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1.  Crystal packing in three related disaccharides: precursors to heparan sulfate oligosaccharides 
The structures of three disaccharide mol­ecules, precursors to novel therapeutics, as determined from weakly diffracting crystals are presented. The crystal packing depends mainly on weak C—H⋯O hydrogen-bond inter­actions, augmented by C—H⋯π contacts in the best-defined structure.
The three title compounds form part of a set of important precursor dissacharides which lead to novel therapeutics, in particular for Alzheimer’s disease. All three crystallize as poorly diffracting crystals with one independent mol­ecule in the asymmetric unit. Two of them are isostructural: 4-meth­oxy­phenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-de­oxy-4-O-(9-fluor­en­yl­methyl­oxycarbon­yl)-α-d-gluco­pyranos­yl]-2-O-benzoyl-3-O-benzyl-6-O-chloro­acetyl-α-l-ido­pyran­oside, C59H56ClN3O16, (I), the ido-relative of a reported gluco-disaccharide [Gainsford et al., 2013 ▶). Acta Cryst. C69, 679–682] and 4-meth­oxy­phenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-de­oxy-4-O-(9-fluorenyl­methyl­oxycarbon­yl)-α-d-gluco­pyranos­yl]-2-O-benzoyl-3-O-benzyl-6-O-meth­oxy­acetyl-α-l-ido­pyran­oside, C60H59N3O17, (II). Both exhibit similar conformational disorder of pendant groups. The third compound 4-meth­oxy­phenyl 4-O-[6-O-acetyl-2-azido-3,4-di-O-benzyl-2-de­oxy-α-d-gluco­pyranos­yl]-2-O-benzoyl-3-O-benzyl-6-O-meth­oxy­oacetyl-β-d-gluco­pyran­oside, C52H55N3O15, (III), illustrates that a slightly larger set of weak inter­molecular inter­actions can result in a less disordered mol­ecular arrangement. The mol­ecules are bound by weak C—H⋯O(ether) hydrogen bonds in (I) and (II), augmented by C—H⋯π inter­actions in (III). The absolute configurations were determined, although at varying levels of significance from the limited observed data.
PMCID: PMC4459361  PMID: 26090127
crystal structure; disaccharide; Alzheimer’s; synthesis; hydrogen bonding; C—H⋯π contacts
2.  Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro 
PLoS ONE  2015;10(4):e0124183.
Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.
PMCID: PMC4409337  PMID: 25909893
3.  Thermodynamic Analysis of Transition State Features in Picomolar Inhibitors of Human 5′ Methylthioadenosine Phosphorylase 
Biochemistry  2013;52(46):10.1021/bi401188w.
Human 5′-methylthioadenosine phosphorylase (MTAP) is solely responsible for 5′-methylthioadenosine (MTA) metabolism to permit S-adenosylmethionine salvage. Transition state (TS) analogues of MTAP are in development as anticancer candidates. TS analogues of MTAP incorporate a cationic nitrogen and a protonated 9-deazaadenine leaving group, mimics of the ribocation transition state. MT-ImmA and MT-DADMe-ImmA are two examples of these TS analogues. Thermodynamic analysis of MTA, inhibitor and phosphate binding reveals the cationic nitrogen to provide −2.6 and −3.6 kcal/mol binding free energy for MT-ImmA and MT-DADMe-ImmA, respectively. The protonated deazaadenine provides an additional −1.3 (MT-ImmA ) to −1.7 kcal/mol (MT-DADMe-ImmA). MT-DADMe-ImmA is a better match in TS geometry than MT-ImmA and is thermodynamically favored. Binding of TS analogues to the MTAP:phosphate complex is fully entropic, in contrast to TS analogue binding to the related human purine nucleoside phosphorylase:phosphate complex, which is fully enthalpic. The binding thermodynamics of phosphate or TS analogues alone to MTAP are fully dominated by enthalpy. Phosphate anchored in the catalytic site forms an ion pair with the cationic TS analogue to cause stabilization of the enzyme structure in the ternary complex. The ternary-induced conformational changes convert the individual enthalpic binding energies to entropy, a presumed shift of the protein architecture toward the transition state. Formation of the ternary TS analogue complex with MTAP induces a remarkable increase in thermal stability (ΔTm 35 °C). The enthalpic, entropic and protein stability features of TS analogue binding to human MTAP are resolved in these studies.
