ABSTRACT
Human interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1ß binders that interfere with IL-1ß signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1ß, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1ß as a target residue suitable for the development of covalent, low-molecular-weight IL-1ß antagonists.
Subject(s)
Interleukin-1beta , Humans , Drug Discovery , Interleukin-1beta/metabolism , Ligands , Receptors, Interleukin-1 Type I/metabolism , Receptors, Interleukin-1 Type I/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , DNA/chemistry , Gene LibraryABSTRACT
Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.
Subject(s)
Cytokines , Thinness , Humans , Interleukin-1beta , Molecular Weight , Binding Sites , BiophysicsABSTRACT
Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1ß antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1ß complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.
Subject(s)
Antibodies , Interleukin-17 , Epitopes/chemistry , Antigens , Binding Sites, AntibodyABSTRACT
Signaling through innate immune receptors such as the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily proceeds via the assembly of large membrane-proximal complexes or "signalosomes." Although structurally distinct, the IL-17 receptor family triggers cellular responses that are typical of innate immune receptors. The IL-17RA receptor subunit is shared by several members of the IL-17 family. Using a combination of crystallographic, biophysical, and mutational studies, we show that IL-17A, IL-17F, and IL-17A/F induce IL-17RA dimerization. X-ray analysis of the heteromeric IL-17A complex with the extracellular domains of the IL-17RA and IL-17RC receptors reveals that cytokine-induced IL-17RA dimerization leads to the formation of a 2:2:2 hexameric signaling assembly. Furthermore, we demonstrate that the formation of the IL-17 signalosome potentiates IL-17-induced IL-36γ and CXCL1 mRNA expression in human keratinocytes, compared with a dimerization-defective IL-17RA variant.
Subject(s)
Interleukin-17 , Receptors, Interleukin-17 , Humans , Receptors, Interleukin-17/genetics , Receptors, Interleukin-17/metabolism , Interleukin-17/metabolism , Dimerization , Cytokines/metabolism , RNA, Messenger/metabolism , Receptors, Interleukin-1/metabolismABSTRACT
The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody-drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin-negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin-expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Biomarkers, Tumor , Cadherins/genetics , Immunoconjugates/pharmacology , Neoplasms/genetics , Amino Acid Sequence , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacokinetics , Binding Sites , Cadherins/chemistry , Cadherins/metabolism , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm , Gene Expression , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacokinetics , Immunohistochemistry , Macaca fascicularis , Mice , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding , Protein Transport , Rats , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases.
Subject(s)
Interleukin-17/immunology , Protein Multimerization/immunology , Receptors, Interleukin-17/immunology , Signal Transduction/immunology , Binding, Competitive , Crystallography, X-Ray , HEK293 Cells , Humans , Interleukin-17/chemistry , Interleukin-17/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Interleukin-17/chemistry , Receptors, Interleukin-17/metabolismABSTRACT
The TGF-ß family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc "trap," to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit.
Subject(s)
Activin Receptors, Type II/antagonists & inhibitors , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Hypertrophy/chemically induced , Muscle, Skeletal/drug effects , Activin Receptors, Type II/metabolism , Activins/metabolism , Animals , Antibodies, Blocking/therapeutic use , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Bone Morphogenetic Proteins/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Growth Differentiation Factors/metabolism , HEK293 Cells , Humans , Hypertrophy/pathology , Male , Mice , Mice, SCID , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myostatin/metabolism , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Wasting Syndrome/drug therapy , Wasting Syndrome/pathologyABSTRACT
IL-17A and IL-17F are prominent members of the IL-17 family of cytokines that regulates both innate and adaptive immunity. IL-17A has been implicated in chronic inflammatory and autoimmune diseases, and anti-IL-17A antibodies have shown remarkable clinical efficacy in psoriasis and psoriatic arthritis patients. IL-17A and IL-17F are homodimeric cytokines that can also form the IL-17A/F heterodimer whose precise role in health and disease remains elusive. All three cytokines signal through the assembly of a ternary complex with the IL-17RA and IL-17RC receptors. Here we report the X-ray analysis of the human IL-17A/F heterodimer that reveals a two-faced cytokine closely mimicking IL-17A as well as IL-17F. We also present the crystal structure of its complex with the IL-17RA receptor. Unexpectedly in view of the much higher affinity of this receptor toward IL-17A, we find that IL-17RA is bound to the "F-face" of the heterodimer in the crystal. Using site-directed mutagenesis, we then demonstrate that IL-17RA can also bind to the "A-face" of IL-17A/F with similar affinity. Further, we show that IL-17RC does not discriminate between the two faces of the cytokine heterodimer either, thus enabling the formation of two topologically-distinct heterotrimeric complexes with potentially different signaling properties.
