Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains.

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.

O(6)-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts.

Tumour resistance to methylating agents is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O(6)-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O(6)-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O(6)-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80-175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1-11.3 days, p < 0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O(6)-benzylguanine (O(6)-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O(6)-BG.

Inactivation of O6-alkylguanine-DNA alkyltransferase. 1. Novel O6-(hetarylmethyl)guanines having basic rings in the side chain.

A number of novel guanine derivatives containing heterocyclic moieties at the O6-position have been synthesized using a purine quaternary salt which reacts with alkoxides under mild conditions. Initially O6-substituents were investigated in which the benzene ring of the known agent, O6-benzylguanine, was replaced by unsubstituted heterocyclic rings. The ability of these agents to inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase), both as pure recombinant protein and in the human lymphoblastoid cell line Raji, has been compared with that of O6-benzylguanine. The present paper focuses on O6-substituents with basic rings, and under standard conditions several of them proved more effective than benzyl for inactivation of both recombinant and Raji ATase. Among the pyridine derivatives, the 2-picolyl compound 7 is not very active in contrast to the 3- and 4-picolyl compounds, and this influenced our choice of isomers of other basic ring systems for study. Since halogen substitution in the thiophene ring considerably increased the activity (17 versus 6), similar modifications in the pyridine series were examined. The more polar O6-substituents in this study are on the whole compatible with the stereochemical requirements of the ATase protein, and their pharmacological properties may be valuable in subsequent in vivo investigations, particularly the thenyl (6), 5-thiazolylmethyl (12), 5-bromothenyl (17), and 2-chloro-4-picolyl (21) derivatives.

Solution structure of the B-Myb DNA-binding domain: a possible role for conformational instability of the protein in DNA binding and control of gene expression.

Double- and triple-resonance heteronuclear NMR spectroscopy have been used to determine the high-resolution solution structure of the minimal B-Myb DNA-binding domain (B-MybR2R3) and to characterize the specific complex formed with a synthetic DNA fragment corresponding to the Myb target site on the Myb-regulated gene tom-1. B-MybR2R3 is shown to consist of two independent protein domains (R2 and R3) joined by a short linker, which have strikingly different tertiary structures despite significant sequence similarities. In addition, the C-terminal region of B-Myb R2 is confirmed to have a poorly defined structure, reflecting the existence of multiple conformations in slow to intermediate exchange. This contrasts with the tertiary structure reported for c-MybR2R3, in which both R2 and R3 have the same fold and the C-terminal region of R2 forms a stable, well-defined helix [Ogata, K., et al. (1995) Nat. Struct. Biol. 2, 309-320]. The NMR data suggest there are extensive contacts between B-MybR2R3 and its DNA target site in the complex and are consistent with a significant conformational change in the protein on binding to DNA, with one possibility being the formation of a stable helix in the C-terminal region of R2. In addition, conformational heterogeneity identified in R2 of B-MybR2R3 bound to the tom-1-A target site may play an important role in the control of gene expression by Myb proteins.

Nucleoside analogs. 14. The synthesis of antitumor activity in mice of molecular combinations of 5-fluorouracil and N-(2-Chloroethyl)-N-nitrosourea moieties separated by a three-carbon chain.

5-fluorouracil (5-FU) seco-nucleosdies having as the "sugar" moiety a two-carbon (C2) side chain carrying a N-(2-chloroethyl)-N-nitrosourea group were designed as molecular combinations of antimetabolite and alkylating agent, but hydrolytic release of free 5-FU was not fast enough for significant contribution to the high activity they showed against colon and breast tumors in mice. In the present study of the synthesis of the more reactive C3 seco-nucleosides, it emerged that, of various groups attached to the aldehydic center in the precursor phthalimides, only the alkoxy/uracil-1-yl type was conveniently obtained by the standard method. The methylthio/uracil-1-yl analog required relatively large amounts of reagent methanethiol, and exploration of alternatives involving alpha-chlorination of alkyl methyl sulfide or Pummerer rearrangement of its S-oxide, or successive hydrolysis and methylation of isothiouronium bromide, gave disappointing yields. For successful preparation of the alkoxy/uracil-3-yl compounds, the route used for C2 homologs required considerable experimental modification. In addition to these O,N- and S,N-acetals, some N,N-acetals bearing two 5-FU residues were prepared. The new drugs have been tested against a panel of experimental tumors in mice. Although it is evident from a parallel study that even these C3 seco-nucleosides release free 5-FU too slowly in vivo, several of them have shown impressive anticancer activity. Reviewing their performance in comparison with earlier molecular combinations, a short list of seven [B.4152 (6), B.4015 (5), B.4030 (10), B.3999 (4), B.3995 (2), B.4083 (3), and B.3996 (the N 3-substituted analog of 1)] should be investigated further. This is particularly appropriate in light of the present understanding of the mode of action of chloroethylating agents. Following a prolonged period of clinical impatience with nitrosoureas because of limited selectivity action, a new era is confidently anticipated as these powerful drugs are increasingly studied in combination with O6-benzylguanine and other more efficient inhibitors of repair enzymes like O6-alkylguanine-DNA-alkyltransferase now being developed.

