Anna Szlachcic

Increased protein stability of FGF1 can compensate for its reduced affinity for heparin

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1·fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1·FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.

Longer action means better drug: Tuning up protein therapeutics

An increasing number of proteins are currently available on the market as therapeutics and this branch of the pharmaceutical industry will expand substantially during the coming years. As many diseases result from dysfunction of proteins forming multicomponent complexes, protein drugs with their inherent high specificity and affinity seem to be optimal medical agents. On the other hand, proteins are often highly instable and sensitive to degradation, which questions their applicability as effective therapeutics. Therefore, redesign and engineering of proteins is usually a required step in the present day drug development. Several approaches have been applied to optimize the protein properties central to their pharmaceutical use. This review focuses on different strategies that improve two crucial factors influencing protein drug efficiency: protein stability and its in vivo half-life. We provide examples of successful genetic and chemical modifications applied in the design of effective protein therapeutics.

FGF1-gold nanoparticle conjugates targeting FGFR efficiently decrease cell viability upon NIR irradiation

Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy.

Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality.

Structure of a highly stable mutant of human fibroblast growth factor 1.

Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).

Site-specific conjugation of fibroblast growth factor 2 (FGF2) based on incorporation of alkyne-reactive unnatural amino acid

Recent advances in site-specific protein modification include the increasingly popular incorporation of unnatural amino acid(s) using amber codon, a method developed by Schultz and coworkers. In this study, we employ this technique to introduce propargyllysine (PrK) in human fibroblast growth factor 2 (FGF2). Owing to an alkyne moiety in its side chain, PrK is compatible with Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). We successfully tested CuAAC-mediated conjugation of FGF2 with two compounds - a fluorophore carboxyrhodamine 110 or a cytotoxic drug monomethyl auristatin E (MMAE). In the case of the MMAE conjugate we improved the initial poor conjugation yield to achieve nearly 100% efficiency after extensive optimization. The detergent-based optimization approach may help overcome problems with the CuAAC reaction yield for protein modification with hydrophobic compounds, such as MMAE.

High-Yield Site-Specific Conjugation of Fibroblast Growth Factor 1 with Monomethylauristatin E via Cysteine Flanked by Basic Residues

Site-specific conjugation is a leading trend in the development of protein conjugates, including antibody-drug conjugates (ADCs), suitable for targeted cancer therapy. Here, we present a very efficient strategy for specific attachment of a cytotoxic drug to fibroblast growth factor 1 (FGF1), a natural ligand of FGF receptors (FGFRs), which are over-expressed in several types of lung, breast, and gastric cancers and are therefore an attractive molecular target. Recently, we showed that FGF1 fused to monomethylauristatin E (vcMMAE) was highly cytotoxic to cells presenting FGFRs on their surface and could be used as a targeting agent alternative to an antibody. Unfortunately, conjugation via maleimide chemistry to endogenous FGF1 cysteines or a cysteine introduced at the N-terminus proceeded with low yield and led to nonhomogeneous products. To improve the conjugation, we introduced a novel Lys-Cys-Lys motif at either FGF1 terminus, which increased cysteine reactivity and allowed us to obtain an FGF1 conjugate with a defined site of conjugation and a yield exceeding 95%. Using FGFR-expressing cancer lines, we confirmed specific cytotoxity of the obtained C-terminal FGF1-vcMMAE conjugate and its selective endocytososis as compared with FGFR1-negative cells. This simple and powerful approach relying on the introduction of a short sequence containing cysteine and positively charged amino acids could be used universally to improve the efficiency of the site-specific chemical modification of other proteins.

Tailoring Small Proteins Towards Biomedical Applications

Over the last two decades proteins have become increasingly important in human therapy and diagnosis. Engineering therapeutic proteins through improving their biological activity and stability has been a major interest in our group. In this mini-review we summarize our research on three proteins with pharmaceutical potential - serine protease inhibitor from squash seeds (CMTI), bovine pancreatic trypsin inhibitor (BPTI), and human fibroblast growth factor 1 (FGF1). To improve the functional properties of these proteins we used multiple techniques such as homology approach, rational design, total chemical synthesis, site-directed mutagenesis and phage display. The physicochemical properties of the obtained protein variants were evaluated using protein crystallography, spectroscopic techniques, enzymatic assays, stability measurements as well as numerous biological tests.

FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1

In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.

Specific Antibody Fragment Ligand Traps Blocking FGF1 Activity

Fibroblast growth factor 1 (FGF1) and its receptors (FGFRs) regulate crucial biological processes such as cell proliferation and differentiation. Aberrant activation of FGFRs by their ligands can promote tumor growth and angiogenesis in many tumor types, including lung or breast cancer. The development of FGF1-targeting molecules with potential implications for the therapy of FGF1-driven tumors is recently being considered a promising approach in the treatment of cancer. In this study we have used phage display selection to find scFv antibody fragments selectively binding FGF1 and preventing it from binding to its receptor. Three identified scFv clones were expressed and characterized with regard to their binding to FGF1 and ability to interfere with FGF1-induced signaling cascades activation. In the next step the scFvs were cloned to scFv-Fc format, as dimeric Fc fusions prove beneficial in prospective therapeutic application. As expected, scFvs-Fc exhibited significantly increased affinity towards FGF1. We observed strong antiproliferative activity of the scFvs and scFvs-Fc in the in vitro cell models. Presented antibody fragments serve as novel FGF1 inhibitors and can be further utilized as powerful tools to use in the studies on the selective cancer therapy.