**7. Other hydrolases**

antibacterial agents [Frase and Lee, 2007], inhibitors of prostate-specific antigen for prostate cancer imaging and therapy [LeBeau et al., 2008], antifungal inhibitors of kexin (regulatory proteins from *Candida*) [Holyoak et al., 2004; Wheatley & Holyoak, 2007], inhibitor of HCV NS3 protease as potential drug against hepatitis [Zhang et al., 2003; Venkatraman et al., 2009], and anticancer and antibacterial inhibitors of proteasome [Hu et al., 2006]. Represen‐

tative examples of these inhibitors are shown in Figure 19.

342 Drug Discovery

**Figure 19.** Boronic acid based inhibitors.

The data considering transition state analogue inhibitors of other hydrolases are practically limited to inhibitors of β–lactamases, arginase and urease.

Antibiotic resistance, especially to widely prescribed β-lactam antibiotics, is a serious threat to public health and is responsible for the increase in morbidity, mortality, and health care costs related to the treatment of bacterial infections. In most cases emergence of antibioticresistant bacteria is primarily driven by overuse of β-lactam antibiotics in food and agricul‐ tural products. The most prominent resistance mechanism is related to the expression of βlactamases, which hydrolyze β–lactam fragment of the drug molecule. In nature, four classes of these enzymes exist. Three of them are serine-based, whereas fourth is zinc-de‐ pendent-hydrolase. To counteract β-lactamases, mechanism-based inhibitors were devel‐ oped to be administered in concert with β-lactam antibiotics. Presently, there are three commercially available β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam). The new approach to obtain such inhibitors is combination of structure of potent β-lactam antibiotics with a boronic [Thomson, et al., 2007; Eidam et al., 2010: Ke, et al., 2011; Chan, et al., 2012] or phosphonic [Nukaga, et al., 2004] acid moieties with the goal of mimicking the transition state and creating a high-affinity, reversible inhibitor that cannot be inactivated by β-lactamases since they do not bear hydrolyzable β-lactam ring.

**Figure 20.** Transition state inhibitors of lactamases.

Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. The hydrolysis of *L*-arginine to *L*-ornithine and urea (Fig. 21) is also the final cytosolic step of the urea cycle in mammali‐ an liver. *S*-(2-Boronoethyl)-*L*-cysteine is one of the most effective inhibitor of the enzyme (Fig 21). The specificity determinants of amino acid recognition by arginase were identified by X-Ray structure of human arginase I enzyme complexed with this inhibitor. These stud‐ ies undoubtedly shown that boronate adopts tetrahedral configuration [Cama et al., 2003 & 2003a; Shishova, et al., 2009]. Also aldehydes and sulfonamides similar to boronic acids ap‐ peared to be promising inhibitors of arginases [Shin et al., 2004].

wise scheme of inhibitor design, shown in Figure 21, led to the synthesis of compounds with low structural complexity, high hydrolytic stability and satisfactory biological activity against various ureases, including cytoplasmic urease from pathogenic *Proteus* species [Vas‐

Transition State Analogues of Enzymatic Reaction as Potential Drugs

http://dx.doi.org/10.5772/52504

345

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds – the peptidyl-transferase center – is com‐ posed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. Peptide bond formation during ribosomal protein synthesis involves an aminolysis reaction between the aminoacyl α-amino group and the carbonyl ester of the growing peptide via a transition state with a developing nega‐ tive charge - the oxyanion. Therefore the observed intermediates and transition states are similar to those observed in proteinases (Fig. 23). Structural and molecular dynamic studies have suggested that the ribosome may stabilize the oxyanion in the transition state of pep‐ tide bond formation via a highly ordered water molecule [Rodnina et al., 2006; Carrasco et

siliou et al., 2008; Berlicki et al., 2012; Vassiliou et al., 2012].

**Figure 22.** Urease hydrolysed reaction and evolution of the structure of its inhibitors.

action [Green & Lorsch, 2002; Weinger et al., 2004; Carrasco, et al., 2011].

To biochemically elucidate how the ribosome stabilizes the developing negative charge in the transition state of peptide bond formation, a series of tetrahedral transition state mimics were synthesized. Their relative binding affinities for the ribosomes also were measured (Fig. 23). The obtained results confirmed high affinity of predicted mechanism of ribosome

**8. Peptide bond formation by ribosome**

al., 2011].

**Figure 21.** Arginase catalyzed reaction and representative inhibitors of the enzyme.

Urease catalyzes hydrolysis of urea in the last step of organic nitrogen mineralization to give ammonia and carbamate, which decomposes to give a second molecule of ammonia and bi‐ carbonate (Fig. 22). The hydrolysis of the reaction products induces an overall pH increase that has negative implications both in human and animal health as well as in the ecosphere. Urease is a virulence factor in infections of urinary (*Proteus mirabilis*, *Ureaplasma urealyticum*) and gastrointestinal tracts (*Helicobacter pylori*), causing severe diseases such as peptic ulcers, stomach cancer, and formation of urinary stones. The efficiency of soil nitrogen fertilization with urea (the most used fertilizer worldwide) decreases due to ammonia volatilization and root damage caused by soil pH increase. Thus, control of the activity of urease through the use of inhibitors could counteract these negative effects [Kosikowska & Berlicki, 2011; Zam‐ belli et al., 2011]. Di- and triamides of phosphoric acid represent a group of urease inhibitors with the highest activity. It is the direct consequence of their similarity to the tetrahedral transition state of the enzymatic reaction of urea hydrolysis. Takeda Chemicals have patent‐ ed a large group of N-acyltriamido phosphates and found over 90 examples with nanomolar activity against *H. pylori* urease, with flurofamide being the most effective (Fig. 22) [Kosi‐ kowska & Berlicki, 2011].

Recently design, synthesis, and evaluation of novel ogranophosphonate inhibitors of bacteri‐ al urease have been described as an attractive alternative to known phosphoramidates. On the basis of the crystal structure of *Bacillus pasteurii* urease, several phosphinic acids and their short peptides have been designed by using the computer-aided techniques. The step‐ wise scheme of inhibitor design, shown in Figure 21, led to the synthesis of compounds with low structural complexity, high hydrolytic stability and satisfactory biological activity against various ureases, including cytoplasmic urease from pathogenic *Proteus* species [Vas‐ siliou et al., 2008; Berlicki et al., 2012; Vassiliou et al., 2012].
