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Proteases: Nature’S Destroyers and the Drugs That Stop Them

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Proteases: Nature’S Destroyers and the Drugs That Stop Them Pharmacy & Pharmacology International Journal Mini Review Open Access Proteases: nature’s destroyers and the drugs that stop them Abstract Volume 2 Issue 6 - 2015 Proteases are found in all life forms and regulate many cellular pathways. Proteases Charles A Veltri have been proven as viable drug targets. This paper reviews the mechanism of Pharmaceutical Sciences, Midwestern University College of hydrolysis of the amino acid based aspartic, cysteine, serine and threonine proteases Pharmacy, USA and will also highlight the current anti protease therapeutics in the clinic. Correspondence: Charles A Veltri, Midwestern University, Keywords: protease, inhibitor, drug development USA, Tel 635723589, Fax 6235723565, Email [email protected] Received: April 17, 2015 | Published: November 30, 2015 Introduction Proteases are found in all life forms. Initially proteases were described from gastric juices and thought to be involved in nonspecific degradation of proteins. While some proteases are nonspecific, others are highly specific both in their substrate selection and cleavage recognition sites. Proteases account for ~2% of the human genome with 553 defined members1,2 and 1-5% of the genomes in infectious organisms such as bacteria, parasites and viruses.3 Proteases regulate many cellular processes, such as protein degradation,4,5 digestion6,7 blood coagulation,8 wound healing,9 ovulation,10 embryonic development,11 bone formation,12 neuronal outgrowth,13 cell-cycle regulation,14 immune and inflammatory response,15 angiogenesis16 and apoptosis.17 Proteases are viable drug targets because of their role in cellular functions of both mammalian cells and pathogens. There are five major classes of proteases categorized by the Figure 1 Cartoon depicting the substrate (P) and the binding pockets (S). catalytic method used to hydrolyze peptide bonds: serine (30%), aspartic (4%), cysteine (26%), threonine (5%) and metallo proteases Nucleophilic proteases (34%).3 Although the specific residues differ between the various classes, they all bind their substrates in a groove where peptide bond Mechanism hydrolysis occurs, and all proteases recognize β-strands in their active Serine, aspartic, threonine and cysteine proteases share common 3 site. The amino acids of the substrate occupy recognition pockets in active site functionality in a nucleophilic residue coupled with a general the groove of the protease. The interactions are designated P3, P2, P1, base. The general base abstracts the proton from the nucleophilic P1′, P2′ and P3′ for the substrate and S3, S2, S1, S1′, S2′ and S3′ for the residue to allow the active site residue to become charged (Figure 2). binding pockets. Prime and nonprime designations are with respect These residues are typically the target for inhibitors. to the cleaved peptide bond (Figure 1). The S pockets of the protease have amino acids complementary to the side chains of the substrate at The mechanism of hydrolysis is highlighted in Figure 3, using the site of cleavage. This complementation allows for selectivity in the the active site of a cysteine proteasesas the example. The protease peptide bonds cleaved by an enzyme, for example trypsin specifically active site residue attacks the carbonyl of the peptide bond to form a cleaves at the carboxyl terminus of arginine or lysine residues. tetrahedral transition state intermediate. Electrons from the oxygen of the carbonyl drive the cleavage of the peptide bond, which abstracts This paper will review the mechanism of how proteases hydrolyze hydrogen from the general base. Water then releases the second half of peptide bonds and discuss the similarities and differences between the product from the active site residue. The oxyanion hole generated the serine, aspartic, cysteine and threonine proteases. All four of during the transition state is stabilized through hydrogen bonding with these classes of proteins share common active site functionality that amino groups of the backbone. The role of the oxyanion hole is not is often the target for inhibitors. The development of inhibitors and fully understood.18 their mechanism of inhibition against proteases will also be discussed. Finally, this paper will highlight the current therapeutics available on While the chemistry of the mechanism of hydrolysis is similar the market to target the various protease classes. for serine, aspartic, cysteine and threonine proteases, the amino acids involved in catalysis are what define the various classes. The different classes of proteases are discussed below. Submit Manuscript | http://medcraveonline.com Pharm Pharmacol Int J. 2015;2(6):222‒230. 222 © 2015 Veltri. