PYRIDINE NUCLEOTIDE-DEPENDENT ENZYMES

Abstract: The invention discloses a bisubstrate analogue effective at inhibiting pyridine nucleotide-dependent enzyme(s). Additionally, disclosed is a method for manufacture of said binding substrate analogue and a complex and drug relating to same. The analogue comprises Diazaborine or part thereof for use in the rational design of bisubstrate enzyme inhibitors which exploit binding energy so as to produce an effective inihibitor, thereby reducing the size of the component of the drug that mimics the substrate. Thus, a compound is produced which is more difficult for an organism to generate resistance against without also compromising the ability of its enzyme to function effectively.

Description: PYRIDINE NUCLEOTIPE - DEPENDENT ENZYMES The invention relates to pyridine nucleotide-dependent enzymes and more particularly, but not exclusively, to the development of inhibitors for binding thereto. Pyridine nucleotide-dependent enzymes, such as those classified as E.C. 1.x.1 or E.C. l.x.3 where x equals a number determined by the substrate range of the relevant enzymes, such as NAD(P)-dependent oxidoreductases, comprise a family of enzymes to which the present invention relates. These enzymes are involved in a large number of biochemical reactions. Advantageously, within this class of enzymes there exists interspecies variation. Thus given the importance of these enzymes in metabolism they represent suitable target sites for drugs. Indeed, antibacterial, antifungal, antiviral, anticancer drugs and other drugs have been developed having regard to these enzymes. In so far as bacteria are concerned the widespread use of antibiotics has provided a powerful selective pressure driving the development of multi drug resistance in many species of bacteria with consequent problems in the treatment of bacterial infections. However, with the increasing availability of structural information on proteins, recent attention has focused on the use of this mformation in strategies for the design of new drugs. Indeed, this data can be used to identify interspecies variations in enzyme structure such as, for example, in the multi functional enzyme complex fatty acids synthase (FAS) where it has been shown that in the type I FAS complex, found in eukaryotes and yeast, all the catalytic domains reside on one or two polypeptides, whereas the molecular structures of the type II FAS, found in plants and most prokaryotes, are considerably different, with the enzymes that catalyse the individual steps being found on separate polypeptides. Recent studies have identified enoyl reductase (ENR), the enzyme that catalyses the final reaction of FAS, as the target for a number of significant therapeutic agents against M. tuberculosis and E. coli. M. tuberculosis ENR is the target for a metabolite of isoniazid, a potent drug that is only active against tubercle bacilli and is used in the front-line chemotherapeutic treatment of tuberculosis, a chronic infectious disease that has afflicted humanity for five millennia and kills more people than any other infection. However, strains of M. tuberculosis are emerging that are resistant to one or more of the main antituberculosis drugs including isoniazid creating potentially severe problems in its treatment. E. coli ENR is inhibited by a range of diazaborines, which are heterocyclic boron containing compounds, whose action is thought to lead to the inhibition of cell growth by preventing lipopolysaccharide synthesis. Biochemical studies on the E. coli enzyme have shown that NAD * is required for diazaborine binding and this has led to the suggestion that the drug either binds to ENR in association with NAD * or that NAD * converts the drug to an active form (1). This application reports that the structure determination and analysis of E. coli ENR complexed with NAD * and with NAD * and a number of diazaborine derivatives to provide a molecular explanation for the inhibitory activities of this class of antibacterial agents. Indeed, we have discovered that agents active against pyridine nucleotide- dependent enzymes exert their effect, at least in part, by binding covalently to the ribose ring of these enzymes and particularly to the 2' hydroxyl group of the mcotinamide ribose and more specifically by binding covalently to the 2' hydroxyl group of the nicotinamide ribose. This is a fortuitous finding given that, conventionally, the design of agents used to block the activity of pyridine nucleotide-dependent enzymes has focused on interaction with the opposite end of the nicotinamide ribose i.e. the side at which oxidation/reduction takes place. This information is therefore of considerable importance in the design and development of new drugs because it clearly implies that the exploitation of this linking mechanism can