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Skoreński et al. Rare Dis Orphan Drugs J 2023;2:6  https://dx.doi.org/10.20517/rdodj.2022.21  Page 11 of 23

               as a pair of donor and acceptor, ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO )-NH with a K  value of 3.2
                                                                                      2
                                                                                 2
                                                                                              M
                                               -1 -1
                                            3
                                                                          [59]
               mM and k /K  value of 1596 × 10  M s , was reported one year later . A structure of simple chromogenic
                           M
                        cat
               PR3 peptide substrates has also been developed, including Ac-Pro-Tyr-Asp-Ala-pNA and Ac-Asp-Tyr-Asp-
                                                                        -1 -1
                                                                                     [60]
                                                      -1 -1
               Ala-pNA, with a k /K  value of 4201 ± 29.7 M s  and 162 ± 12.8 M s  respectively , as well as biotinylated
                              cat
                                  M
               derivative Bt-Val-Tyr-Asp-nVal-pNA with a k /K  value of 80510 ± 2973 M s  .
                                                                               -1 -1[61]
                                                      cat
                                                         M
               PR3 ABPs and Inhibitors
               Azapro-3 (25), ABZ-Val-Ala-Asp-aza(nor)Val-Ala-Asp-Tyr-Gln-Tyr(3-NO ), is an example of the first
                                                                                 2
               selective, non-covalent, reversible azapeptide inhibitor based on a structure of one of the FRET substrates
               for PR3 [Figure 12]. This competitive inhibitor shows high activity and selectivity for PR3 with a K value of
                                                                                                  i
               1.5 µM, with a nonsignificant inhibitory effect of HNE, and without any reactivity towards other proteases,
               including CatG, chymotrypsin, and granzyme B .
                                                       [58]
               Another example of a PR3 inhibitor based on a sequence of its specific FRET substrate is ABZ-Val-Ala-Asp-
               (nor) Val[Ψ](COCH )-Ala-Asp-Tyr-Gln-EDDnp (26), a ketomethylene peptide derivative [Figure 12]. This
                                 2
               compound was characterized as a competitive and reversible inhibitor with an IC  value of 1.91 µM,
                                                                                        50
                                      [62]
               selective for PR3 over HNE .
               Compound 6 (27), an analog of 2-aminobenzaldehyde oxime [Figure 13], was reported as the most potent
               non-peptide inhibitor for PR3 from a synthesized set of compounds, with an IC  value of 0.22 ± 0.02 µM.
                                                                                    50
               However,  this  inhibitor  showed  a  lack  of  selectivity  due  to  better  activity  toward  HNE
               (IC  = 0.05 ± 0.01 µM) .
                                  [63]
                  50
               Structures based on aminophosphonates constitute a well-known group of potent and selective PR3
                                                      [56]
               inhibitors, which have been recently reviewed . The most important advantage of this class of inhibitors is
               their lack of reactivity with other proteases, including threonine, cysteine, aspartyl, and metalloproteinases.
               The first series of peptidyl derivatives α-aminoalkylphosphonate diaryl esters as specific, irreversible
               inhibitors of PR3 was synthesized in 2014. This most potent covalent inhibitor, which acts as the transition
               state analog, was Ac-Pro-Tyr-Asp-Ala (O-C H -4-Cl)  (28) with the k /[I] value of 154 ± 3 M s . A
                                                                                                     -1 -1
                                                  P
                                                       6
                                                               2
                                                                              obs
                                                         4
                                                                           P
               biotinylated derivative of this compound, Bt-[PEG] -Pro-Tyr-Asp-Ala (O-C H -4-Cl)  (29) was an example
                                                          66
                                                                                6
                                                                                            ,
                                                                                        2
                                                                                  4
               of ABP, which can be used to visualize PR3 in native conditions, with k /[I] value of 1163 ± 0.1 M s
                                                                                                       -1 -1
                                                                               obs
               [Figure 14].  However,  this  Bt-ABP  lacked  selectivity  and  showed  activity  toward  HNE
               (k /[I] = 46 ± 0.1 M s ) .
                                -1 -1 [60]
                 obs
               Several peptidyl biotinylated derivatives of α-aminoalkylphosphonate dsiaryl esters with a potential of ABPs
                                                                               F
               f o r   N S P s ,   i n c l u d i n g   P R 3   w e r e   d e s c r i b e d   b y   G r z y w a   e t   al.  i v e   o f   t h e s e   s t r u c t u r e s ,
               Bt-Val-Pro-Abu (O-C H -4-S-CH ) , Bt-Val-Pro-Val (O-C H ) , Bt-Val-Pro-Val (O-C H -4-S-CH ) , Bt-
                                                                                     P
                             P
                                                             P
                                                                  6
                                                                                                    3 2
                                                                    5 2
                                  6
                                                                                          6
                                    4
                                                                                            4
                                            3 2
                                                                             P
               Val-Pro-Leu (O-C H -4-COOCH ) , Bt-LC-Suc-Phe-Val-Thr-(4Gu)Phg (O-C H -4-S-CH ) , show activity
                          P
                                                                                  6
                                                                                            3 2
                                                                                    4
                               6
                                            3 2
                                 4
               toward PR3 with a k /[I] values of 5.1 × 10 ± 350, 1.6 × 10 ± 100, 1.6 × 10 ± 1,200, 1.9 × 10 ± 2,300,
                                                                                   4
                                                                                                  4
                                                       3
                                                                     3
                                  obs
               2.1 × 10 ± 100 [M s ], respectively. However, these ABPs do not show selectivity of action over HNE or
                      3
                              -1 -1
               CatG  [Table 1].
                    [64]
               In 2018, the same research group reported a structure of a new biotinylated PR3 inhibitor, Bt-Val-Tyr-Asp-
                                                                        -1 -1
               nVal (O-C H -4-Cl)  (30), with k /[I] value of 73,258 ± 5,342 M s , and without significant inhibitory
                   P
                        6
                           4
                                 2
                                            obs
               activity toward HNE  [Figure 14].
                                 [61]
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