Biology subjects

Dorogin, J.

Publications and source records attributed to Dorogin, J..

6 recordsLinked to original sources

bioRxiv2026

From Code to Cure: Computationally Designed BMP-2 Binders Using AI-Integrated Pipelines for Controlled Bone Regeneration

Nonunion fractures remain a costly and persistent challenge in regenerative medicine, with current treatments limited by donor-site morbidity, restricted graft availability, and severe adverse effects associated with supraphysiological bone morphogenetic protein 2 (BMP-2) delivery, including ectopic ossification and inflammation. Endogenous BMP-2 signaling is tightly regulated in adult tissues, constraining the precision and scalability of approaches based on transcriptional upregulation or bolus growth factor administration. To address these limitations, we developed a two-phase integrated computational-experimental pipeline for the de novo design of protein binders targeting the BMP-2 knuckle epitope, a receptor-binding surface corresponding to BMPR-II engagement, enabling affinity-tuned modulation of BMP-2 activity rather than uncontrolled pathway activation. Phase I employed PyRosetta-based {beta}-strand motif grafting and physics-based docking protocols to generate 264 candidate binders, followed by deep-learning-driven refinement in Phase II using partial RFDiffusion and ProteinMPNN with AlphaFold2 validation, yielding 22 candidates with stable {beta}-sheet architectures consistent with knuckle-epitope targeting. Experimental validation demonstrated dose-dependent BMP-2 binding, with the lead construct exhibiting an apparent KD of 2.07 nM toward BMP-2. Targeted alanine substitutions revealed differential residue contributions, with mutation of T42 significantly disrupting binding, while other substitutions had more modest effects, indicating a partially hotspot-driven interface supported by other interactions.

bioengineering↗

bioRxiv2026

Discovery and Characterization of Interleukin-4-Specific Affibodies for Affinity-Controlled Protein Release and Macrophage Polarization

Interleukin-4 (IL-4) is a key immunoregulatory cytokine that promotes type 2 inflammation, drives macrophage polarization toward an anti-inflammatory M2 phenotype, and supports tissue repair. However, clinical translation of IL-4 therapies to modulate the immune response is limited by the need for precise control over its delivery to avoid immune dysregulation. Here, we report an affinity-based strategy to modulate IL-4 delivery and bioactivity using engineered affibody proteins. A yeast surface display library was screened via magnetic- and fluorescence-activated cell sorting to identify two IL-4-specific affibodies with moderate binding affinities (dissociation constants, KD = 459 and 141 nM). Circular dichroism confirmed expected alpha-helical folding, and biolayer interferometry characterized the kinetics of IL-4 binding. Structural modeling using AlphaFold3 and RosettaDock and molecular dynamics simulations using GROMACS predicted distinct binding sites for each IL-4-specific affibody on the IL-4 protein and suggested potential interference with receptor complex formation. Bioactivity studies using murine bone marrow-derived macrophages demonstrated that IL-4 complexed with affibodies maintained Ym1 gene expression but significantly reduced Ym1 protein levels, indicating partial inhibition of IL-4 signaling. To enable controlled cytokine delivery via affinity interactions, affibodies were conjugated to polyethylene glycol maleimide (PEG-mal) hydrogels, which were loaded with IL-4. Affibody-conjugated hydrogels achieved high IL-4 loading efficiency (>90%) and exhibited sustained release over 7 days. Increasing affibody-to-IL-4 ratios significantly reduced both the rate and total amount of cytokine release. Overall, this work establishes IL-4-specific affibodies as versatile tools for tuning cytokine presentation and modulating bioactivity and provides a promising approach for regulating inflammatory responses and advancing cytokine-based therapies with improved temporal control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/723637v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@146d4c1org.highwire.dtl.DTLVardef@139dcfeorg.highwire.dtl.DTLVardef@1dbe8f3org.highwire.dtl.DTLVardef@9ac211_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Melt Electrowritten Scaffold-Reinforced Affibody-Conjugated Hydrogels for Controlled Bone Morphogenetic Protein-2 Delivery

