**5. Hydroxyethylene intermediate analogs as inhibitors of proteases**

Aspartic proteases generally bind 6-10 amino acid regions of their polypeptide substrates, which are typically processed with the aid of two catalytic aspartic acid residues in the ac‐ tive site. Thus, there is usually considerable scope for building inhibitor specificity for a par‐ ticular aspartic protease by taking advantage of the collective interactions between a putative inhibitor on both sides of its scissile amide bond, and a substantial portion of the substrate-binding groove of the enzyme [Eder et al., 2007]. A very effective group of their inhibitors are simple hydroxyethylene analogs of tetrahedral oxyanion intermediates of the hydrolysis of peptide bonds. This approach is based on the structure of bestatin, a general inhibitor of aminopeptidases and aspartyl proteinases isolated in 1976 from *Streptomyces oli‐ voreticuli* [Umezawa et al., 1976].

Today, HIV protease inhibitors constitute around 40% of available drugs against HIV. Near‐ ly all of them contain hydroxyethylene unit as transition state analog mimic, to mention on‐ ly Darunavir, Atazanavir, Fosamprenavir, Lopinavir (or Ritonavir) or the oldest one Saquinavir (Fig. 11) [Brik & Wong, 2003; Pokorna et. al, 2009].

Availability of anti-HIV drugs enabled to introduce highly active antiretroviral therapy (HAART), which resulted in dramatic decrease of the mortality and morbidity for a wide variety of opportunistic viral, bacterial, fungal and parasitic infections among HIV-infected individuals in economically developed countries [Andreoni & Perno, 2012]. Thus, the de‐ sign, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. However, both the academias, as well as, the industry need to continue in their effort to develop novel, more potent compounds. This is mainly connected with HIV drug resistance, which in turn results from the high mutation rate, caused by the lack of proofreading activity of the viral reverse transcriptase. The pat‐ tern of mutations associated with the viral resistance is extremely complex as shown in Fig‐ ure 12 [Pokorna et al., 2009]. Taken together, in spite of the indisputable success of the HAART and benefit to patients, new approaches to the antiviral treatment are highly desira‐ ble [Clarke, 2007; Adachi et al., 2009; Pokorna et al., 2009; Alfonso & Mozote, 2011].

shown, that these drugs exhibit marked antiprotozoal activity. For example Saquinavir, Lo‐ pinavir, Indinavir directly inhibited the grown of *Plasmodium falciparum* in vitro at clinically relevant concentrations. This findings suggest that some inhibitors of HIV protease are ac‐ tive against the most virulent human malaria parasite *P. falciparum* that is known to express number of aspartic proteases (plasmepsins) [Skinner-Adams, et al., 2004; Alfonso & Mozote,

Transition State Analogues of Enzymatic Reaction as Potential Drugs

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

337

**Figure 11.** Representative examples of clinical HIV protease drugs.

More than 25 million people are suffering from dementia, and the annual socioeconomic worldwide costs have been estimated to exceed U.S. \$200 billion. γ-Secretase, along with β secretase produces the amyloid β-protein of Alzheimer's disease. Because of its key role in the pathogenesis γ-secretase has been a prime target for drug discovery, and many inhibi‐ tors of this protease have been developed. These enzymes are also effectively inhibited by

2011].

Quite interestingly, small modifications of core structure of the inhibitor results in minute changes in inhibitor affinity to HIV protease as demonstrated in Figure 13 [Wu et al., 2008; Mahalingham et al., 2010].

Studies conducted in order to evaluate the influence of these antiviral drugs on the develop‐ ment of parasites, which are known to co-infect HIV-positive individuals, surprisingly Transition State Analogues of Enzymatic Reaction as Potential Drugs http://dx.doi.org/10.5772/52504 337

**Figure 11.** Representative examples of clinical HIV protease drugs.

act as hydrolytically stable entities. Thus, dialkylsilanol is an effective functional group for the design of active site-directed protease inhibitors. This concept has been successfully test‐ ed by replacing the presumed tetrahedral carbon of thermolysin, HIV-protease and angio‐ tensin converting enzyme substrates with silanediol groups (Fig. 10), which resulted in potent inhibitors of these enzymes [Juers et al., 2005; Sieburth & Chen, 2006; Bo et al., 2011;