be used to advantage. It is interesting to note that the use of this link can be advantageously exploited in the design of new inhibitors because the binding energy associated with the ribose ring, or an analogue thereof, can be used to compensate for a reduction in binding energy between a substrate analogue and the enzyme. This has technical significance in that it enables us to reduce the size of that part of a drug that mimics the substrate. This means it will be more difficult for the organism to generate resistance to the drug without also compromising its ability to act as an effective enzyme for the natural substrate. It is therefore an object of the invention to provide a link for binding a drug to a pyridine nucleotide-dependent enzyme. It is a further object of the invention to provide a binding substrate analogue for use with a pyridine nucleotide-dependent enzyme. It is yet a further object of the invention to provide a method for the manufacture of said analogue employing the use of a link in accordance with the invention. It is a further object still to provide a generic mechanism for the synthesis of a pyridine nucleotide-dependent enzyme inhibitor which mimics a substrate analogue of the enzyme. According to a first aspect of the invention there is therefore provided a bisubstrate analogue effective at inhibiting the activity of a pyridine nucleotide-dependent enzyme comprising a first part, at least a portion of which is adapted to mimic at least a part of a substrate for said enzyme so as to block the activity of same and, bound thereto, a pyridine nucleotide, or analogue thereof, characterised in that said binding comprises a bond between said first part and a functional part of the ribose ring of the pyridine nucleotide of said second part. According to a yet further aspect of the invention there is provided a complex comprising a pyridine nucleotide-dependent enzyme and a bisubstrate analogue in accordance with the invention. According to a yet further aspect of the invention there is provided a complex comprising a pyridine nucleotide-dependent enzyme, a pyridine nucleotide, or analogue thereof, and an analogue of at least a part of a substrate for said enzyme characterised in that said analogue is bound to said pyridine nucleotide via a bond created between said analogue and a functional part of the ribose ring of said pyridine nucleotide. According to a yet further aspect of the invention there is provided a method for the manufacture of a bisubstrate analogue for use in blocking the activity of a pyridine nucleotide-dependent enzyme which method comprises binding a first part of said analogue, which comprises at least a portion that is adapted 5 to mimic at least a part of a substrate for said enzyme, to a second part of said analogue, which comprises a pyridine nucleotide, or analogue thereof, by linking said first part to said second part using a bond between said first part and the ribose ring of said pyridine nucleotide of said second part. In a preferred embodiment of the invention said bond is a covalent bond. More preferably still, said bond, alternatively or in addition, is between said part or analogue and any selected functional group that can form said bond such as, the 2' hydroxyl group of the nicotinamide ribose or an alternative group or functionality located at this position. According to a yet further aspect of the invention there is provided a product produced by the aforementioned method of the invention. According to a yet further aspect of the invention there is provided a drug adapted to block the activity of a pyridine nucleotide-dependent enzyme comprising a portion which is adapted to mimic at least a part of a substrate for said enzyme and a portion which is capable of forming a bond between a functional group of a ribose ring of a pyridine nucleotide, or analogue thereof. In a preferred embodiment of the invention said bond is between the 2' hydroxyl group of said nucleotide or an alternative group or functionality located at this position. The functional group may be selected from any that is capable of forming said bond. In the above preferred embodiments of the invention said analogue or portion includes a Diazaborine, or part thereof. In a yet further preferred embodiment of the invention said enzyme is a NAD(P) - dependent oxidoreductase such as, for example but not limited to, a dehydrogenase or an enoyl reductase. Moreover, reference herein to a substrate for said enzyme is intended to include reference to any such suitable substrate including, without limitation, a nucleotide, including, without limitation, a purine or pyridine nucleotide. In the instance where said substrate of said enzyme comprises the aforementioned nucleotide reference herein to a bond between at least a part of said substrate for said