
Two major goals must be considered during ISS. First is the need to identify compounds that effectively inhibit Dam methylation. Second is the need for those compounds to be specific for bacterial Dam molecules versus other MTases. With respect to the first consideration, we target a number of potential binding sites on EcoDam. From the crystallographic structures, we expect to have many snapshots of Dam molecules proceeding from non-binding pockets to target via ISS. Potential sites include the AdoMet binding pocket and channel(s) into and out of the pocket, the hinge region between the catalytic and DNA binding domains, DNA binding surfaces (specific and non-specific), and unique surface pockets. Notably, conformational changes in the presence of AdoMet/AdoHcy or DNA or flipped target Ade may influence the size or shape or particular cavities; these attributes are checked by structural comparison of the different forms of Dam characterized via crystallography. In addition, by targeting the cofactor-bound Dam structure, inhibitors may be identified that are active even in the presence of high levels of AdoMet (since such high levels can exist intracellularly). Final selection of binding sites includes homology considerations, with the goal of obtaining broad-spectrum antibiotics, as well as the quality of sites for binding of compounds. The latter will be determined by performing preliminary docking against the putative sites, with the quality of each site determined based on docking scores and geometries.
Selectivity of the inhibitors for Dam versus other MTases is a very important criterion for a successful antibiotic. A compound selective for Dam should have a high probability of binding to other Dam proteins but a low probability of binding to non-Dam MTases. For the latter, compounds selected from our initial screen (50,000 compounds, see below) are also screened against the following non-Dam MTases: PRMT1—a protein arginine MTase (Zhang and Cheng, 2003), and DIM-5—a histone H3 Lys9 MTase (Zhang et al., 2002) (Zhang et al., 2003) and the binding energies with these proteins are incorporated into the selectivity score described in the next section. Such selective screening is especially important for inhibitors targeting the cofactor binding region, as the potential for a lack of selectivity is the highest in this functionally similar region of the protein. We also screen the compounds against Salmonella Dam, such that compounds that score favorably against both E. coli and Salmonella Dam, but not to other non-Dam MTases, are preferentially selected for biological testing. Because there is currently no 3D structure of Salmonella Dam, we produce a homology model based on E. coli Dam using the program Modeller (Sali and Blundell, 1993). Accuracy of the modeling is aided by the fact that there are only 22 residues different between the E. coli and Salmonella Dam (92% identity), with almost the same number of amino acids (278 vs. 277) and no gaps between them. Once a crystallographic structure for Salmonella Dam is obtained, the structure can be used for docking in a manner equivalent to Dam from T4 and E. coli.
The target for these screening studies is a Dam-AdoHcy complex. Initially we use the crystal structure of T4Dam-AdoHcy complex as a target. An in silico screen (“ISS”) evaluates a library of compounds for their ability to bind the target via computational calculations based on the structure of the compound and target. In silico screens are advantageous over high throughput screening in that any number of compounds can be readily screened without the need for bench-top time and effort associated with high throughput screens. For example, we have used a relatively “small” library, specifically the National Cancer Institute (NCI) “Diversity Set” library. The NCI Diversity set is a subset of approximately 2000 compounds (see
ISS is useful for identifying target compounds and has been addressed by, for example, Pan et al. (2003); Huang et al. (2004). The same inhibitor of the TGF-β receptor kinase has been identified by both ISS and high-throughput screening (Sawyer et al., 2003; Singhe et al., 2003).
When used herein, the term data representation can comprise chemical and/or structural information of a molecule or molecular complex. For example, a data representation can be a set of structure coordinates, a three-dimensional diagram, a two-dimensional diagram, a chemical formula, or other information for a given molecule, molecular complex, or portion thereof.
