Periodic Reporting for period 1 - BiCyHePepDi (Bicyclic hetero peptidimer - a novel molecule format for therapeutic peptides)
Periodo di rendicontazione: 2016-04-01 al 2017-09-30
The development of a novel molecule format, referred to as “bicyclic hetero peptidimer” (Figure 1), as a new therapeutic molecule format is the first objective in this project. By combination of a moderate active inhibitor of FXIIa with a FXII binding peptide, we hypothesized that this will increase residence time and overall affinity, leading to a more potent inhibitor. The bicyclic hetero peptidimer should serve as potent FXII inhibitor with high inhibitory activity (picomolar range), be easy accessible to chemical synthesis, and have a long circulation half-life.
Coagulation factor XII (FXII, Hageman factor) is a component of the intrinsic pathway of coagulation and FXII is activated by conversion to FXIIa, which serves as serine protease and cleaves plasma prekallikrein (PPK) to generate active plasma kallikrein (PK) which leads in a release of the high molecular weight kininogen (HK). HK liberates the inflammatory mediator bradykinin (BK), which is the fundament of the autosomal dominantly inherited blood disorder hereditary angioedema (HAE). There are three types of HAE, which all lead to an over stimulated production of bradykinin as a result of an increased activity of FXIIa. The patients suffer episodic attacks of swelling, which can affect skin and mucosa as well as the inner organs. This leads to massive pain and can be life threatening in the case of swelling in the throat. The high inhibitory activity is crucial for this target, where only small amounts of activated FXII protease can start a cascade which amplifies its self. FXII plays also an important role in the activation of the intrinsic coagulation pathway. Activation of FXII on artificial surfaces during cardiopulmonary bypass surgery can lead to excessive coagulation and inflammation. Inhibition of FXIIa during this medical procedure could potentially reduce or even eliminate the undesired effects.
The main goal of our project in the end is the development of a potent inhibitor of the intrinsic coagulation cascade, preferably in the picomolar range for FXIIa inhibition. A further goal is the improvement of pharmacokinetic parameters after the successful identification of a potent bicyclic peptidic inhibitor.
For in vitro and in vivo experiments, corn trypsin inhibitor (CTI, Ki = 24 nM) is available, but this molecule would most likely raise an immune reaction if applied as a therapeutic. FXIIa inhibitory bicyclic peptides developed until recently showed too low inhibitory activity. Small molecule FXIIa inhibitors such as PCK (IC50 = 180 nM) and coumarin-based molecuels have been reported but show weak activities. Monoclonal antibodies inhibiting FXIIa are in development. Neither small molecule FXIIa inhibitors nor FXIIa antibodies have reached the market yet.
Coagulation factor XII (FXII, Hageman factor) is a component of the intrinsic pathway of coagulation and FXII is activated by conversion to FXIIa, which serves as serine protease and cleaves plasma prekallikrein (PPK) to generate active plasma kallikrein (PK) which leads in a release of the high molecular weight kininogen (HK). HK liberates the inflammatory mediator bradykinin (BK), which is the fundament of the autosomal dominantly inherited blood disorder hereditary angioedema (HAE). There are three types of HAE, which all lead to an over stimulated production of bradykinin as a result of an increased activity of FXIIa. The patients suffer episodic attacks of swelling, which can affect skin and mucosa as well as the inner organs. This leads to massive pain and can be life threatening in the case of swelling in the throat. The high inhibitory activity is crucial for this target, where only small amounts of activated FXII protease can start a cascade which amplifies its self. FXII plays also an important role in the activation of the intrinsic coagulation pathway. Activation of FXII on artificial surfaces during cardiopulmonary bypass surgery can lead to excessive coagulation and inflammation. Inhibition of FXIIa during this medical procedure could potentially reduce or even eliminate the undesired effects.
The main goal of our project in the end is the development of a potent inhibitor of the intrinsic coagulation cascade, preferably in the picomolar range for FXIIa inhibition. A further goal is the improvement of pharmacokinetic parameters after the successful identification of a potent bicyclic peptidic inhibitor.
For in vitro and in vivo experiments, corn trypsin inhibitor (CTI, Ki = 24 nM) is available, but this molecule would most likely raise an immune reaction if applied as a therapeutic. FXIIa inhibitory bicyclic peptides developed until recently showed too low inhibitory activity. Small molecule FXIIa inhibitors such as PCK (IC50 = 180 nM) and coumarin-based molecuels have been reported but show weak activities. Monoclonal antibodies inhibiting FXIIa are in development. Neither small molecule FXIIa inhibitors nor FXIIa antibodies have reached the market yet.
We started the project with the moderate active inhibitor FXII618, which has been previously identified by modified phage display selection and was published as well as the “binder”. The work performed can be divided into different steps: At first we selected binder out of the already published sequences and confirmed their binding to the target by ELISA. In the next step, a synthesis strategy was elaborated to synthesize a set of bicyclic hetero peptidimers differing in binder, orientation and linker between inhibitor and binder part. We had tested these compounds in our enzymatic activity assay and identified FXIIa inhibitor in the low nanomolar range. But we were not able to improve the activity by varying the linker length. A further characterization revealed that some of the binders actually inhibited FXIIa. At this point, we developed two strategies to still achieve our goal of a potent inhibitor of the intrinsic coagulation cascade. In the first strategy we further improved the FXIIa inhibitor by using unnatural amino acids in activity and stability in plasma.
As a second strategy we developed a compound class inhibiting two essential proteases (FXII and PK) at the initial step of the intrinsic coagulation cascade. With the dual activity we hypothesized that we can inhibit the cascade in more efficient way. To identify those bispecific inhibitor we used the phage display approach, where we select compounds with affinity for the targets. We synthesized the identified compounds and determined their inhibitory activity against both targets with our already mentioned enzymatic assay. We were able to identify compounds which had inhibitory activity for both targets, but unfortunately not in the same potency range. To achieve this, we used also the approach based on unnatural amino acids and we were able to develop compounds with inhibitory activity for both targets in the lower nanomolar range.
With both new strategies, we were able to identify lead compounds which allowed us to approach the nest step being the improvement of the pharmacokinetic parameters. We were able to improve the stability in plasma and the circulation half-life.
As a second strategy we developed a compound class inhibiting two essential proteases (FXII and PK) at the initial step of the intrinsic coagulation cascade. With the dual activity we hypothesized that we can inhibit the cascade in more efficient way. To identify those bispecific inhibitor we used the phage display approach, where we select compounds with affinity for the targets. We synthesized the identified compounds and determined their inhibitory activity against both targets with our already mentioned enzymatic assay. We were able to identify compounds which had inhibitory activity for both targets, but unfortunately not in the same potency range. To achieve this, we used also the approach based on unnatural amino acids and we were able to develop compounds with inhibitory activity for both targets in the lower nanomolar range.
With both new strategies, we were able to identify lead compounds which allowed us to approach the nest step being the improvement of the pharmacokinetic parameters. We were able to improve the stability in plasma and the circulation half-life.
The here conducted research helped to identify limitations of a technology and indicated that the combination with unnatural amino acid SAR can overcome these limitations. Furthermore a FXIIa inhibitor with market potential was further improved, which both have the potential to improve European excellence and competitiveness in a worldwide context.