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: Proteases: nature’s destroyers and the drugs that stop them ©2015 Veltri 223 Figure 2 Active site residues of serine and cysteine proteases. The dashed lines represent hydrogen bonds. Figure 3 Mechanism of hydrolysis using the cysteine protease as the model. The thiol of the Cys attacks the carbonyl of the peptide bond. The electrons from the carbonyl oxygen drive the cleavage of the peptide bond. Water then attacks the carbonyl to release the protein from the protease. Serine proteases trypsin-like proteases cleave positively charged residues (Lys or Arg are preferred at P ), elastase-like proteases prefer small hydrophobic Serine proteases19‒21 use an active site serine to hydrolyze a peptide 1 residues (Ala or Val are preferred at P1) while chymotrypsin-like bond. The other amino acids involved in catalysis vary between proteases prefer large hydrophobic residues (Phe, Tyr, or Leu).2,19,20 the different serine protease groups. The Ser-His-Asp triad can be replaced with Ser-His-Glu, Ser-His-His or Ser-Lys depending on the Aspartic proteases 20,21 family of serine protease. The serine proteases are categorized 19,23‒26 by tertiary structures into the super families of the trypsin-like or Aspartic proteases use two aspartic acid residues to subtilisin-like.2,19,20 Examples of the trypsin-like serine proteases, hydrolyze a peptide bond. These proteases are categorized into the super families of pepsin-like or viral retropepsin-like due to the the larger of the super families, are trypsin, chymotrypsin, thrombin, 23,24,26,27 2,19,20 tertiary structure. The pepsin-like include pepsin, cathepsin D factor IX a, and factor Xa. Interestingly, some cysteine proteases 23,27 22 and chymosin. The viral retropepsin-like include the retro pepsins adopt the trypsin-like structure. Examples of the subtilisin-like 24,26 serine proteases are proteinase K and thermitase.2,19,20 of the immune viruses HIV, FIV and SIV. Along with tertiary structure, serine proteases are designed against Aspartic proteases are unique in the fact they do not have a His residue in their active site. The general acid-base mechanism the nature of the P1 residue recognized by the protease types. The Citation: Veltri CA. Proteases: nature’s destroyers and the drugs that stop them. Pharm Pharmacol Int J. 2015;2(6):222‒230. DOI: 10.15406/ppij.2015.02.00044 Copyright: Proteases: nature’s destroyers and the drugs that stop them ©2015 Veltri 224 proposed for peptide hydrolysis involves two Asp residues and uses the deprotonated Asp residue. The protonated Asp residue undergoes water to attack the targeted peptide bond (Figure 4). The peptide nucleophilic attack by the amide of the peptide bond, resulting in the bond undergoes nucleophilic attack by water which is activated by cleavage of the peptide bond.19,23‒26 Figure 4 Mechanism of hydrolysis of aspartic proteases. The dashed line is a hydrogen bond between the two Asp active site residues. Water is used to attack the carbonyl of the peptide bond. The protonated Asp is then attacked by the amide of the peptide bond, resulting in cleavage of the peptide bond. Cysteine proteases Inhibitor design background Cysteine proteases12,18,19,22,25,28‒30 use a cysteine residue to Early inhibitors of nucleophilic proteases were designed as peptide hydrolyze a peptide bond. These proteases are categorized into three substrates to deliver a warhead to the active site.25 This approach super families of viral, papain-like or caspase-like.19,22 The viral was favorable because a specific substrate could be synthesized for proteases have examples from many viral origins.22 As stated earlier, the target protease. The early warheads were diazo- or halo-ketone the viral cysteine proteases take on the serine protease trypsin-like compounds, which are excellent alkylating agents because diazo and tertiary structure.22 The papain-like proteases include the papain halogens represent good leaving groups.25 The active site residue and cathepsins. The caspase-like proteases include the caspases and attacks the carbonyl of the warhead rather than that of the peptide gingipain.22 bond (Figure 5).25,33 Threonine proteases While many proteases are not highly substrate specific (only a few residues are recognized for cleavage), some proteases have extended The only enzymes that are threonine proteases are the substrate binding grooves and require longer peptides (up to 30 4,5,18,31,32 proteasomes. Proteasomes are multifunctional and possess residues) for effective binding.25 The caspases, thrombins, neutrophil three distinct cleavage recognition sites: trypsin-like (Lys or Arg are elastases and deubiquitinating enzymes are examples of proteases that preferred at P ), chymotrypsin-like which prefer large hydrophobic 4,9,25,34‒37 1 recognize longer peptide fragments. Therefore, the traditional residues (Phe, Tyr, or Leu is preferred at P ), and caspase-like (Asp 1 method of attaching a warhead onto a substrate can become quite or Glu are preferred at P ).4,5,18,31 Inhibition of the proteasomes causes 1 difficult due to the size of the peptide-warhead library necessary to cell cycle arrest and apoptosis. identify the best inhibitors. The proteasomes are all barrel shaped protein complexes that Two approaches have been taken to overcome this barrier.
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