RESUMEN
Acute myeloid leukemia (AML) is marked by significant unmet clinical need due to both poor survival and high relapse rates where long-term disease control for most patients with relapsed or refractory AML remain dismal. Inspired to bring novel therapeutic options to these patients, we envisioned protein degradation as a potential therapeutic approach for the treatment of AML. Following this course, we discovered and pioneered a novel mechanism of action which culminated in the discovery of CC-90009. CC-90009 represents a novel protein degrader and the first cereblon E3 ligase modulating drug to enter clinical development that specifically targets GSPT1 (G1 to S phase transition 1) for proteasomal degradation. This manuscript briefly summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and efficacy data for CC-90009, which is currently in phase 1 clinical development.
Asunto(s)
Acetamidas/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos/uso terapéutico , Isoindoles/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Terminación de Péptidos/antagonistas & inhibidores , Piperidonas/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , Acetamidas/química , Acetamidas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Isoindoles/química , Isoindoles/farmacología , Macaca fascicularis , Masculino , Ratones , Estructura Molecular , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Piperidonas/química , Piperidonas/farmacología , Proteolisis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Mieloma Múltiple/patología , Recurrencia , Estereoisomerismo , Insuficiencia del Tratamiento , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The discovery of 5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC50 <0.10 microM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Piridonas/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Animales , Antivirales/síntesis química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Macaca fascicularis , Microsomas Hepáticos/metabolismo , Piridonas/síntesis química , Piridonas/farmacología , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Estructura-ActividadRESUMEN
5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; IC(50) (1a)<25nM, EC(50) (1b)=16nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F=24%).
Asunto(s)
ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piridonas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Haplorrinos , Piridonas/administración & dosificación , Piridonas/química , Piridonas/farmacocinética , Relación Estructura-ActividadRESUMEN
A novel series of non-nucleoside small molecules containing a tricyclic dihydropyridinone structural motif was identified as potent HCV NS5B polymerase inhibitors. Driven by structure-based design and building on our previous efforts in related series of molecules, we undertook extensive SAR studies, in which we identified a number of metabolically stable and very potent compounds in genotype 1a and 1b replicon assays. This work culminated in the discovery of several inhibitors, which combined potent in vitro antiviral activity against both 1a and 1b genotypes, metabolic stability, good oral bioavailability, and high C(12) (PO)/EC(50) ratios.
Asunto(s)
Disponibilidad Biológica , Diseño de Fármacos , Relación Estructura-Actividad , Antivirales/farmacocinética , Química Farmacéutica , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Genotipo , Hepacivirus/efectos de los fármacos , Hepatitis C , Estructura Molecular , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
Abstract: A general affinity-based screening assay for discovery of lead compounds binding to potential protein drug targets that is based upon protein thermal unfolding and aggregation is described. ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a simple, homogeneous, and high-throughput affinity-based screening technology that can identify compounds that bind and protect the target protein from thermal unfolding, denaturation, and subsequent aggregation. ATLAS detection of thermally unfolded and aggregated hexahistidine [(His)6]-tagged proteins uses time-resolved fluorescence resonance energy transfer between two anti-(His)6 antibodies, labeled with either a donor or acceptor fluorophore, that are simultaneously bound to the aggregated protein. The ATLAS assay is simple to perform and easily automated for screening large compound libraries. The technology is applicable to lead discovery for soluble proteins of known and unknown functions, and particularly for proteins that are difficult to assay functionally. The ATLAS technology has been evaluated using p38 mitogen-activated protein (MAP) kinase as the target protein. Known inhibitors of p38 MAP kinase were examined by ATLAS and a functional assay; the results showed good correlation between the two methods.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Algoritmos , Anticuerpos Monoclonales/química , Fenómenos Biofísicos , Biofisica , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Calor , Luciferasas/antagonistas & inhibidores , Desnaturalización Proteica , Pliegue de Proteína , Temperatura , Proteínas Quinasas p38 Activadas por Mitógenos/químicaRESUMEN
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as potent inhibitors of genotype 1 HCV NS5B polymerase focusing on the optimization of their drug metabolism and pharmacokinetics (DMPK) profiles. This investigation led to the discovery of potent inhibitors with improved DMPK properties.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacocinética , Hepacivirus/enzimología , Piridazinas/síntesis química , Piridazinas/farmacocinética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Antivirales/química , Técnicas Químicas Combinatorias , Diseño de Fármacos , Haplorrinos , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Piridazinas/química , Relación Estructura-ActividadRESUMEN
The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.
Asunto(s)
Antivirales/síntesis química , Química Farmacéutica/métodos , Óxidos S-Cíclicos/síntesis química , Piridazinas/química , Piridazinas/síntesis química , Tiadiazinas/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Antivirales/farmacología , Células CACO-2 , Cristalografía por Rayos X/métodos , Óxidos S-Cíclicos/farmacología , ARN Polimerasas Dirigidas por ADN/química , Diseño de Fármacos , Genotipo , Humanos , Concentración 50 Inhibidora , Microsomas/metabolismo , Modelos Químicos , Conformación Molecular , Piridazinas/farmacología , Relación Estructura-Actividad , Tiadiazinas/farmacologíaRESUMEN
Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b) <10 nM; EC(50) (1b)=34 nM) as well as good stability towards human liver microsomes (HLM t(1/2) =59 min).