PMCID: PMC3870587  PMID: 24148083
transition state analogues; MTAP; binding energy; thermodynamics; cooperativity; protein stabilization; entropic binding
4.  Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase 
Eukaryotic Cell  2014;13(5):572-579.
The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.
PMCID: PMC4060479  PMID: 24585883
5.  Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase 
Bioorganic & medicinal chemistry  2013;21(17):5629-5646.
The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.
PMCID: PMC3740065  PMID: 23810424
Phosphoribosyltransferase; malaria; HGXPRTase; purine salvage; protozoa
6.  Salmonella enterica MTAN at 1.36 Å resolution. Structure-based design of new transition state analogues 
Accumulation of 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) in bacteria disrupts the S-adenosylmethionine pool to alter biological methylations, synthesis of polyamines and production of quorum sensing molecules. Bacterial metabolism of MTA and SAH depends on MTA/SAH nucleosidase (MTAN), an enzyme not present in humans, and a target for quorum sensing since MTAN activity is essential for synthesis of autoinducer-2 molecules. Crystals of Salmonella enterica MTAN with product and transition state analogues of MTA and SAH explain the pM binding affinity and reveal ‘water-wire’ channel for the catalytic nucleophile. The crystal structure shows an extension of the binding pocket filled with polyethylene glycol. We exploited that discovery by the design and synthesis of new modifications of the currently existing transition state analogues to fill this site. This site was previously unknown in MTANs and reveals powerful inhibitors with solvent access. Novel inhibitors with dissociation constants of 5 to 15 pM are characterized.
PMCID: PMC3690532  PMID: 23685211
7.  Methylthioadenosine Deaminase in an Alternative Quorum Sensing Pathway in Pseudomonas aeruginosa 
Biochemistry  2012;51(45):9094-9103.
Pseudomonas aeruginosa possesses an unusual pathway for 5′-methylthioadenosine (MTA) metabolism involving deamination to 5′-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-α-D-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported (Guan, R., Ho, M. C., Almo, S. C. and Schramm, V. L. (2011) Biochemistry 50, 1247–1254) and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The kcat/Km value of 1.6 × 107 M−1s−1 for MTA is the highest catalytic efficiency known for an MTA deaminase. 5′-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC50 of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-α-D-ribose are involved in quorum sensing pathways, thus PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8-R-hydroxyl group in contact with a catalytic site Zn2+, the 5′-methylthio group in a hydrophobic pocket and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.
PMCID: PMC3567251  PMID: 23050701
5′-methylthioadenosine; 5′-methylthioinosine; quorum sensing; transition state analogue; MTA deaminase; 5′-methylthiocoformycin; tight-binding inhibitor; Pseudomonas
8.  A picomolar transition state analogue inhibitor of MTAN as a specific antibiotic for H. pylori 
Biochemistry  2012;51(35):6892-6894.
Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpM-TAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpM-TAN and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC90 value of < 8 ng/ml for H. pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.
PMCID: PMC3437630  PMID: 22891633
transition state analogue; BuT-DADMe-ImmA; 5′-methylthioadenosine nucleosidase; MTAN; Helicobacter pylori; Campylobacter jejuni; ulcers; antibiotics
9.  Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics 
Bioorganic & medicinal chemistry  2012;20(17):5181-5187.
Several acyclic hydroxy-methylthio-amines with 3 to 5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and E. coli and N. meningitidis MTANs and gave Ki values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-d-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon–carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts.
PMCID: PMC3438226  PMID: 22854195
Human MTAP; Bacterial MTANs; Ribooxacarbenium ion mimics; Inhibitors; Acyclic hydroxy-methylthio-amines
10.  Design and Synthesis of Potent “Sulfur-free” Transition State Analogue Inhibitors of 5′-Methylthioadenosine Nucleosidase and 5′-Methylthioadenosine Phosphorylase 
Journal of medicinal chemistry  2010;53(18):6730-6746.
5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM)-related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability we were interested in synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free” transition state analogues inhibitors, of E. coli MTAN and human MTAP that have low nano- to pico-molar dissociation constants and are potentially novel bacterial anti-infective and anti-cancer drug candidates.
