Mammalian proteases

Escherichia coli. The genetics of this gram-negative bacterium are very well known. For this reason, many of the first efforts to produce recombinant products from this microorganism were successful. However, because of the importance of the other criteria Hsted above, many efforts failed. E. co/i is only used to produce the milk-clotting mammalian protease chymosin [9001-98-3] (rennin). 

Sequence 1, in Table I, is the sequence of trypsin, chymotrypsin, pancreatic elastase, thrombin, and other mammalian proteases, and it occurs throughout the animal kingdom down to invertebrates as primitive as the sea anemone (4). The Streptomyces griseus enzymes are from Pronase, a commercial enzyme preparation. Two of its components, Streptomyces griseus trypsin and protease A, not only have the Asp.Ser.Gly sequence, but they show several other homologies in sequence with the mammalian enzymes (5, 6), The same is true for the sequence of a-lytic protease, the Myxobacter 495 enzyme (7). 

The strategy used by the cysteine proteases is most similar to that used by the chymotrypsin family. In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the nucleophile that attacks the peptide bond (see Figure 9.18). in a manner quite analogous to that of the serine residue in serine proteases. An ideal example of these proteins is papain, an enzyme purified from the fruit of the papaya. Mammalian proteases homologous to papain have been discovered, most notably the cathepsins, proteins having a role in the immune and other systems. The cysteine-based active site arose independently at least twice in the course of evolution the caspases, enzymes that play a major role in apoptosis (Section 2.4.3). have active sites similar to that of papain, but their overall structures are unrelated. 

By this point, multiple crystal structures of the enzyme had revealed something unexpected a bound water not seen in mammalian proteases that came between and interacted with two of the carbonyl oxygen atoms of the substrate or inhibitor, and two NHs (from Ile50 and IleSO ) on the flap region of the enzyme. Researchers at DuPont Merck proposed to build in selectivity and take advantage of the entropy released upon displacing this water by... 

The chemistry of P-lactams, p-sultams and P-phospholactams has been compared and the potential of the last two as mechanism based inhibitors of bacterial and mammalian proteases... 

Researchers at different companies used different reasoning in developing drugs. For example, chemists at Hoffinan-La Roche imitated the hydrolysis site of the gag-pol protein, that is, Phe-Pro (see above). They modified the surroundings of the site and discovered that saquinavir is the most effective (see the structure in Fig. 7.4). In Abbot s Lab, the scientists made use of the symmetrical nature of the HlV-1 protease. Because most of other mammalian proteases are not symmetrical, a compound with a symmetrical structure about the mimicking site would inhibit the HlV-1 protease, but not other mammalian proteases, thus hopefully reducing side effects. With this notion in mind, they synthesized a number of compounds and discovered that ritonavir was most effective (see the structure in Fig. 7.4). As you see in Fig. 7.4, the... 

Even though library 2 (Fig. 1) was designed for E. coli leader peptidase, it was found to be a useful library for screening against other proteases. In the example given, it was used to screen for napsin A substrates, a mammalian protease expressed in kidney and lung (8,9). The protease inhibitors E64 and... 

When the first inhibitors for this protease were developed, very litde was known about the enzyme other than its aspartate-catalysed mechanism of action there was no crystal struchire to propose inhibitors based on modelling, and differences with the host proteases were unclear. All that was known was that the HIV protease was a much smaller enzyme than the equivalent host aspartate proteases (arotmd 100 amino acids as opposed to 200), and was able to cleave substrates N-terminal to proline residues, and since mammalian proteases were unable to carry out such cleavages, the possibility of selectivity for the virus over the host arose. Additionally, even before the protease had been isolated, a study of peptides obtained from infected cells suggested that the Met-Met and Tyr-Pro sites were the likely positions of cleavage in the fusion protein. 

---