**4. Inhibitors of proteinases**

First inhibitors being transition state analogs were designed for proteolytic enzymes. The design was based on the resemblance of transition state of phosphonamidates and phos‐ phonic, phosphinic (Fig. 4) acids to the sp3 intermediate of the hydrolysis of peptide bond. Because the lengths of oxygen-to-phosphorus and carbon-to-phosphorus bonds are signifi‐ cantly longer than the corresponding carbon-to-carbon and carbon-to-oxygen bonds, orga‐ nophosphorus fragment of the molecule might be considered as "swollen" tetrahedral intermediate and thus can be treated similar to the transition state of this reaction.

**Figure 5.** Representative organophosphorus inhibitors of metalloproteinases.

Simple phosphonic acid analogs of amino acids and pseudopeptides, containing phosphi‐ nate moiety replacing scissile peptide bond, are acting via this mechanism and rank amongst most potent inhibitors of metalloproteinases (Fig. 5). Inhibitors of neutral alanyl (M1) and leucine (M17) aminopeptidases are among the most recognized and most inten‐ sively studied representatives of metal-containing exopeptidases of biomedical significance [Lowther & Matthews, 2002; Grembecka et al., 2003; Vassiliou et. al. 2007]. Functions related to tumorigenesis and invasion makes these enzymes molecular targets for the development of potential anticancer drugs [Grembecka & Kafarski, 2001; Zhang & Xu, 2008; Fournié-Za‐ luski et al., 2009; Grzywa et al., 2010]. The recognized role of neutral aminopeptidase in the pathogenesis of hypertension provides also an opportunity for regulating arterial blood

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**Figure 4.** Organophosphoprus compounds as transition state analog inhibitors of hydrolysis of peptide bonds.

Crystallography of enzyme-inhibitor complexes and molecular modeling studies had shown that their potent inhibitory activities result from both: resemblance to the transition state and strong electrostatic interactions between positively charged active-site metal ions (pre‐ dominantly zinc ions) and negatively charged phosphonic acid (or related) group [Mucha et al., 2010; Mucha et al., 2011]. Although the phosphonate/phosphinate group is a rather weak zinc complexing moiety, it offers other advantageous structural and electronic features [Col‐ linsova & Jiráček, 2000].

Transition State Analogues of Enzymatic Reaction as Potential Drugs http://dx.doi.org/10.5772/52504 331

**Figure 5.** Representative organophosphorus inhibitors of metalloproteinases.

**4. Inhibitors of proteinases**

330 Drug Discovery

linsova & Jiráček, 2000].

phonic, phosphinic (Fig. 4) acids to the sp3

First inhibitors being transition state analogs were designed for proteolytic enzymes. The design was based on the resemblance of transition state of phosphonamidates and phos‐

Because the lengths of oxygen-to-phosphorus and carbon-to-phosphorus bonds are signifi‐ cantly longer than the corresponding carbon-to-carbon and carbon-to-oxygen bonds, orga‐ nophosphorus fragment of the molecule might be considered as "swollen" tetrahedral

intermediate and thus can be treated similar to the transition state of this reaction.

**Figure 4.** Organophosphoprus compounds as transition state analog inhibitors of hydrolysis of peptide bonds.

Crystallography of enzyme-inhibitor complexes and molecular modeling studies had shown that their potent inhibitory activities result from both: resemblance to the transition state and strong electrostatic interactions between positively charged active-site metal ions (pre‐ dominantly zinc ions) and negatively charged phosphonic acid (or related) group [Mucha et al., 2010; Mucha et al., 2011]. Although the phosphonate/phosphinate group is a rather weak zinc complexing moiety, it offers other advantageous structural and electronic features [Col‐

intermediate of the hydrolysis of peptide bond.

Simple phosphonic acid analogs of amino acids and pseudopeptides, containing phosphi‐ nate moiety replacing scissile peptide bond, are acting via this mechanism and rank amongst most potent inhibitors of metalloproteinases (Fig. 5). Inhibitors of neutral alanyl (M1) and leucine (M17) aminopeptidases are among the most recognized and most inten‐ sively studied representatives of metal-containing exopeptidases of biomedical significance [Lowther & Matthews, 2002; Grembecka et al., 2003; Vassiliou et. al. 2007]. Functions related to tumorigenesis and invasion makes these enzymes molecular targets for the development of potential anticancer drugs [Grembecka & Kafarski, 2001; Zhang & Xu, 2008; Fournié-Za‐ luski et al., 2009; Grzywa et al., 2010]. The recognized role of neutral aminopeptidase in the pathogenesis of hypertension provides also an opportunity for regulating arterial blood pressure by their inhibitors [Banegas et al., 2006; Bodineau et al., 2008]. Additionally, two of these pseudodipeptides appear to be excellent inhibitors when applied to *Plasmodium falcipa‐ rum* M1 and M17 aminopeptidases (Fig. 5), the protozoan counterparts of neutral and leu‐ cine aminopeptidases [Stack et al., 2007; Cunningham et al, 2008; McGowan et al., 2009; McGowan et al., 2010]. They efficiently controlled the growth of *P. falciparium* in cultures, including those of malaria cells lines resistant to chloroquine, and significantly reduced ma‐ laria infections in murine model (*Plasmodium chabaudi*) [Skinner-Adams et al., 2007]. These findings positively validated *P. falciparum* M1 and M17 aminopeptidases as promising tar‐ gets for a novel treatment of malaria and identify new leads with anti-parasite potential [Skinner-Adams et al., 2010; Thivierge et al., 2012].