enzyme and said pyridine nucleotide is intended to include reference to the 2' hydroxyl group of either, or both, the said substrate or the said nucleotide. An embodiment of the invention will now be described with reference to the following figures wherein: Figure 1 Schematic representation of the interactions made by the NAD * -thieno- diazaborine complex with the enzyme surface and ordered solvent molecules. For the NAD * , only the nicotinamide ring and the nicotinamide ribose are shown. Hydrogen bonds are represented by dashed lines and hydrophobic contacts shown as "highlighted" semi-circular arcs (Produced using Ligplot (38)). Figure 2a 7 Initial Fourier map of the NAD * -thieno-diazaborine complex at 2.2A resolution, with the refined structure superimposed. The density (contoured at 1.2σ) was calculated with coefficients 2 ' F 0 ' - ! F c j and α c phases calculated from the refined structure with no inhibitor atoms (Produced using the program Molscript (39)). Figure 2b Initial Fourier map of the NAD * -benzo-diazaborine complex at 2.5A resolution with the refined structure superimposed. The density (contoured at 0.9σ) was calculated with coefficients 2 JFJ-J FJ and α c phases calculated from the refined ENR-NAD * structure with no inhibitor atoms (Produced using the program Molscript (39)). Figure 3 Stereoview of the local superposition of the active site of the nucleotide/inhibitor complexes of ENR and DHFR (PDB entry 3DFR, 26). The Cα backbone trace, NAD * and thieno-diazaborine are shown in green for ENR and the Cα backbone trace, NADP * and methotrexate are shown in red for DHFR (produced using FRODO (76)). Table 1 X-ray data collection and phasing statistics. The Structure of E. coli ENR E. coli ENR is a homo-tetramer of subunit M R of approximately 28,000 and was prepared from an over-expressing E. coli strain (10,11). Crystals of the ENR- NAD* complex (crystal form A) belong to spacegroup P2 j with a tetramer in the 8 asymmetric unit (12) and the structure was initially solved by a combination of isomorphous replacement and molecular replacement (13) using the Brassica napus ENR structure (14) as a search model. The statistics of the structure determination are presented in Table 1. The initial map was improved by four-fold averaging and solvent flattening (IS) and a model was constructed using the graphics program FRODO (16) . This structure was submitted to least-squares refinement using the program TNT (17) with data to 2.1 A and further cycles of model building and refinement were performed. In the final map of the ENR-NAD + complex, the electron density is of high quality for most of the protein atoms. However, the density for the nicotinamide moiety and its associated ribose ring in the cof actor is noticeably weaker than that seen for the rest of the NAD + . There is a break in the density for a stretch of 10 amino acid residues from Leul95 to Met206, which forms a loop between strand β6 and helix A6 (the secondary structure elements have been assigned as in (14)) which borders the nucleotide binding site. In the B. napus structure, residues in the equivalent region (residues Ala240 to Thr249) have high temperature factors and have been implicated in substrate binding (14) . Therefore, this disorder may reflect the fact that the acyl substrate is not present in the crystals of the E. coli enzyme. In addition, in this crystal form of the E.coli enzyme, there is no interpretable density for the first residue and the last four residues and thus, the final model comprises coordinates for the nucleotide and for 248 of the expected 262 residues per subunit of the protein, and has an R factor of 0.157 for all data from 10 to 2.1 A resolution (13). Crystals of the ENR complexed with NAD ' * and either thieno-diazaborine or benzo-diazaborine (crystal form B) belong to spacegroup P6 X 22 with a dimer in the asymmetric unit (12) . The structures were solved at 2.2 and 2.5A, respectively, by molecular replacement using the refined structure of the E. coli ENR-NAD + complex as a search model (18). In the structures of both the diazaborine bound complexes there was exceptionally clear electron density for the drugs. Furthermore, in contrast to the structure of the complex of the enzyme with NAD * alone, the electron density for the entire cof actor was well defined as was that for the previously disordered loop joining β6 and a6. However, for both the diazaborine complexes, there was no interpretable density for either the first residue or for the last four residues. Thus, the final refined models for the structures of the thieno-diazaborine or benzo-diazaborine complexes consist of coordinates for the nucleotide, the diazaborines and 257 of the expected 262 amino acid residues per subunit, with R factors of 0.191 and 0.169 for all data from 10 to 2.2A