When used herein, the term structure coordinates will be understood by one of ordinary skill in the art and can refer to mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of an enzyme or enzyme complex. For example, an enzyme complex can include a methylase, a DNA substrate, and a methyl donor. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to establish the positions of individual atoms within the unit cell of the crystal. For a set of structure coordinates determined by X-ray crystallography, those of ordinary skill in the art understand that coordinate data is not without standard error. In embodiments of this invention, any set of structure coordinates that have a root mean square deviation of protein backbone atoms (e.g. N,alpha-C, C and O) of less than 0.75 angstroms when superimposed—using backbone atoms—on the referenced structure coordinates shall be considered identical.
In an embodiment, a small molecule and/or small molecule data bases are screened computationally for chemical entities or compounds that can bind in whole, or in part, to an enzyme or enzyme complex as described herein. In a particular embodiment of screening, the quality of fit of such entities or compounds to a binding site of interest may be evaluated either by shape complementarity or by estimated interaction energy. See Meng, E. C. et al., J. Comp. Chem., 13, pp. 505-524 (1992).
The present invention is not limited to the use of any particular method for carrying out the screen. The invention can utilize any docking software algorithm and any scoring algorithm known in the art. U.S. Pat. App. No. 2005/0170379 (Kita et al.) summarizes different techniques suitable to perform docking simulations, including rigid-body pattern-matching algorithms (based on any of surface correlations, geometric hashing, pose clustering, graph pattern-matching), fragmental-based methods (including incremental construction or “place and join” operators), stochastic optimization methods (including Monte Carlo, simulated annealing, genetic (or memetic) algorithms, molecular dynamics simulations, and/or hybrids of any one or more of these techniques. Numerous docking programs are available and continue to be developed in terms of algorithms and efficiency. The program DOCK (Ewing et al., 2001) is used in our inhibitor screening studies because of its free distribution. The program performs the following computational tasks: first, an orientation search of a small molecule in a chosen site or pocket, which is a fundamental process of docking; second, a conformational search of a molecule, leading to identification of the best conformation to fit in the target site. More importantly, it can utilize a database of compounds for docking tests, meeting the basic need for virtual screening.
In DOCK based screening, sphere centers are generated based on the Connolly surface of the binding site of interest and the compounds from the database are then docked into the binding site by matching sphere centers with compound atoms. D Selection of the site for docking is typically based on biological data, including homology information, as well as based on the quality of a site for binding inhibitors. Such binding capacity may ideally be validated based on the ability of the docking algorithm to reproduce the bound conformation of a known ligand, such as the ability to reproduce the experimentally determined binding mode of AdoHcy (see
Preliminary molecular docking was conducted targeting the AdoHcy binding site (dark), and a cavity between the two domains (dark and gray,
In an embodiment, docking sites are specified based on the experimentally bound AdoHcy and active site. The region within 8A around the ligand is considered. A 0.3 Å grid is used in all the docking studies to compute interaction energy, a grid. Energy scoring grids are obtained by using a united-atom model, a distance-dependent dielectric function (∈=4r), and 6-12 Lennard-Jones van der Waals potentials. The flexible ligand docking is performed using an Anchor-First mechanism with a minimum anchor fragment size of 7 atoms and a sampling of 25 conformations. The maximum orientations are set to 5000 during docking an anchor fragment.
Energy minimization is performed using the grid-based rigid body simplex algorithm. One cycle of 100 simplex minimization steps are applied to adjust the compound's orientation and conformation, and to locate the nearest local energy minimum to a convergence of 0.5 kcal/mol. The minimization is calculated on-the-fly in the program DOCK and only the final energy scores are documented.
Further investigation is conducted using related compounds having some chemical similarity to the lead compound (e.g. compound #78 (NCI 659390). Related compounds are identified using SMILES string-based pattern recognition (see
Scoring, After docking all 2000 compounds in the database into the cofactor-binding site or the putative binding cavity in the hinge region, the program DOCK was applied to calculate the ligand-protein interaction energy to rank the docked ligands (see
Specificity against other structurally characterized non-Dam MTases: Following the same approach the 2000 compounds were docked against the cofactor site and a peptide binding site (
It should be noted that the majority of the compounds identified from the cofactor sites of Dim-5 and T4Dam are different, indicating that specificity can be achieved via docking. We also note that Roche et al. (2002) predict a high percentage of frequent hitters in databases are comprised of real drugs; thus removing frequent hitters could miss real marketable drugs. Because the goal of docking is to identify compounds complementary to the binding pocket, we evaluate some of the top, but frequent, hits in the biochemical assays.