Pharmacokinetics and anti-tumour activity of B.4152, a reactive molecular combination of 5-fluorouracil and N-(2-chloroethyl)-N-nitrosourea, and its uracil analogue.

The original design of the previously described molecular combinations of 5-fluorouracil (5-FU) and a chloroethyl nitrosourea (CNU) moiety was on the basis that a single drug would be able to deliver two cytotoxic moieties to tumours following hydrolytic release in vivo of free 5-FU. Subsequently experiments have shown to date that the observed activity is due mainly to the alkylating effect of the CNU. This study investigates a molecular combination of 5-FU/CNU (B.4152) which is the most readily hydrolysed under acid conditions of all the 5-FU seco-nucleosides so far prepared, and compares it with the unsubstituted uracil analogue B.4184. In vitro cytotoxicity studies against three murine colon tumour cell lines showed large differences in IC50 values between the two analogues, those for B.4184 being > 10-fold greater than those for B.4152. These differences could not be accounted for by drug stability as half-lives in tissue culture medium were similar. In vivo, B.4152 was also more active against the tumour lines and was marrow sparing at therapeutic doses. Pharmacokinetic studies demonstrated that improved activity was not due to release of free 5-FU, but differences in activity, toxicity and plasma pharmacokinetics between B.4152 and B.4184 are quite marked, indicating that the 5-FU moiety does have an important role to play.

Structure of the B-Myb DNA-binding domain in solution and evidence for multiple conformations in the region of repeat-2 involved in DNA binding: implications for sequence-specific DNA binding by Myb proteins.

A range of double and triple resonance heteronuclear NMR has been used to obtain nearly complete sequence-specific 15N, 13C and 1H resonance assignments for a 110-residue protein corresponding to the B-Myb DNA-binding domain (B-MybR2R3) and to determine its secondary structure in solution. The protein was found to contain two stable helices in repeat-2 (R2) and three in repeat-3 (R3), involving residues K12-K24 (R2-1), W30-H36 (R2-2), E64-V76 (R3-1), W81-L87 (R3-2) and D93-K105 (R3-3). In addition, the chemical shift and nuclear Overhauser effect data suggest that amino acids Q44-W49 near the C-terminus of R2 form an unstable or nascent helix, which could be stabilised on binding to a specific DNA target site. The two N-terminal helices in R2 and R3 occupy essentially identical positions in the two domains, consistent with the high level of sequence similarity between these regions. In contrast, the C-terminal region forming the third helix in R3 shows low sequence similarity with R2, accounting for the differences in secondary structure. In the case of B-MybR2R3, there is a clear chemical shift and line-broadening evidence for the existence of multiple conformations in the C-terminal region of R2, which is believed to form one half of the DNA-binding site. We propose that conformational instability of part of the DNA-binding motif is a way of increasing the specificity of Myb proteins for a relatively short (6-bp) DNA target site by reducing their affinity for non-specific DNA sequences compared to specific sites.

Nucleoside analogues. 13. The effect on anti-tumour activity of varying the uracil 5-substituent and the point of attachment (N1 or N3) of the uracil moiety in seco-nucleoside nitrosoureas.

The high activity against colon tumours in the mouse of nitrosoureas linked to seco-nucleosides with 5-fluorouracil or uracil as base component led us to synthesize and test similar drugs carrying other 5-substituted uracils attached by either N1 or N3. Not only were some of the new drugs active against the solid MAC 13 and against mammary and lung tumours, but unlike earlier compounds were particularly effective (especially N3 and alkoxy drugs like B.4083) against the ascitic MAC 15A, causing some cures. Further, these agents are valuable tools in the study, using modern techniques, of bifunctional alkylation of biological macromolecules, a vital principle of experimental cancer chemotherapy about which our knowledge is still very incomplete.

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution.

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.

Nitrosoureas from chemist to physician: classification and recent approaches to drug design.

Molecular design of chemotherapeutic nitrosoureas is reviewed in the light of a chemical classification of N-(2-chloroethyl)-N-nitrosoureas (CNUs), particularly those recently introduced and earlier compounds tested in the clinic. Of the six categories, three are rather arbitrarily based on physicochemical properties: the original, lipid-soluble drugs, water-soluble sugar derivatives, and amides of intermediate character. Others deal with more complex drug designs incorporating antimetabolites (5-fluorouracil), steroids, redox delivery systems, or hypoxia-selective 2-nitroimidazoles. Current attempts to modify the standard 2-chloroethyl group, with implications for interstrand cross-linking of DNA, are considered. Two unfortunate factors influencing the choice of drugs for clinical trial have been prejudice from the physician and commercial interests. The latter requires no further comment, but a strong plea is made for recognition of the CNU group as one of comparatively few valuable tools for rational drug design requiring appropriate pharmacokinetic evaluation, rather than as a somewhat boring hallmark of repetitive chemists.

