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D K Stammers

Publications and source records attributed to D K Stammers.

At least 37 records · Page 2Linked to original sources
PubMed2000

Phenylethylthiazolylthiourea (PETT) non-nucleoside inhibitors of HIV-1 and HIV-2 reverse transcriptases. Structural and biochemical analyses.

Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC(50) of 6 nM but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC(50) of 5 nM) and also inhibits HIV-2 RT (IC(50) of 2.2 microM). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC).(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1.

Binding, Competitive↗
PubMed2000

Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase.

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglutarate oxygenase, catalyzes three separate oxidative reactions in the biosynthesis of clavulanic acid, a clinically used inhibitor of serine beta-lactamases. The first CAS-catalyzed step (hydroxylation) is separated from the latter two (oxidative cyclization/desaturation) by the action of an amidinohydrolase. Here, we describe crystal structures of CAS in complex with Fe(II), 2-oxoglutarate (2OG) and substrates (N-alpha-acetyl-L-arginine and proclavaminic acid). They reveal how CAS catalyzes formation of the clavam nucleus, via a process unprecedented in synthetic organic chemistry, and suggest how it discriminates between substrates and controls reaction of its highly reactive ferryl intermediate. The presence of an unpredicted jelly roll beta-barrel core in CAS implies divergent evolution within the family of 2OG and related oxygenases. Comparison with other non-heme oxidases/oxygenases reveals flexibility in the position which dioxygen ligates to the iron, in contrast to the analogous heme-using enzymes.

Arginine↗
PubMed1999

Design of MKC-442 (emivirine) analogues with improved activity against drug-resistant HIV mutants.

Two analogues of the nonnucleoside inhibitor of HIV-1 RT, MKC-442 (emivirine), containing different C6 substituents have been designed to be less susceptible to the commonly found drug-resistance mutation of Tyr181Cys. Compound TNK-6123 had a C6 thiocyclohexyl group designed to have more flexibility in adapting to the mutated drug-binding site. GCA-186 had additional 3',5'-dimethyl substituents aimed at forming close contacts with the conserved residue Trp229. Both compounds showed approximately 30-fold greater inhibitory effect than MKC-442 to the Tyr181Cys mutant virus as well as to the clinically important Lys103Asn virus. X-ray crystallographic structure determination of complexes with HIV-1 RT confirmed the predicted binding modes. These strategies might be used to improve the resilience of other NNRTI series against common drug-resistance mutations.

Animals↗
PubMed1999

Crystallographic analysis of the binding modes of thiazoloisoindolinone non-nucleoside inhibitors to HIV-1 reverse transcriptase and comparison with modeling studies.

We have determined the crystal structures of thiazoloisoindolinone non-nucleoside inhibitors in complex with HIV-1 reverse transcriptase to high-resolution limits of 2.7 A (BM +21.1326) and 2. 52 A (BM +50.0934). We find that the binding modes of this series of inhibitors closely resemble that of "two-ring" non-nucleoside reverse transcriptase inhibitors. The structures allow rationalization of stereochemical requirements, structure-activity data, and drug resistance data. Comparisons with our previous structures suggest modifications to the inhibitors that might improve resilience to drug-resistant mutant forms of reverse transcriptase. Comparison with earlier modeling studies reveals that the predicted overlap of thiazoloisoindolinones with TIBO was largely correct, while that with nevirapine was significantly different.

Crystallography, X-Ray↗
PubMed1999

2.0 A X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue.

The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents.

Adenosine Triphosphate↗
PubMed1999

A family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance.

To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance was associated with a diverse family of amino acid insertions in addition to "conventional" point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Treatment history information showed that a common factor for the development of these variants was AZT (3'-azido-3'-deoxythymidine, zidovudine) therapy in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the "fingers" domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.

Anti-HIV Agents↗
PubMed1998

Crystal structures of HIV-1 reverse transcriptase in complex with carboxanilide derivatives.