Summary of DOCK results: Currently we have identified 82 compounds (36 identified for DIM-5, 40 identified for T4Dam, and 6 known inhibitors for histamine MTase, a small molecule MTase (Horton et al., 2001)) (
It is interesting to note that an alternative functional core structure is defined by two of the nine compounds identified in Table 7 (compound numbers 55 and 58). Similar analysis can identify other functional core structures. In addition to compound Dam-iZ1, compound Dam-iZ2 can function as a Dam inhibitor:
M can be an aryl or heteroaryl, wherein aryl is one or more rings, preferably one or two aromatic rings wherein each ring is optionally and independently substituted. A heteroaryl has an aromatic ring containing one or two heteroatoms. In an embodiment, M is:
In an embodiment, the “*” indicates the attachment location of M to the nitrogen atom.
Computer aided drug design (CADD) lead identification via database screening: Identification of novel lead compounds with the potential to bind to Dam is performed via database searching of the virtual NCI Diversity Set and a 3D chemical database of over 3 million commercially available compounds. The 3-million-compound database has been compiled and converted from 2D structures to 3D structures (Huang et al., 2004; Pan et al., 2003) in the University of Maryland CADD Center headed by Dr. MacKerell. The majority of the compounds in the database have recently been shown to have drug-like properties (Sirois et al., 2005). The target for the initial database search is the catalytic site of EcoDam; later searches target novel binding sites as determined via the proposed crystallographic studies. Database searching is performed using the program Dock (Kuntz et al., 1982) using the anchor based search approach to account for ligand flexibility (DesJarlais et al., 1986; Ewing and Kuntz, 1997). Prescreening is performed against select compounds that contain 10 or less rotatable bonds and between 10 and 40 non-hydrogen atoms and energy scoring is based on the GRID (Goodford, 1984) method implemented in Dock. From the initial docking, the top 50,000 compounds are selected based on normalized van der Waals (vdW) attractive interactive energies. Use of the vdW attractive energy, versus total energy or electrostatic energy, forces the procedure to select compounds with structures that sterically complement the binding site (Huang et al., 2004). The normalization procedure is designed to control the molecular weight (MW) of the selected compounds (Pan et al., 2003); use of N1/2 normalization where N is the number of non-hydrogen atoms in the compounds, selects compounds with an average MW of 320 daltons. Such compounds are smaller than the average MW of pharmaceutically active compounds based on the World Drug index. Smaller MW compounds are desirable at this stage of a drug design project as they are more amenable to modification at later stages of the project (Oprea et al., 2001).