and 10 to 2.5A, respectively (18). The E. coli ENR tetramer is made up of four subunits each consisting of a single domain of approximate dimensions 55 x 45 x 45A composed of a parallel β sheet of seven strands (β\-β7) , flanked on one side by helices al , a2 and al and on the other by helices a3-a5, with a further helix, a6, lying along the top of the β sheet. The fold of the polypeptide chain is highly reminiscent of the Rossmann fold and the cof actor is bound in a similar and extended conformation to that observed in other NAD(P)H-dependent oxidreductases, at the COOH-terminal end of the β sheet with the nicotinamide ring lying deep in a pocket on the enzyme surface and bounded by the side chains of Ile20, Tyrl46, Alal89, Ilel92, Phe203 and the main chain polypeptide from Alal89 to Ilel92. Studies Involving Diazaborines The electron density for both diazaborines enabled the unambiguous positioning of the bicyclic thieno- or benzo-diazaborine rings, the sulphonyl groups and the respective propyl or tosyl substituents. Both diazaborine compounds bind in a closely related manner, adjacent to the nicotinamide ring of the cofactor, in a pocket formed by the side chains of Tyrl46, Tyrl56, Metl59, Ile200, Phe203, LeulOO, Lysl63 and the main chain peptide between Gly93 and Ala95. The bicyclic rings of the diazaborines form a face to face interaction with the 10 nicotinamide ring, allowing the formation of an extensive π-π stacking interaction with additional van der Waals interactions between the rings and the side chains of Tyrl56, Tyrl46, Phe203 and Ile200. The only difference between the binding of the two diazaborines is that their respective tosyl and propyl groups occupy subtly modified positions on the enzyme. In the benzo-diazaborine, the tosyl moiety lies perpendicular to the bicyclic ring and interacts with the main chain peptide between Gly93 and Ala95 and the side chain of LeulOO, whereas the propyl moiety of the thieno-diazaborine folds back onto the planar bicyclic ring system in a manner reminiscent of a scorpion's tail and forms interactions with the side chain of Metl59 and Ile200 and the main chain peptide of both Gly93 and Phe 94. It is interesting to note that the loop between Bό and A6, which is ordered in this crystal form, provides two residues, Ile200 and Phe203, whose side chains are in van der Waals contact with the non-boron containing 5- and 6- membered rings of the thieno- and benzo-diazaborines, respectively. Additional interactions made by the drug include a hydrogen bond between a nitrogen in the boron containing ring and an ordered solvent molecule which in turn hydrogen bonds to the pyrophosphate moiety of the NAD * . In the thieno-diazaborine complex the same solvent molecule forms a hydrogen bond to an oxygen of the sulphonyl group. Also the boron hydroxyl and the phenolic hydroxyl of Tyrl56 are 2.όA apart in both complexes and presumably form a hydrogen bond (Figure 1). The high quality of the preliminary electron density maps, particularly for the thieno-diazaborine complex at 2.2 A, permitted accurate positions to be assigned to both the NAD* and the drug. Furthermore, and totally unexpectedly, we observed that the distance between the boron atom of the diazaborine and the 2 'OH of the nicotinamide ribose was 1.7A. This is comparable with a B-O covalent bond length of 1.6 A and, given that the errors in coordinates are very small, we can be confident that the interaction between these two atoms is covalent. This is further supported by the continuous density between the 2 'OH of the nicotinamide ribose and the boron and the unambiguous identification of the position of the hydroxyl 11 oxygen to which the boron is linked, which can be seen to form part of a tetrahedral, rather than a trigonal, arrangement as required if the boron forms four covalent bonds (Figures 2a and b). This finding provides a clear explanation for the strong inhibitory properties of the diazaborines and why they only bind to the enzyme in the presence of NAD* . The position of the aromatic bicyclic ring of the diazaborine above the mcotinamide ring, strongly resembles the proposed model for the binding of the enoyl substrate suggested from studies on B. napus ENR (14). The reaction mechanism for this enzyme is proposed to proceed through the formation of an enolate anion with Tyrl56, acting as a proton donor to the negatively charged alkoxide of the enolate intermediate (14). The coordination of the boron demands that it must possess a full negative charge which will be partially distributed between the four atoms to which it is attached. It is therefore interesting to note that the proposed position for the negatively charged oxygen of the alkoxide lies close to the observed position for the boron atom in the drug. The amino group of the putative catalytic