Second Generation Triple-Helical Peptide Inhibitors of Matrix Metalloproteinases

Abstract

The design of selective matrix metalloproteinase (MMP) inhibitors that also possess favorable solubility properties has proved to be especially challenging. A prior approach using collagen-model templates combined with transition state analogs produced a first generation of triple-helical peptide inhibitors (THPIs) that were effective in vitro against discrete members of the MMP family. These THPI constructs were also highly water-soluble. The present study sought improvements in the first generation THPIs by enhancing thermal stability and selectivity. A THPI selective for MMP-2 and MMP-9 was redesigned to incorporate non-native amino acids (Flp and mep), resulting in an increase of 18 °C in thermal stability. This THPI was effective in vivo in a mouse model of multiple sclerosis, reducing clinical severity and weight loss. Two other THPIs were developed to be more selective within the collagenolytic members of the MMP family. One of these THPIs was serendipitously more effective against MMP-8 than MT1-MMP and was utilized successfully in a mouse model of sepsis. The THPI targeting MMP-8 minimized lung damage, increased production of the anti-inflammatory cytokine IL-10, and vastly improved mouse survival.

Development of matrix metalloproteinase-13 inhibitors – A structure-activity/structure-property relationship study

“…We have already demonstrated that a combination of hydrophobic interactions of the isopropyl group and hydrogen bonding interactions between the amide group in compound 2 with the backbone amide groups of Gly183 and Tyr244 in MMP-13 is responsible for a 40-fold higher inhibition potency compared to its enantiomeric counterpart. 13 A similar trend is observed for the enantiomeric pairs 26 / 27 and 28 / 29 , respectively, which possess hydrophobic groups in their R positions (Table 3). However, in 9 / 10 , 11 / 12 , 13 / 14 , and 15 / 16 , where the terminal methyl group of 2 is substituted by hydrophilic moieties (R1-groups, Table 2), which are able to form additional hydrogen bonding interactions with Tyr214 and/or Ser182 as shown in our docking models (Figure 3A-D), an analogous drop in inhibition potency was not observed.…”

“…Alternatively, the ( R )-enantiomers could bind to MMP-13 in the same binding pose as ( R )- 2 in the X-ray co-crystal structure (PDB code: 5UWM). 13 In this binding mode (Figures 3E, 3F), the polar R1-groups can form hydrogen bonding interactions with Glu223 or backbone amides of MMP-13, and the loss of hydrogen bonds of the amide units of the ( R )-enantiomers can be compensated by the polar interactions of the corresponding R1- group. Consequently, the enantiomeric pairs 9 / 10 , 11 / 12 , 13 / 14 , and 15 / 16 exhibit high inhibition potency regardless of their stereoconfiguration.…”

“…Subsequent amino acid coupling with different valine derivatives resulted in the synthesis of 15 analogs (Table 2). 13 …”

“…9d Using this cocrystal structure for design purposes, we recently reported a three-step approach for the development of highly potent and selective MMP-13 inhibitors. 13 In steps one and two we applied comparative structural analyses followed by molecular design to obtain non-selective but highly potent Zn 2+ -chelating compounds. In the third step the chelating moiety was removed to increase selectivity.…”

Characterization and regulation of MT1‐MMP cell surface‐associated activity

“…Antibodies were purchased from EMD Millipore and Pierce. The triple‐helical substrate fTHP‐9 [(Gly‐Pro‐Hyp) 5 ‐Gly‐Pro‐Lys(Mca)‐Gly‐Pro‐Gln‐Gly~Cys(Mob)‐Arg‐Gly‐Gln‐Lys(Dnp)‐Gly‐Val‐Arg‐(Gly‐Pro‐Hyp) 5 ‐NH 2 ] and the triple‐helical peptide inhibitor GlyΨ{PO 2 H‐CH 2 }Ile‐Tyr THPI [(Gly‐Pro‐Hyp) 4 ‐Gly‐mep‐Flp‐Gly‐Pro‐Gln‐[GlyΨ(PO 2 H‐CH 2 )Ile]‐Tyr‐Phe‐Gln‐Arg‐Gly‐Val‐Arg‐Gly‐mep‐Flp‐(Gly‐Pro‐Hyp) 4 ‐Tyr‐NH 2 , where mep = 4‐methylproline and Flp = 4‐fluoroproline] were synthesized in house using methods described previously (Bhowmick & Fields, ; Bhowmick et al., ; Lauer‐Fields et al., ; Minond, Lauer‐Fields, Nagase, & Fields, ; Minond et al., ). Marimastat, a nonselective inhibitor of MMPs (Beckett & Whittaker, ; Rasmussen & McCann, ), was purchased from Sigma.…”