Secondary virtual searching of the top 50,000 compounds selected from the initial screen includes simultaneous energy minimization of the anchor during the iterative build-up procedure (Chen et al., 2000; Huang et al., 2004). The secondary screening is performed against the non-Dam MTases, DpnII (Tran et al., 1998), PRMT1 (Zhang and Cheng, 2003), and DIM-5 (Zhang et al., 2002; Zhang et al., 2003) as well as EcoDam in order to include specificity in the compound selection. The final score for each compound is obtained by summing the total interaction energies for each compound with EcoDam and the weighted sum of the difference between EcoDam and non-Dam MTase interaction energies, as follows:
I.E.Final=I.E.Dam+Σw(I.E.DAM−I.E.i)
where I.E. is the total interaction energy, i represents each of the non-Dam MTases being used for the selectivity screen, and w is a weighting factor equivalent to 1/n, where n is the number of non-Dam MTases. In this scheme, the absolute binding to EcoDam is combined with the relative binding to EcoDam with respect to the non-Dam MTases. Thus, if a compound binds very favorably to EcoDam as well as to the non-Dam MTases its overall score will be relatively low, while a compound that binds less favorably to non-Dam MTases but binds even less favorably to the on-Darn MTases will score higher. If deemed necessary the weighting of the EcoDam interaction energy versus that of the non-Dam MTases can be adjusted. For example, if specificity problems are particularly problematic with respect to one of the non-Dam MTases, its weighting can be increased relative to the others, causing selectivity with respect to it to have a larger impact on the final score. The use of the total interaction energy, versus the vdW interaction energy used in the initial, method 1 screen, allows both electrostatic and vdW contributions to be taken into account during the second stage of the screening process. This is appropriate as compounds whose binding is dominated by non-specific electrostatics are eliminated in the initial screen. From the method 2 selectivity screen the top 1000 compounds are chosen for the chemical similarity analysis (Butina, 1999), a step that maximizes the chemical diversity of the final compounds selected for biological assay that has been shown to improve screening hit rates (Huang et al., 2004). In this process, chemical similarity is quantified based on chemical fingerprints in combination with the Tanimoto index yielding approximately 100 clusters of chemically similar compounds. One or two compounds are selected from each cluster for biological assay. This final selection process considers stability, potential toxicity, and solubility [i.e. Lipinski's rule of 5 (Lipinski, 2000)], where solubility is estimated via calculated log P values using the Molecular Operating Environment (MOE, Chemical Computing Group). Selected compounds are purchased from the appropriate vendors.
Lead identification potential pitfalls and alternatives. ISS via database searching makes a number of simplifications in order to minimize computer requirements, allowing for the database of 3 million compounds to be searched. Of these simplifications the two most important are (1) the lack of conformational flexibility in the protein (Carlson, 2002) and (2) the simplified scoring function. If either of these assumptions is indicated to be problematic due to a low number of active compounds identified in method 2, the following steps are taken.
To account for protein flexibility multiple structures are used for the method 2 docking. Additional conformations (typically 5) for EcoDam are obtained from a molecular dynamics (MD) simulation and included in the method 2 search, such that each of the 50,000 compounds are screened against each conformation, with the most favorable score for each compound used for the final ranking. The additional conformations are generated via molecular dynamics (MD) simulations of the region of the protein being targeted, using the molecular modeling program CHARMM (Brooks et al., 1983; MacKerell et al., 1998b). These simulations are performed as previously described (Huang et al., 2003) for 5 ns in explicit solvent using the CHARMM22 protein force field (MacKerell et al., 1998a) that includes the recent revision to the treatment of the protein backbone (MacKerell et al., 2004).
Alternate scoring methods are attempted if the hit rate (i.e. number of active compounds selected) is deemed inadequate. One alternate approach is consensus scoring (Charifson et al., 1999), a method that applies multiple scoring function to rank compounds. This approach includes knowledge-based scoring methods that have been shown to yield improvements in the selection of correct orientations of ligands and have the advantage that they implicitly include certain aspects of salvation effects. Additional alternate approaches include generalized linear response methods (Åqvist et al., 1994; Lamb et al., 1999) and free energy of salvation based on the Generalized Born (GB) model (Feig and Brooks, 2004), including a GB version recently implemented in the program DOCK (Kang et al., 2004; Zou et al., 1999).
Each lead compound identified by ISS is evaluated for its potential to be chemically optimized (guided by the Lipinski parameters for the most desirable properties of lead-like molecules). The toxicity is determined first in cells, then worm (Anyanful et al., 2005), then mice. In vitro and in vivo efficacy is evaluated by the ability to prevent and treat disease caused by particular infections pathogens, like pathogenic E. coli and Salmonella, using mouse models. The primary criteria for optimization are potency against the target enzyme (Dam), negative selectivity against the mammalian MTases, no or low host toxicity. Lastly, co-crystal structures of Dam with lead inhibitors are determined and an iterative approach used to design derivative analogs around the core structure with more desirable properties.