Nucleoside analogues. 9. Seco-nucleoside analogues of some 5-fluorouracil/nitrosourea molecular combinations having uracil as base: synthesis and anti-tumour activity.

The synthesis of representative seco-nucleoside analogues of 5-fluorouracil (5-FU)/nitrosourea molecular combinations having uracil (U) as base instead of 5-FU is described. The anti-tumour activity of corresponding pairs of drugs is compared in experimental tumours of the mouse colon, lung and breast. These studies have demonstrated that the presence of a hydrogen or fluorine atom at pyrimidine C-5 is unimportant for the activity shown against most of the experimental tumours employed, rather that the pyrimidine cyclic urea and/or alkylthio functionalities may constitute the critical factors. One exception is the prototypical compound B.3839 and its U analogue B.3912. B.3839 is highly active against colon 38 adenocarcinoma, a tumour which is highly responsive to 5-FU, whereas most of the activity is lost in B.3912. The 5-FU release profile of some of these molecular combinations could be adequate or effective in treatment of slow-growing clinical tumours.

Nucleoside analogues: 8. Some isomers of B.3839, the original 5-fluorouracil/nitrosourea molecular combination, and their effect on colon, breast and lung tumours in mice.

This study describes the manipulation of secondary products arising from the synthesis of the prototypical molecular combination of 5-fluorouracil (5-FU) and chloroethylnitrosourea (CNU), B.3839, in order to investigate the effects produced by connecting the C-S-C-C-CNU chain to the 5-FU ring in different ways. The isolation of phthalimide precursors of these compounds and the transformation into CNUs is described. Anti-tumour activity of these molecular combinations against a series of experimental murine colon, lung and mammary tumours is presented. The spectrum of anti-tumour activity displayed is interesting but defies simple explanation without further detailed in vivo pharmacokinetic and metabolism studies in order to define optimal profiles for activity.

Nucleoside analogues: 7. Effect on colon, breast and lung tumours in mice of 5-fluorouracil/nitrosourea molecular combinations incorporating alkoxy and oxidized sulphur functions.

This study considers further changes in the carrier moiety of molecular combinations of the pyrimidine antimetabolite 5-fluorouracil (5-FU) and the alkylating group chloroethylnitrosourea (CNU). Detailed chemical syntheses are described of compounds incorporating (a) a simpler alkoxy group in the 'sugar' fragment, (b) attachment of the 5-FU residue to the C-X-C-C chain on the side of the heteroatom X distal from the CNU group, and (c) higher oxidation states of the sulphur atom in the prototypical compound B.3839. Anti-tumour activity of these analogues against a series of experimental murine colon, lung and mammary tumours is described. The pattern of activity reveals that the carrier moiety is important but further pharmacokinetics and metabolism studies are required to determine structure-activity relationships.

Nucleoside analogues. 4. Molecular combinations of anti-tumour drugs: synthesis of 5-fluorouracil seco-nucleosides with N-(2-chloroethyl)-N-nitrosourea residues by using aryl N-nitrosocarbamates.

The biological study of molecular combination of anti-tumour drugs remains unexplored. Some drugs prepared earlier, seco-nucleosides with 5-fluorouracil as base and incorporating a N-(2-chloroethyl)-N-nitrosourea (CNU) residue in the linear 'sugar' moiety, have now been obtained much more readily by using aryl N-nitrosocarbamates. These include the more reactive S-oxide of the original combination, B 3839. The structure of a compound with the CNU in the 'alcohol' arm of the seco-nucleoside has been clarified. Isomeric combinations having hydroxyl groups in either the 'alcohol' or the 'aldehyde' arm, with different hydrolysis rates, have been synthesized. Results of anti-tumour testing are reported in the proceeding paper.

Nucleoside analogues. 5. Molecular combination of anti-cancer drugs: activity of 5-fluorouracil/nitrosourea combinations against mouse colon tumours.

Anti-tumour activity of a novel series of molecular combinations, seco-nucleosides where the carrier is a sugar-like fragment linking the pyrimidine anti-metabolite 5-fluorouracil (5-FU) with the alkylating agent N-chloroethyl-N-nitrosourea (CNU), is presented. Three tumour lines, from the mouse adenocarcinoma of the colon (MAC) series, with different sensitivities to 5-FU and CNU were employed. All four molecular combinations tested showed some activity in this system. B 3839, in which 5-FU is linked by a SC--N bond, showed greatest activity against the ascitic tumour MAC 15A but was inactive at non-toxic doses against the solid tumour MAC 13. Activity against MAC 15A was of the same order as that achieved with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea. In contrast B 3958, with an OC--N bond, proved inactive against the ascitic tumour but was highly active against MAC 13. The factors responsible for this reversal are as yet unknown.