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Piridazinas/síntesis química , Piridazinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Estructura Molecular , Piridazinas/química , Relación Estructura-ActividadRESUMEN
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacocinética , Hepacivirus/efectos de los fármacos , Piridazinas/síntesis química , Piridazinas/farmacocinética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Antivirales/sangre , Antivirales/química , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Microsomas Hepáticos/efectos de los fármacos , Estructura Molecular , Piridazinas/sangre , Piridazinas/química , Ratas , Relación Estructura-ActividadRESUMEN
A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC(50) values. The most potent compound exhibited IC(50) less than 10nM against the genotype 1b HCV polymerase and EC(50) of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Tiazoles/síntesis química , Tiofenos/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Genotipo , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , ARN Viral/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacocinética , Tiofenos/farmacocinéticaRESUMEN
Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).
Asunto(s)
Antivirales/farmacología , Piridazinas/farmacología , Pirroles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Sitios de Unión/efectos de los fármacos , Línea Celular , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Piridazinas/síntesis química , Piridazinas/química , Pirroles/síntesis química , Pirroles/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/químicaRESUMEN
4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).
Asunto(s)
Química Farmacéutica/métodos , Hepacivirus/enzimología , Microsomas Hepáticos/enzimología , Piridazinas/síntesis química , Piridazinas/farmacología , Tiazinas/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Células CACO-2 , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Hepacivirus/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Piridazinas/química , Relación Estructura-Actividad , Tiazinas/química , Tiazinas/farmacología , Factores de TiempoRESUMEN
Previous studies on mammalian peroxidases and cytochrome P450 family 4 enzymes have shown that a carboxylic group positioned close to a methyl group of the prosthetic heme is required for the formation of a covalent link between a protein carboxylic acid side chain and the heme. To determine whether there are additional requirements for covalent bond formation in the P450 enzymes, a glutamic acid or an aspartic acid has been introduced into P450(cam) close to the heme 5-methyl group. Spectroscopic and kinetic studies of the resulting G248E and G248D mutants suggest that the carboxylate group coordinates with the heme iron atom, as reported for a comparable P450(BM3) mutant [Girvan, H. M., Marshall, K. R., Lawson, R. J., Leys, D., Joyce, M. G., Clarkson, J., Smith, W. E., Cheesman, M. R., and Munro, A. W. (2004) J. Biol. Chem. 279, 23274-23286]. The two P450(cam) mutants have low catalytic activity, but in contrast to the P450(BM3) mutant, incubation of the G248E (but not G248D) mutant with camphor, putidaredoxin, putidaredoxin reductase, and NADH results in partial covalent binding of the heme to the protein. No covalent attachment is observed in the absence of camphor or any of the other reaction components. Pronase digestion of the G248E P450(cam) mutant after covalent attachment of the heme releases 5-hydroxyheme, establishing that the heme is covalently attached through its 5-methyl group as predicted by in silico modeling. The results establish that a properly positioned carboxyl group is the sole requirement for autocatalytic formation of a heme-protein link in P450 enzymes, but also show that efficient covalent binding requires placement of the carboxyl close to the methyl but in a manner that prevents strong coordination to the iron atom.
Asunto(s)
Sustitución de Aminoácidos/genética , Alcanfor 5-Monooxigenasa/genética , Hemo/metabolismo , Secuencia de Aminoácidos , Animales , Alcanfor/metabolismo , Alcanfor 5-Monooxigenasa/metabolismo , Catálisis , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Ácido Glutámico/genética , Glicina/genética , Humanos , Hidroxilación , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/genética , RatasRESUMEN
Histidine-51 in horse liver alcohol dehydrogenase (ADH) is part of a hydrogen-bonded system that appears to facilitate deprotonation of the hydroxyl group of water or alcohol ligated to the catalytic zinc. The contribution of His-51 to catalysis was studied by characterizing ADH with His-51 substituted with Gln (H51Q). The steady-state kinetic constants for ethanol oxidation and acetaldehyde reduction at pH 8 are similar for wild-type and H51Q enzymes. In contrast, the H51Q substitution significantly shifts the pH dependencies for steady-state and transient reactions and decreases by 11-fold the rate constant for the transient oxidation of ethanol at pH 8. Modest substrate deuterium isotope effects indicate that hydride transfer only partially limits the transient oxidation and turnover. Transient data show that the H51Q substitution significantly decreases the rate of isomerization of the enzyme-NAD(+) complex and becomes a limiting step for ethanol oxidation. Isomerization of the enzyme-NAD(+) complex is rate limiting for acetaldehyde reduction catalyzed by the wild-type enzyme, but release of alcohol is limiting for the H51Q enzyme. X-ray crystallography of doubly substituted His51Gln:Lys228Arg ADH complexed with NAD(+) and 2,3- or 2,4-difluorobenzyl alcohol shows that Gln-51 isosterically replaces histidine in interactions with the nicotinamide ribose of the coenzyme and that Arg-228 interacts with the adenosine monophosphate of the coenzyme without affecting the protein conformation. The difluorobenzyl alcohols bind in one conformation. His-51 participates in, but is not essential for, proton transfers in the mechanism.
