Thermodynamic studies of interactions of calf spleen PNP with acyclic phosphonate inhibitors

Breer, Katarzyna; Wielgus‐Kutrowska, Beata; Hashimoto, Motomu; Hikishima, Sadao; Yokomatsu, Tsutomu; Szczepanowski, Roman H.; Bochtler, Matthias; Girstun, Agnieszka; Staroń, Krzysztof; Bzowska, Agnieszka

doi:10.1093/nass/nrn335

Cited by 5 publications

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“…The entropy changes vary from significantly unfavorable (Immucillins 1-9) to near zero (10)(11)(12) and slightly favorable (13)(14)(15)(16)(17) with inhibitors of closely related chemical structures. The entropic pattern suggests that the entropic term originates in protein dynamic structure rather than the conformational flexibility states of the inhibitors or the order parameters for water.…”

Section: Discussionmentioning

confidence: 99%

“…Immucillins 4, 6, and 8 exhibit lower-than-expected unfavorable entropies and are the most tightly bound species. Ligands with favorable entropic contributions(12)(13)(14)(15)(16)(17) lack the ability to form a normal 5 0 -hydroxyl interaction at the catalytic site.…”

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confidence: 99%

“…X-ray structures of PNPs with substrate, product, or inhibitor complexes reveal that all sites are filled under ligand saturation − . Thermodynamic parameters for Immucillin binding have not been reported; however, those for the binding of product and multisubstrate analogue inhibitors to bovine PNP have been published (e.g., ref ). Here, four generations of transition state analogues and related inhibitors were titrated against human PNP using isothermal titration calorimetry (ITC).…”

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confidence: 99%

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Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

Edwards

Mason²,

Clinch³

et al. 2009

Biochemistry

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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.Human purine nucleoside phosphroylase (PNP) is required for the catabolism of 6-oxy-(and 2'-deoxy)-nucleosides to free nucleobases for recycling by phosphoribosyl transferases or oxidation to uric acid and excretion. Genetic deficiency of PNP in humans causes deoxyguanosine accumulation in the blood and its conversion to dGTP causes arrest of DNA synthesis and apoptosis specifically in activated T-cells. Human PNP is therefore a target for the treatment of autoimmune disorders and T-cell cancers (1-2).The transition state structure of human PNP has permitted the design of several transition state analogues with picomolar dissociation constants (3-7). The transition state is characterized by a fully-developed ribocation with a C1' -N9 distance of ≥ 3.0 Å and the phosphate nucleophile at a similar distance to C1' of the ribosyl cation (Figure 1) (3). Four structurally distinct generations of transition state analogues have been synthesized (Figure 2).

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Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

Edwards

Mason²,

Clinch³

et al. 2009

Biochemistry

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“…However, the effect is not large as a result of enthalpy‐entropy compensation. The gain in enthalpic contribution to the Gibbs binding energy, when compared with DFPP‐G binding, is balanced by an entropic effect [17].…”

Section: Resultsmentioning

confidence: 99%

9‐Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow‐binding picomolar inhibitors of trimeric purine nucleoside phosphorylase

Breer¹,

Glavaš‐Obrovac

Suver

et al. 2010

The FEBS Journal

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Genetic deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1) activity leads to a severe selective disorder of T‐cell function. Therefore, potent inhibitors of mammalian PNP are expected to act as selective immunosuppressive agents against, for example, T‐cell cancers and some autoimmune diseases. 9‐(5′,5′‐difluoro‐5′‐phosphonopentyl)‐9‐deazaguanine (DFPP‐DG) was found to be a slow‐ and tight‐binding inhibitor of mammalian PNP. The inhibition constant at equilibrium (1 mm phosphate concentration) with calf spleen PNP was shown to be = 85 ± 13 pm (pH 7.0, 25 °C), whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher ( = 4.4 ± 0.6 nm). The rate constant for formation of the enzyme/inhibitor reversible complex is (8.4 ± 0.5) × 105 m−1·s−1, which is a value that is too low to be diffusion‐controlled. The picomolar binding of DFPP‐DG was confirmed by fluorimetric titration, which led to a dissociation constant of 254 pm (68% confidence interval is 147–389 pm). Stopped‐flow experiments, together with the above data, are most consistent with a two‐step binding mechanism: E + I ↔ (EI) ↔ (EI)*. The rate constants for reversible enzyme/inhibitor complex formation (EI), and for the conformational change (EI) ↔ (EI)*, are kon1 = (17.46 ± 0.05) × 105 m−1·s−1, koff1 = (0.021 ± 0.003) s−1, kon2 = (1.22 ± 0.08) s−1 and koff2 = (0.024 ± 0.005) s−1, respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of Ki = 12.1 nM (68% confidence interval is 8.7–15.5 nm) and = 237 pm (68% confidence interval is 123–401 pm), respectively. At a concentration of 10−4 m, DFPP‐DG exhibits weak, but statistically significant, inhibition of the growth of cell lines sensible to inhibition of PNP activity, such as human adult T‐cell leukaemia and lymphoma (Jurkat, HuT78 and CCRF‐CEM). Similar inhibitory activities of the tested compound were noted on the growth of lymphocytes collected from patients with Hashimoto’s thyroiditis and Graves’ disease. The observed weak cytotoxicity may be a result of poor membrane permeability.

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1.45Å resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

Chojnowski

Breer

Narczyk

et al. 2010

Biochemical and Biophysical Research Communications

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Thermodynamic studies of interactions of calf spleen PNP with acyclic phosphonate inhibitors

Cited by 5 publications

References 0 publications

Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

9‐Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow‐binding picomolar inhibitors of trimeric purine nucleoside phosphorylase

1.45Å resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

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