Subject(s)
Interleukin-17/chemistry , Interleukin-17/metabolism , Protein Multimerization , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Conserved Sequence , Cytokines/chemistry , Cytokines/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Receptors, Interleukin-17/metabolism , Structure-Activity RelationshipABSTRACT
Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs.
Subject(s)
Bone and Bones/drug effects , Drug Delivery Systems , Administration, Oral , Biological Availability , Bone and Bones/enzymology , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Geranyltranstransferase/antagonists & inhibitors , HumansABSTRACT
Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Geranyltranstransferase/antagonists & inhibitors , Quinolines/pharmacology , Salicylic Acid/pharmacology , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Geranyltranstransferase/metabolism , Humans , Molecular Structure , Quinolines/chemical synthesis , Quinolines/chemistry , Salicylic Acid/chemical synthesis , Salicylic Acid/chemistry , Structure-Activity RelationshipABSTRACT
Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.
Subject(s)
Diphosphonates , Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Geranyltranstransferase/antagonists & inhibitors , Allosteric Regulation , Allosteric Site , Bone and Bones/chemistry , Bone and Bones/metabolism , Crystallography, X-Ray , Diphosphonates/analysis , Diphosphonates/chemistry , Diphosphonates/metabolism , Diphosphonates/pharmacology , Enzyme Inhibitors/chemistry , Geranyltranstransferase/metabolism , Humans , Imidazoles/analysis , Imidazoles/chemistry , Imidazoles/pharmacology , Magnetic Resonance Spectroscopy , Soft Tissue Neoplasms/drug therapy , Zoledronic AcidABSTRACT
To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen-containing bisphosphonate (N-BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N-BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS-N-BP complexes rather than to compete with and displace the N-BP inhibitor. Stabilization of the FPPS-N-BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N-BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N-BP drugs. Moreover, our data form the basis for structure-guided design of optimized N-BPs with improved pharmacological properties.
Subject(s)
Diphosphonates/chemistry , Diphosphonates/pharmacology , Calorimetry, Differential Scanning , Crystallography, X-Ray , Humans , Models, Molecular , Molecular StructureABSTRACT
Breast helical computed tomography (CT) was evaluated for use in assessing response to neoadjuvant chemotherapy and residual tumor volume. Forty-three patients with large, inflammatory breast cancers (stage IIA, 12; IIB, 13; IIIA, 9; IIIB, 9), all histologically confirmed by core biopsy, were evaluated prior to and following neoadjuvant chemotherapy. The breast helical CT procedure involved patients in the prone position using single acquisition during quiet respiration following intravenous injection of nonionic contrast material. Helical CT results (3.2-mm slices and maximum intensity projections) were compared to clinical and mammographic evaluations, as well as to pathologic findings. All tumors were clearly visible by breast helical CT, showing important tumor enhancement. Helical CT evaluation of response to chemotherapy (using World Health Organization criteria) corresponded better with mammography (78%, Cohen's kappa statistic (kappa) = 0.65) than with clinical examination (53%, kappa = 0.30). Helical CT measurement of residual tumor volume after neoadjuvant chemotherapy and correlation with pathologic findings were globally satisfactory. The intraclass correlation coefficient was 0.69 (excellent for rounded opacities [0.97], but not as good for diffuse, scattered or multinodular opacities [0.60]). By contrast, clinical and mammographic correlations were globally unsatisfactory (0.49 and 0.28, respectively). Breast helical CT can be very useful in the quantitative assessment of response to neoadjuvant chemotherapy and preoperative determination of residual tumor volume. For this reason, it can be considered an alternative to breast magnetic resonance imaging because of its simplicity, rapidity, and accessibility.