lysine (Lysl63) is only 4.1 A from the boron atom and therefore this residue may well afford partial stabilization of the negatively charged boron, in a manner similar to its role in the stabilization of the transition state during catalysis. We anticipate that there must be subtle differences in the mode of binding of the substrate and the drug as the covalent bond formation observed between the ribose 2 'OH and the boron atom allows close approach of these atoms in a manner that cannot be mirrored in the enzyme/substrate complex. Nevertheless, it is clear that the inhibitory action of the diazaborines derives in part from their structural resemblance to the enzymes substrate. Analysis of the structure of an S94A mutant of mycobacterial ENR (19), which is resistant to isoniazid, has given rise to the suggestion that the mutation weakens the binding affinity of this enzyme for NAD* through the modification of a hydrogen bonding network associated with its pyrophosphate moiety and that this then leads 12 to reduced affinity of the enzyme for the derivative of isoniazid which forms the active compound. Based upon this finding, a similar mechanism has been proposed for diazaborine resistance in a G93S mutation in E. coli ENR (11, 19). Examination of the structure of the ENR/diazaborine complexes reveals that Gly93 does indeed lie close to the nicotinamide binding site with its alpha carbon 3.4 A from the diazaborine. However, modelling studies show that in the absence of changes to the main chain torsion angles in the G93S mutant, the Cβ atom of the serine side chain would be unacceptably close to the two oxygens of the sulphonyl group of the diazaborine (2.lA and 2.6A, respectively). Thus, the resistance to diazaborine is explained by the serine side chain of the G93S mutant encroaching into the drug binding site and causing severe steric hindrance. The structure of the E. coli enzyme is closely related to that of its mycobacterial counterpart (Protein Data Bank (PDB) entry 1ENY, 19), particularly in the region of the active site and 199 Cα atoms from these two enzymes can be superimposed with a root mean square deviation of 1.06 A. Of the eight amino acids which have one or more atoms lying within a 4 A radius of any part of the thieno-diazaborine, five are conserved in the M. tuberculosis ENR. In addition, there is a conservative change of Tyrl46 to Phe and since the phenolic hydroxyl group of Tyrl46 does not contact the drug this change is not expected to interfere with the protein/drug interactions. The remaining two residues (Leu200 and Phe203) lie in the loop between /36 and ah which orders on diazaborine binding, providing interactions with the bicyclic ring of both drugs. This loop is considerably modified in the mycobacterial enzyme due to the insertion of an extra helix but residue Metl99 lies in a comparable position to Phe203 and could provide similar interactions with the bicyclic rings and residue Metl03 could form interactions with the propyl group. The overall similarity between the structures of the E.coli and mycobacterial enzymes suggests that the appropriate diazaborine derivative or a bisubstrate analogue, which mimics the combined diazaborine/NAD + structure, could be designed to fit in the active site pocket of the mycobacterial enzyme. Therefore, the insights provided by our analysis of E. coli ENR may well be important in driving the development of new antituberculosis agents. Furthermore, the difference between the mammalian and bacterial FAS enzyme complexes may well provide opportunities to exploit ENR as a target for the design of antibacterial agents against other organisms such as the multi drug resistant strains of staphylococcus that are proving to be a problem for the current range of antibiotics (20). In ENR we can see that the diazaborines form a covalent bond with an enzyme- bound substrate to generate a tight, non-covalently bound bisubstrate analogue. In this respect they are similar to inhibitors of pyridoxal phosphate containing enzymes (e.g. gabaculine (21)) which covalently modify the cofactor. The best analogy is perhaps with 5-fluro-2-deoxyuridylic acid which acts as a potent inhibitor of thymidylate synthase (22) . This also mimics one of the substrates (the deoxynucleoside) to covalently modify the other (methylene tetrahydrofolate) to form a bisubstrate analogue. Given that the chemical reactivity of boron might make it difficult to obtain a boron-containing drug free of undesirable side effects, it is obviously important to consider the alternative possibility of designing a pre¬ formed bisubstrate analogue without boron as an approach to inhibiting this enzyme. Potent bisubstrate inhibitors of other enzymes with nucleotide substrates have been described (e.g. the polyoxin inhibitors of chitin synthase (23) and various