The design and development of pseudopeptidic inhibitors of aminopeptidases are greatly facilitated by two factors. First, the results of extensive structure-activity relationship stud‐ ies, available for a wide collection of fluorogenic substrates, have defined the requirements of the S1 binding pockets of these enzymes [Drag & Salvesen, 2009; Drag et al., 2010; Gajda et al., 2012; Poręba et al., 2011; Poręba 2012]. Second, computer-aided analysis of numerous crystal structures available for leucine aminopeptidase has pointed to this enzyme as a pri‐ mary molecular target for extending and optimizing interactions within the S1' pocket [Grembecka et al., 2001; Jørgensen et al., 2002; Evdokimov et al., 2007; Khandelwal et al., 2005; Khaliullin et al. 2010; Li et al., 2010].

Phosphinic pseudopeptides have also clearly revealed their potential for the regulation of matrix metalloproteinases (MMPs, matrixins), zinc-dependent endopeptidases implicated in the breakdown of the extracellular matrix [Yiotakis at al., 2004; Fisher & Mobashery, 2006]. Cleavage of the matrix component (collagen, lamanin, elastin, gelatin, etc.) is physiologically essential for tissue remodeling processes such as morphogenesis, embryogenesis and repro‐ duction [Overall & Kleifield, 2006; Sang et. al., 2006]. Overexpression or inadequate level of matrix metalloproteinases leads to pathological states such as osteoarthritis, rheumatoid ar‐ thritis and inflammation, but it is most associated with tumor growth, invasion, and meta‐ stasis. Angiogenetic process favored by these enzymes is essential for vascoularization and growth of tumors. Thus, they were the first proteinase targets seriously considered for com‐ bating cancer. Despite that preliminary clinical/preclinical studies on MMP inhibition in tu‐ mor models brought positive results the outcome in the drug market has been so far unsatisfactory. The spectacular failure of the last-step clinical trials is mainly due to a lack of selectivity and serious side effects [Fisher & Mobashery, 2006]. The field is now resurging with careful reinvestigation of the precise roles of each particular MMP member and a focus on the development of selective inhibitors that fully discriminate between different mem‐ bers of the MMP family [Reiter et al., 2003; Matziari et al., 2007; Zucker & Cao, 2009; Devel et al., 2010; Johnson et al., 2011]. Such selectivity had been reached by variation of peptide scaffold by means of combinatorial pseudopeptide synthesis [Buchardt et al, 2000; Dive et al., 2004] or by application of molecular modeling based on crystallographic studies of these enzymes [Rao, 2005; Pirard, 2007; Verma & Hansch, 2007; Anzellotti & Farrell, 2008; Kalva et al., 2012]. Representative selective inhibitors of this class are shown in Figure 6.

**Figure 6.** Representative inhibitors selective against chosen matrix metalloproteinases.

uer-Fields et al., 2008].

Lazcano et al. 2012].

Quite interesting approach is preparation of hybrid systems as this composed of a phosphi‐ nate transition state analogue that has been incorporated within a triple-helical peptide tem‐ plate. The template sequence was based on the α1(V)436-450 collagen region, which is hydrolyzed at the Gly439-Val440 bond selectively by MMP-2 and MMP-9. In that manner high‐ ly selective inhibitor towards these two gelatinases was found [Lauer-Fields et al., 2007; La‐

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Phosphinic transition state analog approach has been also recently applied for the design and synthesis of novel potent inhibitors of other proteinases of medicinal importance. Thus, inhibitors of angiotensin converting enzyme [Mores et al., 2008; Julien et al. 2010; Akif et al., 2011] are potential drugs against hypertension, aspartyl aminopeptidase as antimalarial agent [Teuscher et al., 2007], inhibitors of cathepsin C and renal dipeptidase may be consid‐ ered as potential anticancer agents [Gurulingappa et al., 2003: Mucha et al., 2004], inhibitors of sortase, which is bacterial virulence protein [Kruger et al., 2004], whereas inhibition of py‐ roglutamyl peptidase II enhances the analeptic effect of thyrotropin [Matziari et al., 2008;

