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Transition state analogs (transition state analogues), are chemical compounds with a chemical structure that resembles the transition state of a substrate molecule in an enzyme-catalyzed chemical reaction. Enzymes interact with a substrate by means of strain or distortions, moving the substrate towards the transition state.^[1] Transition state analogs can be used as inhibitors in enzyme-catalyzed reactions by blocking the active site of the enzyme. Theory suggests that enzyme inhibitors which resembled the transition state structure would bind more tightly to the enzyme than the actual substrate.^[2] Examples of drugs that are transition state analog inhibitors include flu medications such as the neuraminidase inhibitor oseltamivir and the HIV protease inhibitors saquinavir in the treatment of AIDS.

Transition state analogue

The transition state of a structure can best be described in regards to statistical mechanics where the energies of bonds breaking and forming have an equal probability of moving from the transition state backwards to the reactants or forward to the products. In enzyme-catalyzed reactions, the overall activation energy of the reaction is lowered when an enzyme stabilizes a high energy transition state intermediate. Transition state analogs mimic this high energy intermediate but do not undergo a catalyzed chemical reaction and can therefore bind much stronger to an enzyme than simple substrate or product analogs.

Designing transition state analogue

To design a transition state analogue, the pivotal step is the determination of transition state structure of substrate on the specific enzyme of interest with experimental method, for example, kinetic isotope effect. In addition, the transition state structure can also be predicted with computational approaches as a complementary to KIE. We will explain these two methods in brief.

Kinetic isotope effect

Kinetic isotope effect (KIE) is a measurement of the reaction rate of isotope-labeled reactants against the more common natural substrate. Kinetic isotope effect values are a ratio of the turnover number and include all steps of the reaction.^[3] Intrinsic kinetic isotope values stem from the difference in the bond vibrational environment of an atom in the reactants at ground state to the environment of the atom's transition state.^[3] Through the kinetic isotope effect much insight can be gained as to what the transition state looks like of an enzyme-catalyzed reaction and guide the development of transition state analogs.

Computational simulation

Computational approaches have been regarded as a useful tool to elucidate the mechanism of action of enzymes.^[4] Molecular mechanics itself can not predict the electron transfer which is the fundamental of organic reaction but the molecular dynamics simulation provide sufficient information considering the flexibility of protein during catalytic reaction. The complementary method would be combined molecular mechanics/ quantum mechanics simulation (QM/MM)methods.^[5] With this approach, only the atoms responsible for enzymatic reaction in the catalytic region will be reared with quantum mechanics and the rest of the atoms were treated with molecular mechanics.^[6]

Examples of transition state analogue design

After determining the transition state structures using either KIE or computation simulations, the inhibitor can be designed according to the determined transition state structures or intermediates. The following three examples illustrate how the inhibitors mimic the transition state structure by changing functional groups correspond to the geometry and electrostatic distribution of the transition state structures.

Methylthioadenosine nucleosidase inhibitor

Methylthioadenosine nucleosidase are enzymes that catalyse the hydrolytic deadenylation reaction of 5'-methylthioadenosine and S-adenosylhomocysteine. It is also regarded as an important target for antibacterial drug discovery because it is important in the metabolic system of bacteria and only produced by bacteria.^[7] Given the different distance between nitrogen atom of adenine and the ribose anomeric carbon (see in the diagram in this section), the transition state structure can be defined by early or late dissociation stage. Based on the finding of different transition state structures, Schramm and coworkers designed two transition state analogues mimicking the early and late dissociative transition state. The early and late transition state analogue shown binding affinity (Kd) of 360 and 140 pM, respectively.^[8]

Thermolysin inhibitor

Thermolysin is an enzyme produced by Bacillus thermoproteolyticus that catalyses the hydrolysis of peptides containing hydrophobic amino acids.^[9] Therefore, it is also a target for antibacterial agents. The enzymatic reaction mechanism starts form the small peptide molecule and replaces the zinc binding water molecule towards Glu143 of thermolysin. The water molecule is then activated by both the zinc ion and the Glu143 residue and attacks the carbonyl carbon to form a tetrahedral transition state (see figure). Holden and coworkers then mimicked that tetrahedral transition state to design a series of phosphonamidate peptide analogues. Among the synthesized analogues, R = L-Leu possesses the most potent inhibitory activity (K_i = 9.1 nM).^[10]