PMCID: PMC3742014  PMID: 20718423
11.  Acyclic Immucillin Phosphonates: Second generation inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase 
Chemistry & biology  2012;19(6):721-730.
Plasmodium falciparum, the primary cause of deaths from malaria, is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5′-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. Here we present a new class of HGXPRT inhibitors, the acyclic Immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.
PMCID: PMC3397391  PMID: 22726686
12.  Entropy-Driven Binding of Picomolar Transition State Analogue Inhibitors to Human 5′-Methylthioadenosine Phosphorylase† 
Biochemistry  2011;50(47):10408-10417.
Human 5′-methylthioadenosine phosphorylase (MTAP) links the polyamine biosynthetic and S-adenosyl-L-methionine salvage pathways and is a target for anticancer drugs. p-Cl-PhT-DADMe-ImmA is a 10 pM, slow-onset tight-binding transition state analogue inhibitor of the enzyme. Titration of homotrimeric MTAP with this inhibitor established equivalent binding and independent catalytic function of the three catalytic sites. Thermodynamic analysis of MTAP with tight-binding inhibitors revealed entropic-driven interactions with small enthalpic penalties. A large negative heat capacity change of −600 cal/mol•K upon inhibitor binding to MTAP is consistent with the loss of hydrophobic interactions and release of water. Crystal structures of apo MTAP and MTAP in complex with p-Cl-PhT-DADMe-ImmA were determined at 1.9 A and 2.0 A resolution, respectively. Inhibitor binding caused condensation of the enzyme active site, reorganization at the trimer interfaces, the release of water from the active sites and subunit interfaces, and compaction of the trimeric structure. These structural changes cause the entropy-favored binding of transition state analogues. Homotrimeric human MTAP is contrasted to the structurally related homotrimeric human purine nucleoside phosphorylase. p-Cl-PhT-DADMe- ImmA binding to MTAP involves a favorable entropy term of −17.6 kcal/mol with unfavorable enthalpy of 2.6 kcal/mol. In contrast, binding of an 8.5 pM transition state analogue to human PNP has been shown to exhibit the opposite behavior, with an unfavorable entropy term of 3.5 kcal/mol and a favorable enthalpy of −18.6 kcal/mol. Transition state analogue interactions reflect protein architecture near the transition state and the profound thermodynamic differences for MTAP and PNP suggest dramatic differences in contributions to catalysis from protein architecture.
PMCID: PMC3222737  PMID: 21985704
13.  A synchrotron radiation study of the one-dimensional complex of sodium with (1S)-N-carboxyl­ato-1-(9-deaza­adenin-9-yl)-1,4-dide­oxy-1,4-imino-d-ribitol, a member of the ’immucillin’ family 
The sodium salt of [immucillin-A–CO2H]− (Imm-A), namely catena-poly[[[triaqua­disodium(I)](μ-aqua)[μ-(1S)-N-car­box­yl­ato-1-(9-deaza­adenin-9-yl)-1,4-dide­oxy-1,4-imino-d-ribi­tol][triaqua­disodium(I)][μ-(1S)-N-carboxyl­ato-1-(9-deaza­aden­in-9-yl)-1,4-dide­oxy-1,4-imino-d-ribitol]] tetra­hydrate], {[Na2(C12H13N4O6)2(H2O)7]·4H2O}n, (I), forms a polymeric chain via Na+—O inter­actions involving the carboxyl­ate and keto O atoms of two independent Imm-A mol­ecules. Extensive N,O—H⋯O hydrogen bonding utilizing all water H atoms, including four waters of crystallization, provides crystal packing. The structural definition of this novel compound was made possible through the use of synchrotron radiation utilizing a minute fragment (volume ∼2.4 × 10−5 mm−3) on a beamline optimized for protein data collection. A summary of intra-ring conformations for immucillin structures indicates considerable flexibility while retaining similar intra-ring orientations.
PMCID: PMC2855577  PMID: 20203397
14.  Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work and with Transition State Analogues† 
Biochemistry  2010;49(9):2058-2067.
Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH, Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH, Kd = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP•PO4•Inosine ↔ PNP•Hx•R-1-P) and inhibited complexes (PNP•PO4•DATMe-ImmH and PNP•PO4•ImmH) show protection from HDX at 9, 13 and 15 sites per subunit relative to resting PNP (PNP•PO4) in extended incubations. The PNP•PO4•ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely to entropy of binding than to affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH, and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.
PMCID: PMC2832846  PMID: 20108972
15.  Transition State Analogues Rescue Ribosomes from Saporin-L1 Ribosome Inactivating Protein† 
Biochemistry  2009;48(41):9941-9948.
Ribosome-inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves and demonstrate robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (Ki*) of 2.3 to 8.7 nM at physiological conditions and bind up to 40,000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1 linked immunotoxins.
PMCID: PMC2762729  PMID: 19764816
16.  Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases† 
Biochemistry  2009;48(40):9618-9626.
Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA (Tyler, P. C., Taylor, E. A., Fröhlich, R. G. G. and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872–6879). The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation (Larson, E. T. et al. (2008) J. Mol. Biol. 381, 975–988). Here, the crystal structure of ADA from Plasmodium vivax in complex with MT-coformycin reveals an unprecedented binding geometry for 5’-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5’-methylthioribosyl groups are rotated 130°. A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5’-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic and structural analyses of PvADA and kinetic analysis of five other plasmodial ADAs establishes the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.
PMCID: PMC2771402  PMID: 19728741
17.  Altered Enthalpy-Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase† 
Biochemistry  2009;48(23):5226-5238.
Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetery indicate that binding of a structurally rigid first-generation Immucillin (K d = 56 pM) is driven by large negative enthalpy values (ΔH = −21.2 kcal/mol) with a substantial entropic (-TΔS) penalty. The tightest-binding inhibitors (K d = 5 to 9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP·inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5’-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the seventeen Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. By introducing flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.
PMCID: PMC2711852  PMID: 19425594
18.  p-Tolyl 2-O-benzoyl-3-O-benzyl-4,6-O-benzyl­idene-1-thio-α-l-idopyran­oside 
The title compound, C34H32O6S, is an ido-configured thio­glycoside building block for heparan sulfate fragments. It contains disordered tolyl and O-benzyl groups with occupancy ratios of 0.539 (13):0.461 (13) and 0.613 (13):0.387 (13), respectively, as determined from a weakly diffracting crystal. The fused rings adopt chair conformations with the mol­ecules packing into a three-dimensional network via C—H⋯O and three C—H⋯π inter­actions. The former inter­actions, occuring between mol­ecules related by a twofold axis, define an R 2 2(26) motif.
PMCID: PMC3007053  PMID: 21587835
19.  Third-Generation Immucillins: Syntheses and Bio-Activities of Acyclic Immucillin Inhibitors of Human Purine Nucleoside Phosphorylase 
Journal of medicinal chemistry  2009;52(4):1126-1143.
ImmH (1) and DADMe-ImmH (2) are potent inhibitors of human purine nucleoside phoshorylase (PNP), developed by us and currently in clinical trials for the treatment of a variety of T-cell related diseases. Compounds 1 and 2 were used as templates for the design and synthesis of a series of acyclic immucillin analogues (8–38) in order to identify simplified alternatives to 1 and 2. SerMe-ImmG (8) and DATMe-ImmG (9) displayed the lowest inhibition constants of 2.1 and 3.4 pM, respectively, vs PNP. It was postulated that the flexible natures of 8 and 9 enabled them to adopt conformations resembling those of 1 and 2 within the active site of PNP and that the positioning of two hydroxyl groups was critical for picomolar activity. SerMe-ImmH (10, Kd = 5.2 pM) was shown to be orally available in mice with a long biological residence time on blood PNP.
PMCID: PMC2698043  PMID: 19170524
20.  Immucillins in Custom Catalytic-Site Cavities 
Neighboring-group participation in the reaction catalyzed by purine nucleoside phosphorylase involves a compression mode between the 5'- and 4'-ribosyl oxygens, facilitated by His257. The His257Gly mutant opens a space in the catalytic site. Hydrophobic 5′-substituted Immucillins are transition-state analogue inhibitors of this mutant enzyme. Dissociation constants as low as 2 pM are achieved, with Km/Kd as high as 400,000,000.