**Figure 6.** Representative inhibitors selective against chosen matrix metalloproteinases.

pressure by their inhibitors [Banegas et al., 2006; Bodineau et al., 2008]. Additionally, two of these pseudodipeptides appear to be excellent inhibitors when applied to *Plasmodium falcipa‐ rum* M1 and M17 aminopeptidases (Fig. 5), the protozoan counterparts of neutral and leu‐ cine aminopeptidases [Stack et al., 2007; Cunningham et al, 2008; McGowan et al., 2009; McGowan et al., 2010]. They efficiently controlled the growth of *P. falciparium* in cultures, including those of malaria cells lines resistant to chloroquine, and significantly reduced ma‐ laria infections in murine model (*Plasmodium chabaudi*) [Skinner-Adams et al., 2007]. These findings positively validated *P. falciparum* M1 and M17 aminopeptidases as promising tar‐ gets for a novel treatment of malaria and identify new leads with anti-parasite potential

The design and development of pseudopeptidic inhibitors of aminopeptidases are greatly facilitated by two factors. First, the results of extensive structure-activity relationship stud‐ ies, available for a wide collection of fluorogenic substrates, have defined the requirements of the S1 binding pockets of these enzymes [Drag & Salvesen, 2009; Drag et al., 2010; Gajda et al., 2012; Poręba et al., 2011; Poręba 2012]. Second, computer-aided analysis of numerous crystal structures available for leucine aminopeptidase has pointed to this enzyme as a pri‐ mary molecular target for extending and optimizing interactions within the S1' pocket [Grembecka et al., 2001; Jørgensen et al., 2002; Evdokimov et al., 2007; Khandelwal et al.,

Phosphinic pseudopeptides have also clearly revealed their potential for the regulation of matrix metalloproteinases (MMPs, matrixins), zinc-dependent endopeptidases implicated in the breakdown of the extracellular matrix [Yiotakis at al., 2004; Fisher & Mobashery, 2006]. Cleavage of the matrix component (collagen, lamanin, elastin, gelatin, etc.) is physiologically essential for tissue remodeling processes such as morphogenesis, embryogenesis and repro‐ duction [Overall & Kleifield, 2006; Sang et. al., 2006]. Overexpression or inadequate level of matrix metalloproteinases leads to pathological states such as osteoarthritis, rheumatoid ar‐ thritis and inflammation, but it is most associated with tumor growth, invasion, and meta‐ stasis. Angiogenetic process favored by these enzymes is essential for vascoularization and growth of tumors. Thus, they were the first proteinase targets seriously considered for com‐ bating cancer. Despite that preliminary clinical/preclinical studies on MMP inhibition in tu‐ mor models brought positive results the outcome in the drug market has been so far unsatisfactory. The spectacular failure of the last-step clinical trials is mainly due to a lack of selectivity and serious side effects [Fisher & Mobashery, 2006]. The field is now resurging with careful reinvestigation of the precise roles of each particular MMP member and a focus on the development of selective inhibitors that fully discriminate between different mem‐ bers of the MMP family [Reiter et al., 2003; Matziari et al., 2007; Zucker & Cao, 2009; Devel et al., 2010; Johnson et al., 2011]. Such selectivity had been reached by variation of peptide scaffold by means of combinatorial pseudopeptide synthesis [Buchardt et al, 2000; Dive et al., 2004] or by application of molecular modeling based on crystallographic studies of these enzymes [Rao, 2005; Pirard, 2007; Verma & Hansch, 2007; Anzellotti & Farrell, 2008; Kalva et

al., 2012]. Representative selective inhibitors of this class are shown in Figure 6.

[Skinner-Adams et al., 2010; Thivierge et al., 2012].

332 Drug Discovery

2005; Khaliullin et al. 2010; Li et al., 2010].

Quite interesting approach is preparation of hybrid systems as this composed of a phosphi‐ nate transition state analogue that has been incorporated within a triple-helical peptide tem‐ plate. The template sequence was based on the α1(V)436-450 collagen region, which is hydrolyzed at the Gly439-Val440 bond selectively by MMP-2 and MMP-9. In that manner high‐ ly selective inhibitor towards these two gelatinases was found [Lauer-Fields et al., 2007; La‐ uer-Fields et al., 2008].