Arginase inhibitor

Arginase is a binuclear manganese metalloprotein that catalyses the hydrolysis of L-arginine to L-ornithine and urea. It is also regarded as a drug target for the treatment of asthma.^[11] The mechanism of hydrolysis of L-arginine is carried out via nucleophilic attack on the guanidino group by water, forming a tetrahedral intermediate. Studies shown that a boronic acid moiety adopts a tetrahedral configuration and serves as an inhibitor. In addition, the sulfonamide functional group can also mimic the transition state structure.^[12] Evidence of boronic acid mimics as transition state analogue inhibitors of human arginase I was elucidated by x-ray crystal structures.^[13]

References

^ Silverman RB (2004). The Organic Chemistry of Drug Design and Drug Action. San Diego, CA: Elsevier Academic Press. ISBN 0-12-643732-7.
^ Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA (January 1996). "The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase". Biochemistry. 35 (4): 1270–1273. doi:10.1021/bi952168g. PMID 8573583.
^ ^a ^b Schramm VL (2011). "Enzymatic transition states, transition-state analogs, dynamics, thermodynamics, and lifetimes". Annual Review of Biochemistry. 80 (1): 703–732. doi:10.1146/annurev-biochem-061809-100742. PMC 5502542. PMID 21675920.
^ Kollman P, Kuhn B, Peräkylä M (2002). "Computational Studies of Enzyme-Catalyzed Reactions: Where Are We in Predicting Mechanisms and in Understanding the Nature of Enzyme Catalysis?". J. Phys. Chem. B. 106 (7): 1537–1542. doi:10.1021/jp012017p.
^ Hou G, Cui Q (January 2012). "QM/MM analysis suggests that Alkaline Phosphatase (AP) and nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily". Journal of the American Chemical Society. 134 (1): 229–246. doi:10.1021/ja205226d. PMC 3257412. PMID 22097879.
^ Saen-Oon S, Quaytman-Machleder S, Schramm VL, Schwartz SD (October 2008). "Atomic detail of chemical transformation at the transition state of an enzymatic reaction". Proceedings of the National Academy of Sciences of the United States of America. 105 (43): 16543–16548. Bibcode:2008PNAS..10516543S. doi:10.1073/pnas.0808413105. PMC 2575456. PMID 18946041.
^ Singh V, Lee JE, Núñez S, Howell PL, Schramm VL (September 2005). "Transition state structure of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues". Biochemistry. 44 (35): 11647–11659. doi:10.1021/bi050863a. PMID 16128565.
^ Gutierrez JA, Luo M, Singh V, Li L, Brown RL, Norris GE, et al. (November 2007). "Picomolar inhibitors as transition-state probes of 5'-methylthioadenosine nucleosidases". ACS Chemical Biology. 2 (11): 725–734. doi:10.1021/cb700166z. PMID 18030989.
^ Endo S (1962). "Studies on protease produced by thermophilic bacteria". J. Ferment. Technol. 40: 346–353.
^ Holden HM, Tronrud DE, Monzingo AF, Weaver LH, Matthews BW (December 1987). "Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues". Biochemistry. 26 (26): 8542–8553. doi:10.1021/bi00400a008. PMID 3442675.
^ Maarsingh H, Zaagsma J, Meurs H (October 2009). "Arginase: a key enzyme in the pathophysiology of allergic asthma opening novel therapeutic perspectives". British Journal of Pharmacology. 158 (3): 652–664. doi:10.1111/j.1476-5381.2009.00374.x. PMC 2765587. PMID 19703164.
^ Cama E, Shin H, Christianson DW (October 2003). "Design of amino acid sulfonamides as transition-state analogue inhibitors of arginase". Journal of the American Chemical Society. 125 (43): 13052–13057. doi:10.1021/ja036365b. PMID 14570477.
^ Shishova EY, Di Costanzo L, Emig FA, Ash DE, Christianson DW (January 2009). "Probing the specificity determinants of amino acid recognition by arginase". Biochemistry. 48 (1): 121–131. doi:10.1021/bi801911v. PMC 2665027. PMID 19093830.