Activity testing encompasses biochemical, in vitro and in vivo assays. These assays are available to test Dam inhibitors identified by ISS and/or computer-aided drug design. For example, the assay can assess DNA methylation in a biochemical system, pedestal formation in whole cells in vitro, or the mouse pathogen Citrobacter rodentium as a model of pathogenic E. coli disease in vivo. See, for example, Swimm et al. (2004); Wei et al. (2005).
High throughput assays (HTA) that allow screening of several hundred to thousand compounds are used. In particular, two HTA assays are used: (1) in vitro HTA in microplate format; and (2) cell-based high throughput virulence inhibition assay.
In vitro high throughput assay in microplate format: For the analysis of DNA methylation, we use a microplate assay that utilizes a biotinylated oligonucleotide substrates for analysis of DNA methylation (Roth 2000) The assay uses labeled [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radioactivity determined by liquid scintillation counting. As an example, we studied methylation of DNA by the EcoDam shown in
82 compounds were screened, at a compound concentration of 200 μM, in a microplate assay to assess their ability to inhibit Dam. The test was repeated using only the positives (with > 2 fold inhibition). All 9 positive compounds (Table 8) were “identified” by DOCK as potential inhibitors of Dam. This is a good validation of the ISS. The microplate assay uses labeled [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radioactivity determined by liquid scintillation counting. Compound #78 (e.g. NCI 659390) was found also to inhibit actin pedestal formation in a cell-based virulence inhibition assay (see
Cell and animal models for pathogenic E. coli infection: Enteropathogenic E. coli (EPEC), which is closely related to enterohemorrhagic E. coli O157:H7 (EHEC), and the closely related mouse pathogen Citrobacter rodentium all cause attaching and effacing (A/E) lesions, characterized by flattening of intestinal microvilli, adherence of the bacteria to epithelial cells, and reorganization of the host actin cytoskeleton, which result in the formation of an actin-filled membrane protrusion or “pedestal” beneath each bacterium (Goosney et al., 2000; Knutton et al., 1989). Pedestal formation is readily detected on cultured fibroblasts (see
To determine whether formation of actin pedestals by EPEC could be used to screen for drugs which inhibit virulence, we tested each of the 82 compounds. 3T3 cells were plated in 96 well optical dishes. In this “proof of principle” experiment, each of the 82 compounds was added to a well at 20 μM, and cells were infected with EPEC for 6 hrs. The optical density (OD600) of the supernatant was assessed to estimate effects on bacterial growth, and the cells were fixed and stained with DAPI to recognize bacteria (and cell nuclei), and FITC-phalloidin to recognize filamentous actin. Inhibition of actin pedestals is readily identifiable as the loss of intense actin staining (Kalman et al., 1999; Swimm et al., 2004a). The plate was then scanned visually on an inverted Zeiss 200M fluorescence microscope with a 20× objective. All the wells on the dish were examined with 5 minutes (a high throughput format). At low power, actin pedestals are seen as intense fluorescence apposed to groups of bacteria (see
Formation of pedestals is highly correlated with the development of diarrhea, but its relationship to the onset of disease is poorly understood. Of importance here, pedestal formation is an indicator of pathogenic E. coli virulence and is readily amenable to high throughput drug screening protocols. To make pedestals, EPEC initially attaches loosely to epithelial cells and then inserts its Type III secretion system into the host cell plasma membrane, and secretes several virulence factors into the host cytoplasm and membrane (Goosney et al., 2000), including the translocated intimin receptor (Tir) (Kenney et al., 1999). We also assessed effect of compound G6 on expression of the bacterial virulence factor Tir. As seen in