**5. Hydroxyethylene intermediate analogs as inhibitors of proteases**

Aspartic proteases generally bind 6-10 amino acid regions of their polypeptide substrates, which are typically processed with the aid of two catalytic aspartic acid residues in the ac‐ tive site. Thus, there is usually considerable scope for building inhibitor specificity for a par‐ ticular aspartic protease by taking advantage of the collective interactions between a putative inhibitor on both sides of its scissile amide bond, and a substantial portion of the substrate-binding groove of the enzyme [Eder et al., 2007]. A very effective group of their inhibitors are simple hydroxyethylene analogs of tetrahedral oxyanion intermediates of the hydrolysis of peptide bonds. This approach is based on the structure of bestatin, a general inhibitor of aminopeptidases and aspartyl proteinases isolated in 1976 from *Streptomyces oli‐*

Today, HIV protease inhibitors constitute around 40% of available drugs against HIV. Near‐ ly all of them contain hydroxyethylene unit as transition state analog mimic, to mention on‐ ly Darunavir, Atazanavir, Fosamprenavir, Lopinavir (or Ritonavir) or the oldest one

Availability of anti-HIV drugs enabled to introduce highly active antiretroviral therapy (HAART), which resulted in dramatic decrease of the mortality and morbidity for a wide variety of opportunistic viral, bacterial, fungal and parasitic infections among HIV-infected individuals in economically developed countries [Andreoni & Perno, 2012]. Thus, the de‐ sign, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. However, both the academias, as well as, the industry need to continue in their effort to develop novel, more potent compounds. This is mainly connected with HIV drug resistance, which in turn results from the high mutation rate, caused by the lack of proofreading activity of the viral reverse transcriptase. The pat‐ tern of mutations associated with the viral resistance is extremely complex as shown in Fig‐ ure 12 [Pokorna et al., 2009]. Taken together, in spite of the indisputable success of the HAART and benefit to patients, new approaches to the antiviral treatment are highly desira‐

ble [Clarke, 2007; Adachi et al., 2009; Pokorna et al., 2009; Alfonso & Mozote, 2011].

Quite interestingly, small modifications of core structure of the inhibitor results in minute changes in inhibitor affinity to HIV protease as demonstrated in Figure 13 [Wu et al., 2008;

Studies conducted in order to evaluate the influence of these antiviral drugs on the develop‐ ment of parasites, which are known to co-infect HIV-positive individuals, surprisingly

Meanwell, 2011].

336 Drug Discovery

*voreticuli* [Umezawa et al., 1976].

Mahalingham et al., 2010].

Saquinavir (Fig. 11) [Brik & Wong, 2003; Pokorna et. al, 2009].

shown, that these drugs exhibit marked antiprotozoal activity. For example Saquinavir, Lo‐ pinavir, Indinavir directly inhibited the grown of *Plasmodium falciparum* in vitro at clinically relevant concentrations. This findings suggest that some inhibitors of HIV protease are ac‐ tive against the most virulent human malaria parasite *P. falciparum* that is known to express number of aspartic proteases (plasmepsins) [Skinner-Adams, et al., 2004; Alfonso & Mozote, 2011].

More than 25 million people are suffering from dementia, and the annual socioeconomic worldwide costs have been estimated to exceed U.S. \$200 billion. γ-Secretase, along with β secretase produces the amyloid β-protein of Alzheimer's disease. Because of its key role in the pathogenesis γ-secretase has been a prime target for drug discovery, and many inhibi‐ tors of this protease have been developed. These enzymes are also effectively inhibited by

peptidomimetics containing hydroxyethylene fragment replacing hydrolyzed peptide bond. Only one drug (Semagacestat, Fig. 14) reached phase III clinical studies so far, however, un‐ covered evidence of cognitive worsening in treated patients compared with placebo led to suspension of the trials in 2010. Anyway, design, synthesis and evaluation of new low-mo‐ lecular, nanomolar inhibitors of secretases, structure of which significantly drifted away from peptidic transition state analogs (Fig. 14), is still challenging and brought new promis‐ ing results [Osterman et al., 2006; Wolfe, 2012]. Due to rapid technological progress in chem‐ istry, bioinformatics, structural biology and computer technology, computer-aided drug design plays more and more important role in this respect [Avram et al., 2006; Fujimoto et

Transition State Analogues of Enzymatic Reaction as Potential Drugs

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

339

Hypertension is a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, and heart failure, the leading causes of death in the Western world. Inhibitors of the renin–angiotensin system have proven to be successful treatments for hypertension. As renin specifically catalyses the rate limiting step of this system, it represents the optimal tar‐

al., 2008; Xu et al., 2009; Al-Tel et al. 2011; Hamada et al., 2012].