[1] Silverman RB (2004). The Organic Chemistry of Drug Design and Drug Action. San Diego, CA: Elsevier Academic Press. ISBN 0-12-643732-7.

[2] Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA (January 1996). "The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase". Biochemistry. 35 (4): 1270–1273. doi:10.1021/bi952168g. PMID 8573583.

[Schramm2011-3] Schramm VL (2011). "Enzymatic transition states, transition-state analogs, dynamics, thermodynamics, and lifetimes". Annual Review of Biochemistry. 80 (1): 703–732. doi:10.1146/annurev-biochem-061809-100742. PMC 5502542. PMID 21675920.

[4] Kollman P, Kuhn B, Peräkylä M (2002). "Computational Studies of Enzyme-Catalyzed Reactions: Where Are We in Predicting Mechanisms and in Understanding the Nature of Enzyme Catalysis?". J. Phys. Chem. B. 106 (7): 1537–1542. doi:10.1021/jp012017p.

[5] Hou G, Cui Q (January 2012). "QM/MM analysis suggests that Alkaline Phosphatase (AP) and nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily". Journal of the American Chemical Society. 134 (1): 229–246. doi:10.1021/ja205226d. PMC 3257412. PMID 22097879.

[6] Saen-Oon S, Quaytman-Machleder S, Schramm VL, Schwartz SD (October 2008). "Atomic detail of chemical transformation at the transition state of an enzymatic reaction". Proceedings of the National Academy of Sciences of the United States of America. 105 (43): 16543–16548. Bibcode:2008PNAS..10516543S. doi:10.1073/pnas.0808413105. PMC 2575456. PMID 18946041.

[7] Singh V, Lee JE, Núñez S, Howell PL, Schramm VL (September 2005). "Transition state structure of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues". Biochemistry. 44 (35): 11647–11659. doi:10.1021/bi050863a. PMID 16128565.

[8] Gutierrez JA, Luo M, Singh V, Li L, Brown RL, Norris GE, et al. (November 2007). "Picomolar inhibitors as transition-state probes of 5'-methylthioadenosine nucleosidases". ACS Chemical Biology. 2 (11): 725–734. doi:10.1021/cb700166z. PMID 18030989.

[9] Endo S (1962). "Studies on protease produced by thermophilic bacteria". J. Ferment. Technol. 40: 346–353.

[10] Holden HM, Tronrud DE, Monzingo AF, Weaver LH, Matthews BW (December 1987). "Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues". Biochemistry. 26 (26): 8542–8553. doi:10.1021/bi00400a008. PMID 3442675.

[11] Maarsingh H, Zaagsma J, Meurs H (October 2009). "Arginase: a key enzyme in the pathophysiology of allergic asthma opening novel therapeutic perspectives". British Journal of Pharmacology. 158 (3): 652–664. doi:10.1111/j.1476-5381.2009.00374.x. PMC 2765587. PMID 19703164.

[12] Cama E, Shin H, Christianson DW (October 2003). "Design of amino acid sulfonamides as transition-state analogue inhibitors of arginase". Journal of the American Chemical Society. 125 (43): 13052–13057. doi:10.1021/ja036365b. PMID 14570477.

[13] Shishova EY, Di Costanzo L, Emig FA, Ash DE, Christianson DW (January 2009). "Probing the specificity determinants of amino acid recognition by arginase". Biochemistry. 48 (1): 121–131. doi:10.1021/bi801911v. PMC 2665027. PMID 19093830.

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PROFILPELAJAR.COM

Transition state analog

Transition state analogue

Designing transition state analogue

Kinetic isotope effect

Computational simulation

Examples of transition state analogue design

Methylthioadenosine nucleosidase inhibitor

Thermolysin inhibitor

Arginase inhibitor

See also

References

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