Abstract
The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin–associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI.
Highlights
The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions
The secondgeneration compounds were developed on the scaffold of the 35-amino acid Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), yielding SGMI-1 and SGMI-2 [24, 25]. We revealed that both MASP-1 and Mannan-binding lectin–associated serine proteinase 2 (MASP-2) are essential for LP activity in human serum [23,24,25], and both enzymes are promising targets for drug development
TFMI-2a is significantly less potent with a KI value of 640 nM. These results show that the hMASP-2– binding consensus P1–P4Ј region (RAVKR) shared in all three TFPI1-based MASP-2 inhibitor (TFMI-2) variants is compatible with rMASP-2 inhibition, but as expected, P3 Phe of TFMI-2a is detrimental for binding to the enzyme, causing an almost 100-fold affinity drop
Summary
We followed the same strategy we already applied for developing TFPI1 D2– based MASP-3 inhibitors [32]. TFMI-2a is significantly less potent with a KI value of 640 nM These results show that the hMASP-2– binding consensus P1–P4Ј region (RAVKR) shared in all three TFMI-2 variants is compatible with rMASP-2 inhibition, but as expected, P3 Phe of TFMI-2a is detrimental for binding to the enzyme, causing an almost 100-fold affinity drop. Wieslab experiments demonstrated that the TFMI-2 variants are potent LP inhibitors, with IC50 values of 35–384 nM (Fig. 2 and Table 2), whereas none of them inhibit the CP and the AP. Assays, we used 100 g/ml sodium polyanethole sulfonate (SPS) to selectively suppress the activation of the CP and the AP [40, 41] and detected C4 deposition In this assay, all TFMI-2 variants were significantly, 13.4 –35.8-fold more potent than SGMI-2 (Fig. 4 and Table 2). Even at the highest concentration, TFMI-2 variants have no effect in the PT and TT tests (Fig. 6, A and B) and have only a negligible effect in the APTT test (Fig. 6C)
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