synthetic inhibitors of protein kinase C (24)). However, hitherto, no such good bisubstrate inhibitors have been described for NAD(P)H-dependent reductases or dehydrogenases. Examples of this type designed to inhibit HMG Co A reductase were only very weak inhibitors possibly not only because of the lack of a moiety to mimic the adenosine diphosphoribose but also, quite probably, because of steric problems in the active site associated with the nature of the linkage (25). In the light of this, an important feature of the current study is that it provides clear 14 evidence for the type of linkage that may need to be created in order to synthesize a bisubstrate analogue with the necessary geometry to occupy the active site cleft within pyridine nucleotide-dependent enzymes in general. Applications for this Technology The similarities in chemistry catalysed by the family of NAD (P) -dependent oxidoreductases gives rise to generic opportunities for the creation of a series of novel enzyme inhibitors based on a related chemistry. Across the family of enzymes which belong to this class, the catalytic cycle of oxidation/reduction necessarily leads to a situation where the π electron system of a substrate is presented to the face of the rύcotinamide ring. Furthermore, the glycosidic bond between the nicotinamide ring and its associated ribose moiety generally adopts one of only two conformations which differ by a rotation of approximately 180° and which lead to the presentation of either the pro-AR or the pro-AS hydrogen of NADH to the active site. These conformations merely result in a shift in the position of the carboxyamide moiety of the nicotinamide ring on the enzyme surface and do not effect the relative positions of the nicotinamide ring to its ribose group. Thus, for those dehydrogenases where there is sufficient space around the 2 ' OH group of the nicotinamide ribose, there is an excellent opportunity to form a bisubstrate analogue using a bridging atom attached to this hydroxyl. As can be seen from figure 3, the local superposition of the active site of the nucleotide/inhibitor complexes of ENR and dihydrofolate reductase (DHFR)(PDB entry 3DFR, 26) suggest that this approach might be utilized for the design of new anti-cancer agents targeted against the latter. Some other examples of NAD(P)- dependent oxidoreductases that are known to be drug targets are Aldose Reductase a target for therapeutic agents used for the treatment of diabetic complications ( 27) , steroid 5α-reductase the target for finasteride used to treat benign prostatic hyperplasia (28) and inosine monophosphate dehydrogenase the target for mycophenolic acid an immunosuppressant (29). Thus, across the family of oxidoreductases, there would appear to be opportunities to copy the chemistry seen in the complex of ENR with diazaborines in the rational design of bisubstrate enzyme inhibitors which couple the binding energy provided by the nucleotide or a related derivative and a substrate analogue to produce an effective inhibitor. In this manner we hope to be able to reduce the size of the component of the drug that mimics the substrate, with the possibility of producing a compound which would be more difficult for the organism to generate resistance against without also compromising the ability of its enzyme to function effectively. Such a concept may well have widespread applications in many areas of pharmaceutical chemistry. 16 References 10 M.M. Kater, G.M. Koningstein, H.J.J. Nijkamp and A.R. Stuitje, Plant Mol. Biol. 25, 771 (1994). 11 H. Bergler, G. Hogenauer and F. Turnowsky, J. General Microbiology 138, 2093 (1992). 12 C. Baldock et al., Acta Cryst. D in press (1996). 13 Crystallisation of the ENR-NAD* complex has been reported elsewhere (12). Data were collected from two form A crystals to 2.5A (native 1 ) on a twin San Diego multiwire systems (SDMS) area detector using a Rigaku RU-200 rotating anode source (30,31) and to 2.1 A (native 2 ) at the DRAL Synchrotron. The data sets were processed using SDMS software (32) and the MOSFLM package (33) respectively and scaled and merged using ROTAPREP, ROT A VAT A and AGROVATA (J4;Tablel). An attempt was made to use the model of the Brassica napus ENR as a basis for a molecular replacement solution but the map calculated following refinement of the model using TNT (17) was not of sufficient quality to confidently assign residues. However, the phases were sufficient to reveal the positions of the heavy atoms in a mercury derivative by difference Fourier methods. The heavy- atom derivative was obtained by soaking an ENR-NAD* (form A) crystal for one hour in O.lmM ethylmercuriphosphate, lOmM NAD*, 20% PEG400, lOOmM acetate pH5.0. Data were collected at the DRAL Synchrotron to a resolution of 3 A (Hg) and were processed as above (see Tablel). Refinement