Phosphinic transition state analog approach has been also recently applied for the design and synthesis of novel potent inhibitors of other proteinases of medicinal importance. Thus, inhibitors of angiotensin converting enzyme [Mores et al., 2008; Julien et al. 2010; Akif et al., 2011] are potential drugs against hypertension, aspartyl aminopeptidase as antimalarial agent [Teuscher et al., 2007], inhibitors of cathepsin C and renal dipeptidase may be consid‐ ered as potential anticancer agents [Gurulingappa et al., 2003: Mucha et al., 2004], inhibitors of sortase, which is bacterial virulence protein [Kruger et al., 2004], whereas inhibition of py‐ roglutamyl peptidase II enhances the analeptic effect of thyrotropin [Matziari et al., 2008; Lazcano et al. 2012].

It is worth mentioning that *Monopril®*, the sodium salt of fosinopril [Fig. 7], the ester prodrug of an angiotensin-converting enzyme (ACE) inhibitor fosinoprilat, is perhaps one of the most effective implementation of transition state analogy in medicine [Powell et al., 1984].

Begacestat (Fig. 8), an effective and potent inhibitor of γ-secretase is an exception here [May‐ er et al., 2008; Martone et al., 2009]. γ-Secretase catalyzes the final step in the generation of amyloid β peptides from amyloid precursor protein. Amyloid β-peptides aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Begacestat appeared to be well tolerated in mouse and dog toxicity studies and has been advanced to human clinical trials for the treat‐

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Dialkylsilanediols are tetrahedral functional groups that can mimic hydrated carbonyls and thus might be also considered as "swollen" intermediates of peptide bond hydrolysis. When silanediols are embedded in a peptide-like structure, they are recognized by proteinases and

ment of this neurological disease.

**Figure 9.** Mode of binding of sulfonamide HIV protease inhibitor.

**Figure 10.** Silenediols as proteinase inhibitors.

**Figure 7.** Fosinopril and fosinoprilat.

Sulfonamides also mimic both shape and electronic environment of the transition state of peptide bond hydrolysis. This approach was used for introduction of transition state inhibi‐ tors of HIV-protease, thermolysin and thrombine as well as haptens for generation of cata‐ lytic antibodies (Fig. 8) [Moree, et al., 1993; Moree, et al., 1995; Löwik et al., 2000; Liskamp & Kruijzer, 2004; Turcotte et al., 2012]. Unfortunately most of them appeared to be ineffective. This might be explained by non-typical bonding of potent inhibitor of this class with HIV protease (Fig. 9). It appeared that sulfonamide moiety displaces water molecule from active site and forms hydrogen bonds with two isoleucines, not as expected with catalytic aspartic acids [Meanwell, 2011]. Thus, sulfonamide group does not act as transition state analogue.

**Figure 8.** Sufonamides as inhibitors of proteases.

Begacestat (Fig. 8), an effective and potent inhibitor of γ-secretase is an exception here [May‐ er et al., 2008; Martone et al., 2009]. γ-Secretase catalyzes the final step in the generation of amyloid β peptides from amyloid precursor protein. Amyloid β-peptides aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Begacestat appeared to be well tolerated in mouse and dog toxicity studies and has been advanced to human clinical trials for the treat‐ ment of this neurological disease.

**Figure 9.** Mode of binding of sulfonamide HIV protease inhibitor.

It is worth mentioning that *Monopril®*, the sodium salt of fosinopril [Fig. 7], the ester prodrug of an angiotensin-converting enzyme (ACE) inhibitor fosinoprilat, is perhaps one of the most effective implementation of transition state analogy in medicine [Powell et al., 1984].

Sulfonamides also mimic both shape and electronic environment of the transition state of peptide bond hydrolysis. This approach was used for introduction of transition state inhibi‐ tors of HIV-protease, thermolysin and thrombine as well as haptens for generation of cata‐ lytic antibodies (Fig. 8) [Moree, et al., 1993; Moree, et al., 1995; Löwik et al., 2000; Liskamp & Kruijzer, 2004; Turcotte et al., 2012]. Unfortunately most of them appeared to be ineffective. This might be explained by non-typical bonding of potent inhibitor of this class with HIV protease (Fig. 9). It appeared that sulfonamide moiety displaces water molecule from active site and forms hydrogen bonds with two isoleucines, not as expected with catalytic aspartic acids [Meanwell, 2011]. Thus, sulfonamide group does not act as transition state analogue.

**Figure 7.** Fosinopril and fosinoprilat.

334 Drug Discovery

**Figure 8.** Sufonamides as inhibitors of proteases.

**Figure 10.** Silenediols as proteinase inhibitors.

Dialkylsilanediols are tetrahedral functional groups that can mimic hydrated carbonyls and thus might be also considered as "swollen" intermediates of peptide bond hydrolysis. When silanediols are embedded in a peptide-like structure, they are recognized by proteinases and 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; Meanwell, 2011].
