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Obtaining a family of high-affinity, high-specificity protein inhibitors of plasmin and plasma kallikrein

Summary

Human lipoprotein-associated coagulation inhibitor (LACI) is a serum protein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this domain. We iteratively varied 13 residues in the region corresponding to the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round best binder, EPI-P211, had KD=2 nM. Using information from the first selection, we made a PLA-biased library containing ∼ 500 000 proteins and selected from these a protein, EPI-P302, having a KD for PLA of 87 pM. EPI-P302 inhibits pKAL with KD=250 nM (∼2800-fold higher than for PLA) and KD values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a KD for pKAL of 287 pM. We used information from this selection to construct a pKAL-biased library from which we selected EPI-K502, which has a KD for pKAL of 40 pM. EPI-K502 inhibits PLA with KD∼20 nM (500-fold higher than for pKAL); KD values for other proteases are much higher. For both targets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These proteins are candidate drugs and imaging agents for indications involving excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for these targets.

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Abbreviations

APP-I-D1:

Kunitz domain of amyloid precursor protein-I

BPTI:

bovine pancreatic trypsin inhibitor

F.VIIa :

Factor VIIa

F.XIa :

Factor XIa

FIR:

fraction of input recovered

hNE:

human neutrophil elastase

LACI-D1:

lipoprotein-associated coagulation inhibitor, first Kunitz domain

pKAL:

human plasma kallikrein

PLA:

human plasmin

RT:

room temperature

THBN:

human thrombin

Vg:

variegated

w.t.:

wild-type

%RIA:

percent residual inhibitory activity

:::

fusion (gene or protein)

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Correspondence to Robert Charles Ladner.

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Ley, A.C., Markland, W. & Ladner, R.C. Obtaining a family of high-affinity, high-specificity protein inhibitors of plasmin and plasma kallikrein. Mol Divers 2, 119–124 (1996). https://doi.org/10.1007/BF01718709

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Keywords

  • Combinatorial library
  • Screening
  • Kunitz domain
  • LACI
  • Specificity
  • Plasmin
  • Plasma kallikrein