PMCID: PMC2614083  PMID: 18778937
PNP; Immucillin; ImmH; Mutant; Binding; Transition-state analogue
21.  Transition state analogues in quorum sensing and SAM recycling 
Transition state structures can be derived from kinetic isotope effects and computational chemistry. Molecular electrostatic potential maps of transition states serve as blueprints to guide synthesis of transition state analogue inhibitors of target enzymes. 5’-Methylthioadenosine phosphorylase (MTAP) functions in the polyamine pathway by recycling methylthioadenosine (MTA) and maintaining cellular S-adenosylmethionine (SAM). Its transition state structure was used to guide synthesis of MT-DADMe-ImmA, a picomolar inhibitor that shows anticancer effects against solid tumors. Biochemical and genomic analysis suggests that MTAP inhibition acts by altered DNA methylation and gene expression patterns. A related bacterial enzyme, 5’-methylthioadcnosine nucleosidase (MTAN), functions in pathways of quorum sensing involving AI-1 and AI-2 molecules. Transition states have been solved for several bacterial MTANs and used to guide synthesis of powerful inhibitors with dissociation constants in the femtomolar to picomolar range. BuT-DADMe-ImmA blocks quorum sensing in Vibrio cholerae without changing bacterial growth rates. Transition state analogue inhibitors show promise as anticancer and antibacterial agents.
PMCID: PMC2725438  PMID: 18776260
22.  l-Enantiomers of Transition State Analogue Inhibitors Bound to Human Purine Nucleoside Phosphorylase 
Human purine nucleoside phosphorylase (PNP) was crystallized with transition state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.
PMCID: PMC2531256  PMID: 18154341
23.  Synthesis of 5′-Methylthio Coformycins: Specific Inhibitors for Malarial Adenosine Deaminase 
Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Since transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2′-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5’-methylthioadenosine. In contrast, human adenosine deaminase (HsADA) does not deaminate 5’-methylthioadenosine. 5′-Methylthio coformycin and 5’-meththio-2′-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to cause toxicity in humans and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA.
PMCID: PMC2522312  PMID: 17488013
Coformycin; Deoxycoformycin; Pentostatin; transition state analogueue; ADA inhibitors; adenosine deaminase; malaria; purine salvage; isozyme specificity; Plasmodium falciparum
24.  Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae 
Biochemistry  2006;45(43):12929-12941.
Streptococcus pneumoniae 5′-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative SN1 transition state for E. coli MTAN and transition state analogues resembling the transition state are powerful inhibitors of the enzyme (Singh, V., Lee, J. L., Núñez, S., Howell, P. L. and Schramm, V. L. (2005) Biochemistry 44, 11647-11659). The MTAN from S. pneumoniae has 40% sequence identity to E. coli MTAN, but exhibits remarkably distinct kinetic and inhibitory properties. 5′-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early SN1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a Ki of 1.0 μM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolT-ImmA) or ethyl (EtT-ImmA) groups increases the affinity to give Ki values of 335 nM, 60 nM and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully-dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a Ki value of 24 nM and replacing the 5′-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a Ki* value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 103 to 104 fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since kcat/Km for S. pneumoniae MTAN is <10-2 that of E. coli MTAN.
PMCID: PMC2517848  PMID: 17059210
5′-methylthioadenosine; 5′-methylthioadenosine nucleosidase; quorum sensing; nucleoside hydrolase; transition state; transition state analogue inhibitors; polyamines; MTAN structure; catalytic efficiency
25.  Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues† 
Biochemistry  2007;46(3):659-668.
Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min and a Km/Kd ratio of 203,300. Tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP•ImmA•PO4 and TvPNP•DADMe-ImmA•PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 Å ionic interaction between a PO4 oxygen and the N1’ cation of the hydroxypyrrolidine and is weaker in the TvPNP•ImmA•PO4 structure at 3.5 Å. However, the TvPNP•ImmA•PO4 structure includes hydrogen bonds between the 2’-hydroxyl and the protein that are not present in TvPNP•DADMe-ImmA•PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP•ImmH•PO4, causing an unfavorable leaving-group interaction.
PMCID: PMC2517847  PMID: 17223688

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