**Figure 14.** Inhibitors of secretases.

**Figure 12.** Three-dimensional structure of HIV protease complexed with Darunavir. Mutations associated with resist‐ ance to clinically used inhibitors are depicted as balls.

**Figure 13.** Influence of linker length on activity of HIV protease inhibitors.

peptidomimetics containing hydroxyethylene fragment replacing hydrolyzed peptide bond. Only one drug (Semagacestat, Fig. 14) reached phase III clinical studies so far, however, un‐ covered evidence of cognitive worsening in treated patients compared with placebo led to suspension of the trials in 2010. Anyway, design, synthesis and evaluation of new low-mo‐ lecular, nanomolar inhibitors of secretases, structure of which significantly drifted away from peptidic transition state analogs (Fig. 14), is still challenging and brought new promis‐ ing results [Osterman et al., 2006; Wolfe, 2012]. Due to rapid technological progress in chem‐ istry, bioinformatics, structural biology and computer technology, computer-aided drug design plays more and more important role in this respect [Avram et al., 2006; Fujimoto et al., 2008; Xu et al., 2009; Al-Tel et al. 2011; Hamada et al., 2012].

**Figure 14.** Inhibitors of secretases.

**Figure 12.** Three-dimensional structure of HIV protease complexed with Darunavir. Mutations associated with resist‐

ance to clinically used inhibitors are depicted as balls.

338 Drug Discovery

**Figure 13.** Influence of linker length on activity of HIV protease inhibitors.

Hypertension is a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, and heart failure, the leading causes of death in the Western world. Inhibitors of the renin–angiotensin system have proven to be successful treatments for hypertension. As renin specifically catalyses the rate limiting step of this system, it represents the optimal tar‐ get for antihypertensive drugs. Aliskiren (Fig. 15), a promising drug lowering blood pres‐ sure in sodium-depleted marmosets and hypertensive human patients, was developed using a combination of crystal structure analysis of renin–inhibitor complexes and computa‐ tional methods [Wood et al., 2003]. The therapy was introduced under the names Takturna and Rasilez.

however, an important difference between these two types of inhibitors. The boronic acid derivative possesses a negative charge, whereas the hemiacetal adduct is neutral (Fig 17).

Transition State Analogues of Enzymatic Reaction as Potential Drugs

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

341

Hence, the peptidyl boronic acid adduct is a better transition state analog than the hemiace‐ tal formed from peptidic aldehyde [Polgár, 2005]. Aldehydes typically have a low preva‐ lence in drugs and drug candidates because of their potential chemical reactivity and susceptibility to be engaged in a reduction/oxidation pathways in vivo. Therefore peptidyl boronic acids are considered as far better drug candidates. Additionally slight changes in

In 2003, bortezomib (Fig. 18), a first-in-class therapeutic, gained approval from the US Feder‐ al Drug Administration for the treatment of relapsed multiple myeloma and mantle cell lymphoma. Approval in the UK, for multiple myeloma, followed in 2006. It possesses a unique mode of action. Bortezomib acts as inhibitor of the 26S proteasome, the key regulator of intracellular protein degradation, found in the nucleus and cytosol of all eukaryotic cells, and forming part of the critical ubiquitin-proteasome system. This inhibition results in dis‐

This finding initiated intensive researches on boronic inhibitors of serine and threonine pro‐ teases [Trippier & McGuigan, 2010]. For, example recently inhibitors of Lon proteases (bac‐ terial ATP-dependent protease conferring bacterial virulence) afforded interesting

pH can result in release of the inhibitor from the active site, which is profitous.

ruption of homeostatic mechanisms within the cell that can lead to cell death.

**Figure 17.** Suicidal substrates yielding transition state analogs

**Figure 18.** Structure of bortezomib.

**Figure 15.** Aliskiren (left hand side) and its more potent analog.

Another possibility arose from use of fluoroketone derivatives. α-monofluoroketones are approximately 50% hydrated, whereas the α,α-difluoroketones are 100% hydrated in aque‐ ous solutions (Fig. 16). The latter ones are obviously of choice because of their striking simi‐ larity to phosphinic inhibitors (two hydroxyls placed at terahedral atom). This approach is applied very rarely but gave good inhibitors of fungal endothiapepsin [Tuan et al., 2007] and matrix metalloprotease [Reiter et al., 2000] (Fig. 16).

**Figure 16.** Difluoroketones as transition state inhibitor.