As demonstrated FIGS. 31-33, the formation of actin pedestals by (EPEC) can be used to screen for drugs that inhibit virulence. As a control, we assess pedestal formation with an EPEC strain having a nonpolar deletion of the gene encoding the Dam MTase. To do this, we employ the methods of Datsenko and Wanner, a highly efficient means of inactivating E. colligenes. This method has been used to generate a nonpolar deletion in tnaA. Briefly, the method utilizes PCR and the bacteriophage a Red recombination system, and it avoids the labor-intensive process of creating the gene disruption on a plasmid vector. The Red system encodes three gene products, Gam, Bet, and Exo. Gam inhibits the host RecBCD exonucleaseV so that Bet and Exo can gain access to linear DNA ends to promote recombination. We use PCR to generate linear DNA containing Dam adjacent sequences flanking two FRT (FLP recognition target) sites that surround the chloramphenicol (Cm) resistance gene. The product is treated with DpnI (to eliminate methylated (unamplified) template DNA), re-purified, and then electroporated into an EPEC strain, which contains the Red helper plasmid pKD46. Mutants are selected on LB agar containing chloramphenicol and ampicillin plus 1 mM L-arabinose to induce the Red genes. Mutants are passaged on nonselective medium to allow segregational loss of the Red helper plasmid (rendering them Amps). Recombinational insertion of the deleted gene is verified by PCR analysis utilizing appropriate primers. Elimination of carR is performed using a helper plasmid encoding the FLP recombinase, which is curable by growth at 43° C. PCR products generated from this region are sequenced for verification of the deletion. Dam− phenotype is demonstrated by resistance to DpnI digestion and sensitivity to DpnII digestion. Complementation assays of the deletion strain are performed by expression of the Dam protein from an appropriate plasmid. The Dam-deletion strain is used as a control in the actin pedestal assay and in the mouse virulence assay.
Compounds identified as inhibitory in the in vitro assay, but having no affect on actin pedestal formation in cell-based assay, the likely reason is inability of the compound to enter the bacteria. If this occurs, the compounds can be optimized, as known in the art, to permit entry into the bacteria. To test whether these compounds affect bacterial growth and methylation, E. coli cultures carrying pUC19 plasmid are grown in the presence of various concentrations of the inhibitors. The plasmid is isolated and digested with restriction enzyme DpnI (cuts only methylated DNA) and KpnII (cuts only unmethylated DNA) to analyze its methylation status. Any loss of methylation in this assay indicates that the inhibitor can enter the bacterial cell and inhibit Dam activity. We assay 40 NCI compounds (identified by DOCK for T4Dam) at 50 μM, and some compounds displayed slight incomplete DpnI digestion (I56 in
If more sensitive assays are required arising from the requirement of inhibition of the majority of methylation activity in bacteria, other bacterial-based assays can be employed. One such assay is outlined in
Screening of lead Dam MTase inhibitory compounds in mice. The mouse pathogen Citrobacter nodentium is a model of pathogenic E. coli disease. C. rodentium colonizes the colon, and causes A/E lesions, colonic hyperplasia, and inflammation reminiscent of EPEC effects in humans. C57BL/6 mice have been infected with EPEC using an approach developed for Salmonella infection of mice. Barthel et al. 2003. This approach utilizes short-term streptomycin treatment to reduce, though not eliminate, intestinal flora. Upon infection of streptomycin-treated mice with EPEC (Strr), colons became colonized, developed epithelial hyperplasia at 7 days post infection (=pi), and had elevated neutrophil recruitment as measured by myeloperoxidase (MPO) levels (
Attaching and effacing (A/E) lesions are essential for EPEC and C. rodentium to colonize the colon, so we expect that Dam MTase inhibitors that block A/E lesions in vitro (see preliminary results) to decrease bacterial load in vivo. Oral ingestion of EPEC or C. rodentium results in 109-1010 colony forming units (CFU) per gram of colon tissue by 10 days pi. Typically, the pathogen is cleared by 6 weeks pi in normal adult mice. Initial experiments determine safety of compounds in non-infected mice and bioavailability. We deliver the compound by Alzet osmotic pumps placed subcutaneously (Reeves et al. 2005). These pumps have the capacity to deliver drug continuously, thus minimizing the effects of drug half life on bioavailability. Serum drug levels are measured by Liquid Chromatography Mass Spectroscopy. We conduct a thorough examination of blood enzyme levels and look for other evidence of pathology by autopsy.