of the heavy atom 17 parameters was performed using MLPHARE (35), and resulted in a phase set with an overall mean figure of merit of 0.34 to 3 A resolution. Using a map derived from these phases, molecular masks for the molecule were generated using the program MAMA (36) and 50 cycles of solvent flattening and fourfold molecular averaging using the program DM (15) reduced the free R factor to 24.5%. In the resultant electron-density map, calculated using the averaged phases, clear density could be found for all but the first residue, last four residues and 10 residues from the loop joining ββ and a6 and a model comprising 247 of the 262 amino acids of the E. coli ENR could be built with confidence using the graphics program FRODO (16). Several cycles of rebuilding and refinement, gave a final R factor for the model of 0.157 (52346 reflections in the range 10-2.1 A, 7836 atoms including 324 water molecules) with the root mean square deviation (rmsd) on bonds of 0.017A, and the rmsd on angles of 2.92 2 . The average B factor for the tetramer is 30.4A 2 (24.0A 2 for main chain atoms). 14 J.B. Rafferty et al., Structure 3, 927 (1995). 15 K. Cowtan, unpublished program. 16 T.A. Jones, J. Appl. Cryst. 11, 268 (1978). 17 D.E. Tronrud, L.F. Ten Eyck and B.W. Matthews, Ada Cryst. A43, 489 (1987). 18 Crystallisation of the ENR-NAD * -diazaborine complex has been reported elsewhere (12). These crystals have unit cell dimensions of a=b=80.9A c = 328.3A a=β = 90° y = 120° for the thieno-diazaborine complex and a=b = 80.όA c = 325.3A α=/3=90° y = 120° for the benzo-diazaborine complex and belong to spacegroup P6,22 (form B). Data sets were collected on the ENR- NAD* -thieno-diazaborine complex (thieno) and on the ENR-NAD * -benzo- diazaborine complex (benzo) at the DRAL Synchrotron and were processed as for crystals of form A (7J;Tablel). Cross-rotation and translation function searches were calculated on the ENR-NAD * -thieno-diazaborine data with an AB dimer from the ENR-NAD* structure using the program POLARRFN (34,37) and TFFC (34) and gave a clear solution with a signal to noise ratio of 81σ. Similar cross-rotation and translation function searches were carried out with the ENR-NAD* -benzo-diazaborine data and again a clear solution with a signal to noise ratio of 67σ was obtained. Appropriately orientated and translated models were refined against their respective data sets using the program TNT (17). The electron density maps for the diazaborine complexes were readily interpretable and clear unambiguous density could be observed for the location of the diazaborine compounds which were subsequently incorporated into the refinement. All side chains were fitted except for residue Arg204 in one subunit of the thieno-diazaborine complex and residues Arg204 and Lys205 in both subunits of the benzo-diazaborine complex. The thieno-diazaborine complex gave a final R factor of 0.191 (30 825 reflections in the range 10-2.2A, 3936 atoms), the rmsd on bonds of 0.012A, and the rmsd on angles of 2.9°. The average B factor for the dimer is 26.6 A 2 (22.0 A 2 for main chain atoms). The benzo-diazaborine complex gave a final R factor of 0.169 (20204 reflections in 19 the range 10-2.5A, 3930 atoms), the rmsd on bonds of 0.013A, and the rmsd on angles of 2.7°. The average B factor for the dimer is 23.6A 2 (20.θA 2 for main chain atoms) . 19 A. Dessen, A. Quemard, J.S. Blanchard, W.R. Jacobs Jr, and J.C Sacchettini, Science 267, 1638 (1995). 20 J.H.T. Wagenvoort, B.I. Davies, E.J.A. Westermann, T.J. Werink and H.M.J. Toenbreker, The Lancet 341, 840 (1993). 21 R.R. Rando, Biochemistry 16, 4604 (1977). 22 L.W. Hardy et al., Science 235, 448 (1987). 23 G.W. Gooday, A. de Rousset-Hall and D. Hunsley, Trans. Br. mycol. Soc. 67, 193 (1976). 24 A. Ricouart, J.C. Gesquiere, A. Tartar and C. Sergheraert, J. Med. Chem. 34, 73 (1991). 25 C. Taillefumier, D. de Fornel and Y. Chapleur, Bioorganic and Medicinal Chemistry Letters 6, 615 (1996). 26 J.T. Bolin, D.J. Filman, D.A. Matthews, R.C. Hamlin and J. Kraut, J. Biol. Chem. 257, 13650 (1982). 27 D.K.Wilson, I. Tarle, J.M. Petrash and F.A. Quiocho, Proc. Natl. Acad. Sci. USA 90, 9847 (1993). 28 H.G. Bull et al. , J. Am. Chem. Soc. 118, 2359 (1996). 29 B. Fulton and A. Markham, Drugs 51, 278 (1996). 30 R. Hamlin, Methods Enzymol. 114, 416 (1985). 1 N.H. Xuong, C. Nielson, R. Hamlin, and D. Anderson, J. Appl. Cryst. 18, 342 (1985). 32 A.J. Howard, C. Nielson, and N.H. Xuong, Methods Enzymol. 114, 452 (1985). 33 A.G. W. Leslie, Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, no. 26. SERC Daresbury Laboratory, Warrington, UK, (1992). 34 Collaborative Computational Project No. 4, Acta Cryst. D50, 760 (1994). 35 Z. Otwinowski, Proceedings of the CCP4 Study Weekend. (W. Wolf, P.R. Evans and A.G.W. Leslie, eds), SERC Daresbury Laboratory, Warrington, UK, 80 (1991). 