Asunto(s)
Alcohol Deshidrogenasa/química , Dominio Catalítico , Histidina/química , Hígado/enzimología , Alcohol Deshidrogenasa/genética , Sustitución de Aminoácidos/genética , Animales , Dominio Catalítico/genética , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Glutamina/genética , Histidina/genética , Caballos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , NAD/químicaRESUMEN
We demonstrated earlier that the heme in cytochrome P450 enzymes of the CYP4A family is covalently attached to the protein through an I-helix glutamic acid residue [Hoch, U., and Ortiz de Montellano, P. R. (2001) J. Biol. Chem. 276, 11339-11346]. As the critical glutamic acid residue is conserved in many members of the CYP4F class of cytochrome P450 enzymes, we investigated covalent heme binding in this family of enzymes. Chromatographic analysis indicates that the heme is covalently bound in CYP4F1 and CYP4F4, which have the required glutamic acid residue, but not in CYP4F5 and CYP4F6, which do not. Catalytic turnover of CYP4F4 with NADPH-cytochrome P450 reductase shows that the heme is covalently bound through an autocatalytic process. Analysis of the prosthetic group in the CYP4F5 G330E mutant, into which the glutamic acid has been reintroduced, shows that the heme is partially covalently bound and partially converted to noncovalently bound 5-hydroxymethylheme. The modified heme presumably arises by trapping of a 5-methyl carbocation intermediate by a water molecule. CYP4F proteins thus autocatalytically bind their heme groups covalently in a process that requires a glutamic acid both to generate a reactive (cationic) form of the heme methyl and to trap it to give the ester bond.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/química , Hemo/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/genética , Hemo/análogos & derivados , Hidroxilación , Mutagénesis Sitio-Dirigida , Ratas , Análisis Espectral , Relación Estructura-ActividadRESUMEN
The prosthetic heme group in the CYP4A family of cytochrome P450 enzymes is covalently attached to an I-helix glutamic acid residue. This glutamic acid is conserved in the CYP4 family but is absent in other P450 families. As shown here, the glutamic acid is linked, presumably via an ester bond, to a hydroxyl group on the heme 5-methyl group. Mutation of the glutamic acid to an alanine in CYP4A1, CYP4A3, and CYP4A11 suppresses covalent heme binding. In wild-type CYP4A3 68% of the heme is covalently bound to the heterologously expressed protein, but in the CYP4A3/E318D mutant, 47% of the heme is unchanged, 47% is present as noncovalently bound 5-hydroxymethylheme, and only 6% is covalently bound to the protein. In the CYP4A3/E318Q mutant, the majority of the heme is unaltered, and <2% is covalently linked. The proportion of covalently bound heme in the recombinant CYP4A proteins increases with time under turnover conditions. The catalytic activity is sensitive in some, but not all, CYP4A enzymes to the extent of covalent heme binding. Mutations of Glu(318) in CYP4A3 decrease the apparent k(cat) values for lauric acid hydroxylation. The key conclusions are that (a) covalent heme binding occurs via an ester bond to the heme 5-methyl group, (b) covalent binding of the heme is mediated by an autocatalytic process, and (c) fatty acid oxidation is sensitive in some CYP4A enzymes to the presence or absence of the heme covalent link.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/química , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/química , Cartilla de ADN , Unión Proteica , Espectrofotometría UltravioletaRESUMEN
Synthetic peptides based on amino-acid residues 27-38 of human serum amyloid P component represent a novel type of heparin binders as they do not contain clusters of basic amino acids or other known features associated with protein or peptide heparin binding. Here, we characterize the binding using capillary electrophoresis (CE), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). By CE, heparin-binding activity was readily apparent for both a regular peptide and a slightly N-terminally modified form, while a sequence-scrambled peptide had no measurable binding. Dissociation constants in the 1-15 microm range were estimated, but only a minor part of the binding isotherm was covered by the experiments. SPR measurements using immobilized peptides verified heparin binding, the range of the binding constants, and the reduced binding of the sequence-scrambled peptide. Structurally defined heparin oligosaccharides were used to establish that while the tetrasaccharide is too small to exhibit strong binding, little difference in binding strength is observed between hexa- and tetradeca-saccharides. These experiments also confirmed the almost complete lack of activity of the sequence-scrambled peptide. The amino-acid sequence-dependent binding and the importance of a disulfide bond in the peptide were verified by ITC, but the experimental conditions had to be modified because of peptide precipitation and ITC yielded significantly weaker binding constants than the other methods. While the precise function of the peptide in the intact protein remains unclear, the results confirm the specificity of the glycosaminoglycan interaction with regard to peptide sequence by applying two additional biophysical techniques and showing that the N-terminal part of the peptide may be modified without changing the heparin binding capabilities.