Next we determine the effect of the leading compounds on bacterial levels in EPEC- or C. rodentium-infected mice. C57BL/6 mice are orally infected with 2.5×108 CFU in 200 μL phosphate buffered saline (PBS). The mice are treated with drug or carrier for ten days. At day 10 pi, mice are sacrificed and colons harvested, homogenized mechanically, and serially diluted. The number of viable bacteria is determined by plating on MacConkey agar, which is selective for gram negative organisms. C. rodentium colonies are easily distinguished by their pink centers rimmed with white (Wei et al. 2005). We then determine whether the drug reduces bacterial-associated pathology in infected mice. C. rodentium or EPEC infection in mice causes weight loss, reduced activity, diarrhea, ruffled fur, and a hunched posture (data not shown). Immunocompetent mice are able to resolve the infection by six weeks pi and recover normal appearance and activity. In mice sacrificed prior to recovery, histological analysis of the colon reveals an obvious increase in mass (hyperplasia), crypt heights, and infiltration of lymphocytes and granulocytes (Wei et al. 2005).
Drugs that prevent A/E lesion formation reduce EPEC- or C. rodentium-associated disease parameters in infected mice. Mice are orally infected and treated with drug or carrier. To approximate a more realistic clinical scenario, a second group of mice are treated with drug or carrier upon display of disease symptoms (typically by day 10 pi). Mice are weighed every day and visually observed for signs of physical distress (listlessness, hunched posture, perianal fecal staining). Mice are sacrificed on days 14 and 24 pi and their colons examined histologically for signs of disease (3 μm sections cut and stained with hematoxylin and eosin). Crypt heights are measured by micrometry and appearance of mice are graded by an observer blind to the treatment group: One point is assigned to each condition: listlessness, ruffled coat, prolapsed rectum, perianal fecal staining (maximum score=4; minimum score (robust health)=0). Crypt height and body weight results are expressed as average values+/−one standard error. Treatment groups include at least ten mice. Statistical analysis is calculated by the Mann-Whitney t test, with p<0.01 considered significant. If a drug treated group reduces pathology scores, we can conclude that drug therapy positively affects C. rodentium disease outcome.
Methodologies for drug delivery in mice. Infection models together with drug delivery and detection systems are available. For example, the role of Abl-family tyrosine kinases in poxvirus virulence (Reeves et al., 2005) has been established by examining the effects of Abl-kinase inhibitors (e.g. Gleevec and PD-166326) on viral spread and survival in mice. Methodologies were developed to detect these compounds in mouse serum using Liquid Chromatography Mass Spectroscopy and to measure half-life of these compounds in mice (Wolff et al. 2005). These technologies are readily applicable to the detection of DAM MTase inhibitory compounds in serum samples. Some compounds can be delivered by oral lavage, but for others, the measured half-life in mice is short (about 4 hrs), and require delivery via continuous release Azlet osmotic pumps placed subcutaneously prior to or after infection. Other methodologies that solubilize compounds or otherwise improve their bioavailability can be utilized. Together, these methodologies have allowed successful treatment of infections caused by pathogenic microbes in mice.
Investigation of Inhibition mechanisms. The mechanism(s) of action of the three leading compounds are analyzed kinetically to address the mechanism of inhibition: competitive, uncompetitive or non-competitive with AdoMet or DNA. This information on whether the inhibitor interferes with coenzyme binding, specific DNA binding or conformational changes that can be compared with the predictions based on the docking studies. Finally, co-crystallization of Dam-inhibitor complexes are conducted. The information derived from Dam-inhibitor complex structure is used to identify site(s) of structural variability to generate derivatives around the same functional core structure, via synthesis of a compound library, with more desirable pharmacological properties.
Kinetic studies can be complemented by various additional established assays to further investigate coenzyme and DNA binding:
DNA binding studies. DNA binding by EcoDam and other MTases can be studied using nitrocellulose filter binding and surface plasmon resonance (BiaCore). These assays allow fast and reliable determination of equilibrium binding constants and the effects of inhibitors on the binding equilibrium. SPR BiaCore also permits determination of rate constants of DNA binding and release.