The carboxanilides are nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT), of potential clinical importance. The compounds differ in potency and in their retention of potency in the face of drug resistance mutations. Whereas UC-84, the prototype compound, only weakly inhibits many RTs bearing single point resistance mutations, inhibition by UC-781 is little affected. It has been proposed that UC-38 and UC-781 may form quaternary complexes with RT at a site other than the known binding pocket of other NNIs. X-ray crystal structures of four HIV-1 RT-carboxanilide complexes (UC-10, UC-38, UC-84, and UC-781) reported here reveal that all four inhibitors bind in the usual NNI site, forming binary 1:1 complexes with RT in the absence of substrates with the amide/thioamide bond in cis conformations. For all four complexes the anilide rings of the inhibitors overlap aromatic rings of many other NNIs bound to RT. In contrast, the second rings of UC-10, UC-84, and UC-781 do not bind in equivalent positions to those of other "two-ring" NNIs such as alpha-APA or HEPT derivatives. The binding modes most closely resemble that of the structurally dissimilar NNI, Cl-TIBO, with a common hydrogen bond between each carboxanilide NH- group and the main-chain carbonyl oxygen of Lys101. The binding modes differ slightly between the UC-10/UC-781 and UC-38/UC-84 pairs of compounds, apparently related to the shorter isopropylmethanoyl substituents of the anilide rings of UC-38/UC-84, which draws these rings closer to residues Tyr181 and Tyr188. This in turn explains the differences in the effect of mutated residues on the binding of these compounds.

Anilides↗
PubMed1998

Biaryl acids: novel non-nucleoside inhibitors of HIV reverse transcriptase types 1 and 2.

A series of biaryl acids has been found to show micromolar inhibition of the HIV reverse transcriptase (RT) from types 1 and 2 with IC50S in the micromolar range. The series was discovered by consideration of the polymerase active site and sub-structure searching of the company compound collection. Synthesis of analogues to investigate the SAR is described. Two of these compounds have shown inhibition of HIV-2 RT only.

Anti-HIV Agents↗
PubMed1998

Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration.

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Crystallization↗
PubMed1998

3'-Azido-3'-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long range conformational changes.

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Many of these drugs [e.g., 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI)] are analogues of the nucleoside substrates used by the HIV RT. One of the main problems in anti-HIV therapy is the selection of a mutant virus with reduced drug sensitivity. Drug resistance in HIV is generated for nucleoside analogue inhibitors by mutations in HIV RT. However, most of these mutations are situated some distance from the polymerase active site, giving rise to questions concerning the mechanism of resistance. To understand the possible structural bases for this, the crystal structures of AZT- and ddI-resistant RTs have been determined. For the ddI-resistant RT with a mutation at residue 74, no significant conformational changes were observed for the p66 subunit. In contrast, for the AZT-resistant RT (RTMC) bearing four mutations, two of these (at 215 and 219) give rise to a conformational change that propagates to the active site aspartate residues. Thus, these drug resistance mutations produce an effect at the RT polymerase site mediated simply by the protein. It is likely that such long-range effects could represent a common mechanism for generating drug resistance in other systems.

Binding Sites↗
PubMed1998

Allosteric inhibitors against HIV-1 reverse transcriptase: design and synthesis of MKC-442 analogues having an omega-functionalized acyclic structure.

Based on X-ray crystallographic analysis of MKC-442/human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) complex, analogues in which the N1-substituent is replaced with omega-functionalized alkyl groups were designed to improve the affinity for the enzyme. Synthesis of these compounds was carried out starting from MKC-442 by a sequence of reactions (N3-protection, removal of N1-ethoxymethyl group, alkylation, and N3-deprotection). The compounds were evaluated for anti-HIV activity. Structure-activity relationships are discussed in terms of the possible interaction with the enzyme.

Allosteric Site↗
PubMed1997

Unique features in the structure of the complex between HIV-1 reverse transcriptase and the bis(heteroaryl)piperazine (BHAP) U-90152 explain resistance mutations for this nonnucleoside inhibitor.

The viral reverse transcriptase (RT) provides an attractive target in the search for anti-HIV therapies. The nonnucleoside inhibitors (NNIs) are a diverse set of compounds (usually HIV-1 specific) that function by distorting the polymerase active site upon binding in a nearby pocket. Despite being potent and of generally low toxicity, their clinical use has been limited by rapid selection for resistant viral populations. The 2.65-A resolution structure of the complex between HIV-1 RT and the bis(heteroaryl)piperazine (BHAP) NNI, 1-(5-methanesulfonamido-1H-indol-2-yl-carbonyl)-4- [3-(1-methyl-ethylamino) pyridinyl] piperazine (U-90152), reveals the inhibitor conformation and bound water molecules. The bulky U-90152 molecule occupies the same pocket as other NNIs, but the complex is stabilized quite differently, in particular by hydrogen bonding to the main chain of Lys-103 and extensive hydrophobic contacts with Pro-236. These interactions rationalize observed resistance mutations, notably Pro-236-Leu, which occurs characteristically for BHAPs. When bound, part of U-90152 protrudes into the solvent creating a channel between Pro-236 and the polypeptide segments 225-226 and 105-106, giving the first clear evidence of the entry mode for NNIs. The structure allows prediction of binding modes for related inhibitors [(altrylamino)piperidine-BHAPs] and suggests changes to U-90152, such as the addition of a 6 amino group to the pyridine ring, which may make binding more resilient to mutations in the RT. The observation of novel hydrogen bonding to the protein main chain may provide lessons for the improvement of quite different inhibitors.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Crystal structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase.