36 G.J. Kleywegt and T.A. Jones, ESF/CCP4 Newsletter, no. 28, 56 (1993). 37 W. Kabsch, unpublished program. 38 A.C. Wallace, R.A. Laskowski and J.M. Thornton, Prot. Eng. 8, 127 (1995). 39 P.J. Kraulis, J. Applied Crys. 24, 946 (1991). v β *-. 00 J ° 3 Table 1. X-ray data collection and phasing statistics (Form A crystals) (Form B crystals) Data set " Native' Native 2 Native'* 2 Hg Thieno Benzo Resolution (A) 2.5 2.1 2.1 3.0 2.2 2.5 co c m No. of observed reflections 1 14615 86550 1 13658 20674 76176 45376 co No. of unique reflections 27520 49465 51902 14042 31179 20393 H C H Completeness (%) 78 89 93 73 95 95 m ro co 7.7 5.8 5.7 x Rmerge ? (%) 7.1 3.8 5.8 m m Fractional isomorphous difference* 0.24 H No. of heavy atom sites 6 c m Phasing power (acentric/centric)* 1.4/1.0 σ» Rcuins (acentric/centric) 1 0.76/0.68 * See (13). t R me rge where I, and I m are the observed intensity and mean intensity of related reflections, respectively, fractional isomorphous difference=∑l IFp H l-IF P l l/ΣIF P l, where F PH and F P are the structure factor amplitudes for derivative and native crystals, respectively. 'Phasing power=<F H /lack of cIosure>. l R cu n ls =<lack of closure>/<isomorphous difference>. r> H O » vo e> 00

Claims: CLAIMS 1. A bisubstrate analogue effective at inhibiting the activity of a pyridine nucleotide-dependent enzyme comprising a first part, at least a portion of which is adapted to mimic at least a part of a substrate for said enzyme so as to block the activity of same and, bound thereto, a pyridine nucleotide, or analogue thereof, characterised in that said binding comprises a bond between said first part and a functional part of the ribose ring of the pyridine nucleotide of said second part. 2. A complex comprising a pyridine nucleotide-dependent enzyme and a bisubstrate analogue in accordance with Claim 1. 3. A complex comprising a pyridine nucleotide-dependent enzyme, a pyridine nucleotide, or analogue thereof, and at least a part of a substrate or an analogue of at least a part of a substrate for said enzyme characterised in that said substrate or analogue is bound to said pyridine nucleotide via a bond created between said substrate or analogue and a functional part of the ribose ring of said pyridine nucleotide. 4. A method for the manufacture of a bisubstrate analogue for use in blocking the activity of a pyridine nucleotide-dependent enzyme which method comprises binding a first part of said analogue, which comprises at least a portion that is adapted to mimic at least a part of a substrate for said enzyme, to a second part of said analogue, which comprises a pyridine nucleotide, or analogue thereof, by linking said first part to said second part using a bond between said first part and the ribose ring of said pyridine nucleotide of said second part. 5. An analogue, complex or method according to any preceding claim wherein said bond is a covalent bond. 6. An analogue, complex or method according to any preceding claim wherein said bond, alternatively or in addition, is between said part or substrate or analogue and any selected functional group that can form said bond such as, the 2' hydroxyl group of the nicotinamide ribose or an alternative group or functionality located at this position. 7. A product produced by the aforementioned method according to Claims 4 to 6. 8. A drug adapted to block the activity of a pyridine nucleotide-dependent enzyme comprising a portion which is adapted to mimic at least a part of a substrate for said enzyme and a portion which is capable of forming a bond between a functional group of a ribose ring of a pyridine nucleotide, or analogue thereof. 9. A drug according to Claim 8 wherein said bond is between the 2' hydroxyl group of said nucleotide or an alternative group or functionality located at this position. 10. An analogue, complex or method according to any preceding claim wherein said analogue or portion thereof includes a Diazaborine, or part thereof. 11. An analogue, complex, method or drug according to any preceding claim wherein said enzyme is an NAD(P)-dependent oxidoreductase. 24 12. An analogue, complex, method or drug according to Claim 11 wherein said enzyme is a dehydrogenase. 13. An analogue, complex, method or drug according to Claim 11 wherein said enzyme is an enoyl reductase.

Inventors: RAFFERTY JOHN BERNARD, RICE DAVID WILLIAM, SLABAS ANTONI RYSZARD, STUITJE ANTOINE RAYMOND, RAFFERTY, JOHN, BERNARD, RICE, DAVID, WILLIAM, SLABAS, ANTONI, RYSZARD, STUITJE, ANTOINE, RAYMOND
Applicants: RAFFERTY JOHN BERNARD, RICE DAVID WILLIAM, SLABAS ANTONI RYSZARD, STUITJE ANTOINE RAYMOND, UNIV SHEFFIELD, RAFFERTY, JOHN, BERNARD, RICE, DAVID, WILLIAM, SLABAS, ANTONI, RYSZARD, STUITJE, ANTOINE, RAYMOND, THE UNIVERSITY OF SHEFFIELD
Application year: 1997
Main classification: C07H23
IPC codes: C07H23