Biomedical Engineering Reference
In-Depth Information
constitute an unfavorable peptide bond for cleavage by a trypsin-like
enzyme.
92
The substitution of the cleavable bond and neighboring amino acid
residues is based on established KLK2 peptide substrates identified by phage
display.
70
A series of recombinant ACT molecules have been expressed
and studied. The most selective was found to incorporate the Arg-Gly-Arg-
-
Ser-Glu sequence. The new inhibitor has shown a rapid (within minutes)
association with KLK2 comparedtowild-typeACT(12-16h).
92
Currently,
KLK2 ACT-derived inhibitors, such as the compound MDKP67b, are
in development by Med Discovery (Switzerland). MDKP67b is a mutated
form of ACT with four single amino acid substitutions that, at the time of
writing, is about to undergo clinical trials as a targeted treatment for prostate
cancer (http://med-discovery.webcreatif.net/Templates/Standard.aspx?id_
page
¼
43&flag
¼
0).
k
9.3.2.2 Mutant Serpins Targeting KLK14
A similar approach to that described for KLK2 ACT-based inhibitors has been
followed. Initially, a pentapeptide phage display library was screened to iden-
tify substrates specific for KLK14.
65
Then, the identified peptapeptides (E8:
Leu-Gln-Arg-Ala-Ile and G9: Thr-Val-Asp-Tyr-Ala) were incorporated into
the scaffold of either a
1
-antitrypsin or a
1
-antichymotrypsin.
93
E8-engineered
inhibitors showed relaxed specificity with various proteases with trypsin-like
activity, while a G9-engineered ACT exhibited a very high specificity. The
ACT
E8
inhibitor showed a 1:1.2 (enzyme:inhibitor) stoichiometry of inhibition
and a second-order rate constant K
a
of 575 000. The ACT
G9
has a 1:1.5 stoi-
chiometry and a lower association constant K
a
of 74 000 M
-1
s
-1
. Finally,
ACT
G9
inhibited the liquefaction of human semen, a process that depends on
the enzymatic activity of KLK14.
94
9.4 Synthetic Inhibitors Based on Modified
Peptide-Substrates or Small Molecules
Powers et al.
95
outlined the principles underlying the design of micro-
molecular inhibitors of serine proteases. In brief, a common approach
involves the attachment of a reactive group, often referred to as the warhead
(alkylating, acylating, phosphonylating or sulfonylating functional group, or
others) to the appropriate peptide sequence that is specifically recognized by
the enzyme of interest. The use of phosphonylfluoridate derivatives leads to
very potent inhibitors of serine proteases, but because of concomitant inhi-
bition of acetylcholinesterase, they are highly toxic and, therefore, not the
first choice for inhibitor design. However, recent developments have led to
less toxic variants that have found numerous applications, e.g. variants based
on the general formula RCONHCH(R)-PO(OPh)F that are modified pep-
tides at the C-terminus.
95
Another class of phosphonates includes the