AdoMet binding studies. The kinetics of AdoMet binding to EcoDam can be monitored directly by a change of the intrinsic fluorescence to Trp10 (Liebert and Jeltsch, unpublished). Fluorescence effects are detectable in binary as well as ternary complexes. Therefore, this assay permits measurement of any effect of the inhibitors on AdoMet binding directly and with high sensitivity.
Target base flipping (2AP-based assay). An objective of the present invention is the development of inhibitors that specifically interfere with binding of the Dam enzyme to specific GATC sites and conformational changes. One of the most impressive conformational changes of the enzyme-DNA complex that precedes methylation is the flipping of the target base out of the DNA helix. The mechanism of base flipping has been studied by stopped-flow kinetics using a substrate that contains 2-aminopurine. This base analog provides strong fluorescence signal after DNA bending and base flipping, correlating the 2-aminopurine signal in EcoDam to base flipping (Liebert et al. 2004). Results of this assay indicate that base flipping and DNA recognition are tightly coupled and interwoven processes. Base flipping takes place in a biphasic manner, first the target base is rotated out of the DNA in a very fast reaction and later the target base is tightly contacted by the enzyme and positioned in the active site pocket (Liebert et al. 2004). An inhibitor that binds into the binding pocket of the target base may specifically prevent the base flipping. By following 2-aminopurine fluorescence in equilibrium and using rapid kinetics approaches, the possible influence of MTase inhibitors on this conformational change can be examined.
When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups, including any isomers and enantiomers of the group members, and classes of compounds that can be formed using the substituents are disclosed separately. When a compound is claimed, it should be understood that compounds known in the art including the compounds disclosed in the references disclosed herein are not intended to be included. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.
Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination. One of ordinary skill in the art will appreciate that methods, device elements, starting materials, synthetic methods, structures, libraries and assays other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials, synthetic methods, and structures, libraries and assays are intended to be included in this invention. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g. Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. 1).
It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above also may be used in veterinary medicine.
Depending on the specific conditions being treated and the targeting method selected, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Alfonso and Gennaro (1995). Suitable routes may include, for example, oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, or intramedullary injections, as well as intrathecal, intravenous, or intraperitoneal injections.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection. Appropriate compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions, including those formulated for delayed release or only to be released when the pharmaceutical reaches the small or large intestine.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers: In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material, including Table 9 listing of references; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
E. coli Dam (%)
Escherichla coli
Erwinia chrysanthemi
Salmonella enterica
serovar Typhimurium
Klebsiella pneumoniae
Yersinia
pseudotuberculosis
Vibrio cholerae
Pasteurella multocida
Haemophilis influenzae
Neisseria meningitidis
E. coli Crystallization Summary
enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect Immun
Haemophilus influenzae tetranucleotide repeats. Embo J 21, 1465-1476.
coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640-6645.
Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity. Proc Natl
Salmonella DNA adenine methylase mutants confer cross-protective immunity. Infect Immun 69, 6725-6730.
flexneri shows no significant attenuation of virulence. Microbiology 150, 1073-1078.
pseudotuberculosis and Vibrio cholerae. Infect Immun 69, 7610-7615.
Diplococcus pneumoniae with respect to DNA methylation. J Mol Biol 114, 153-168.
Escherichia coli. Mol Microbiol 45, 673-695.
Typhimurium virulence in Caenorhabditis elegans. FEMS Microbiol Lett. 245(1): 53-9.
Mycobacterium tuberculosis. Biochem Biophys Acta 1545, 67-77.
coli serotype. N Engl J Med 308, 681-685.
Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing
Escherichia coli dam DNA methyltransferase modifies DNA in a highly processive reaction. J Mol Biol
Escherichia coli. Mol Microbiol 11, 605-618.
This invention was made with government support under GM49245 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/44277 | 12/6/2005 | WO | 00 | 8/7/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60634066 | Dec 2004 | US |