Sulfonamides were amongst the first clinically useful antibacterial agents to be discovered. The identification of sulfanilamide as the active component of the dye Prontosil rubrum led to the synthesis of clinically useful analogues. Today sulfamethoxazole (in combination with trimethoprim), is used to treat urinary tract infections caused by bacteria such as Escherichia coli and is also a first-line treatment for pneumonia caused by the fungus Pneumocystis carinii, a common condition in AIDS patients. The site of action is the de novo folate biosynthesis enzyme dihydropteroate synthase (DHPS) where sulfonamides act as analogues of one of the substrates, para-aminobenzoic acid (pABA). We report here the crystal structure of E.coli DHPS at 2.0 A resolution refined to an R-factor of 0.185. The single domain of 282 residues forms an eight-stranded alpha/beta-barrel. The 7,8-dihydropterin pyrophosphate (DHPPP) substrate binds in a deep cleft in the barrel, whilst sulfanilamide binds closer to the surface. The DHPPP ligand site is highly conserved amongst prokaryotic and eukaryotic DHPSs.

Amino Acid Sequence↗
PubMed1996

Complexes of HIV-1 reverse transcriptase with inhibitors of the HEPT series reveal conformational changes relevant to the design of potent non-nucleoside inhibitors.

Crystal structures of HIV-1 reverse transcriptase (RT) complexed with a range of chemically diverse non-nucleoside inhibitors (NNIs) have shown a single pocket in which the inhibitors bind and details of the inhibitor-protein interactions. To delineate the structural requirements for an effective inhibitor, we have determined the structures of three closely related NNIs which vary widely in their potencies. Crystal structures of HIV-1 RT complexed with two very potent inhibitors, MKC-442 and TNK-651, at 2.55 angstroms resolution complement our previous analysis of the complex with the less effective inhibitor, HEPT. These structures reveal conformational changes which correlate with changes in potency. We suggest that a major determinant of increased potency in the analogues of HEPT is an improved interaction between residue Tyr181 in the protein and the 6-benzyl ring of the inhibitors which stabilizes the structure of the complex. This arises through a conformational switching of the protein structure triggered by the steric bulk of the 5-substituent of the inhibitor pyrimidine ring.

Antiviral Agents↗
PubMed1996

Identification of the domains of neuronal nitric oxide synthase by limited proteolysis.

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

Amino Acid Sequence↗
PubMed1996

Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim.

Conformationally restricted analogues of the antibacterial agent trimethoprim (TMP) were designed to mimic the conformation of drug observed in its complex with bacterial dihydrofolate reductase (DHFR). This conformation of TMP was achieved by linking the 4-amino function to the methylene group by one- and two-carbon bridges. A pyrrolo[2,3-d]pyrimidine, a dihydro analogue, and a tetrahydropyrido[2,3-d]pyrimidine were synthesized and tested as inhibitors of DHFR. One analogue showed activity equivalent to that of TMP against DHFR from three species of bacteria. An X-ray crystal structure of this inhibitor bound to Escherichia coli DHFR was determined to evaluate the structural consequences of the conformational restriction.

Animals↗
PubMed1994

The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 A resolution.

BACKGROUND: The fungal pathogen Pneumocystis carinii causes a pneumonia which is an opportunistic infection of AIDS patients. Current therapy includes the dihydrofolate reductase (DHFR) inhibitor trimethoprim which is selective but only a relatively weak inhibitor of the enzyme for P. carinii. Determination of the three-dimensional structure of the enzyme should form the basis for design of more potent and selective therapeutic agents for treatment of the disease. RESULTS: The structure of P. carinii DHFR in complex with reduced nicotinamide adenine dinucleotide phosphate and trimethoprim has accordingly been solved by X-ray crystallography. The structure of the ternary complex has been refined at 1.86 A resolution (R = 0.181). A similar ternary complex with piritrexim (which is a tighter binding, but less selective inhibitor) has also been solved, as has the binary complex holoenzyme, both at 2.5 A resolution. CONCLUSIONS: These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

Amino Acid Sequence↗