Bone morphogenetic protein-2 (BMP-2) is clinically used to promote bone regeneration but suffers from uncontrolled release when delivered from collagen sponges, necessitating high doses that can cause adverse effects. Hydrogels offer tunable protein release but are limited by weak mechanics and poor stability during storage and handling. Here, we introduce a two-part protein delivery platform that integrates mechanical reinforcement with affinity-controlled protein release. We developed a melt electrowritten (MEW) scaffold-reinforced, affibody-conjugated polyethylene glycol maleimide (PEG-mal) hydrogel for affinity-controlled BMP-2 delivery. MEW scaffolds improved hydrogel handling, compressive resistance, and stability during lyophilization and rehydration, without altering bulk stiffness. Engineered BMP-2-specific affibodies provided affinity-based control over BMP-2 release. This ability to control BMP-2 release was preserved after lyophilization and rehydration of the hydrogels. In vivo, affibody conjugation of high-affinity affibodies to the hydrogels significantly enhanced BMP-2 retention in subcutaneous implants, while MEW reinforcement significantly increased bone volume and defect bridging in rat femoral bone defects. This affibody-conjugated, MEW scaffold-reinforced hydrogel system effectively integrates mechanical reinforcement with tunable protein-material affinity interactions, advancing hydrogel-based delivery strategies for BMP-2 and other protein therapeutics in musculoskeletal repair.

bioengineering↗

bioRxiv2024

Applying computational protein design to engineer affibodies for affinity-controlled delivery of vascular endothelial growth factor and platelet-derived growth factor

Effective angiogenesis requires the coordinated presentation of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which play competing roles in stimulating vascular outgrowth and stabilization. Dysregulation in VEGF and PDGF secretion are implicated in abnormal vascular structures, poor vessel stability, and inadequate tissue repair. Current biomaterial delivery vehicles for these proteins have a limited ability to precisely control the kinetics of protein release, preventing systematic exploration of their temporal effects. Here, we combined yeast surface display and computational protein design to engineer eight VEGF-specific and PDGF-specific protein binders called affibodies with a broad range of affinities (dissociation constants = 2.23-9260 nM) for affinity-controlled protein release. Starting from one VEGF- and one PDGF-specific affibody discovered from yeast surface display, computational modeling was used to select disruptive mutations to create VEGF-specific affibodies with lower affinities for VEGF and expand the affinity range of PDGF-specific affibodies. In both cases, the specificity of the affibodies for their target protein was either maintained or enhanced. Soluble protein-specific affibodies modulated protein bioactivity as evidenced by changes in VEGF-induced endothelial cell proliferation and luminescent output of a PDGF-responsive fibroblast cell line. Affibody-conjugated polyethylene glycol maleimide (PEG-mal) hydrogels sustained VEGF and PDGF release compared to hydrogels without affibodies, enabling tunability of protein release over 7 days. VEGF and PDGF released from affibody-conjugated hydrogels exhibited higher bioactivity compared to proteins released from hydrogels without affibodies, highlighting the potential of these engineered affinity interactions to prolong protein bioactivity. This work underscores the power of computational protein design to enhance biomaterial functionality, creating a platform for tunable protein delivery to support angiogenesis and tissue repair.

bioengineering↗

bioRxiv2024

Recombinant and synthetic affibodies function comparably for modulating protein release

PurposeAffibodies are a class of versatile affinity proteins with a wide variety of therapeutic applications, ranging from guided contrast agents for imaging to cell-targeting therapeutics. We have identified several affibodies specific to bone morphogenetic protein-2 (BMP-2) with a range of binding affinities and demonstrated the ability to tune release rate of BMP-2 from affibody-conjugated poly(ethylene glycol) (PEG) hydrogels based on affibody affinity strength. In this work, we compare the purity, structure, and activity of recombinant, bacterially-expressed BMP-2-specific affibodies with affibodies synthesized via solid-phase peptide synthesis. MethodsHigh- and low-affinity BMP-2-specific affibodies were recombinantly expressed using BL21(DE3) E. coli and chemically synthesized using microwave-assisted solid-phase peptide synthesis with Fmoc-Gly-Wang resin. The secondary structures of the affibodies and dissociation constants of affibody-BMP-2 binding were characterized by circular dichroism and biolayer interferometry, respectively. Endotoxin levels were measured using chromogenic limulus amebocyte lysate (LAL) assays. Affibody-conjugated PEG-mal hydrogels were fabricated and loaded with BMP-2 to evaluate hydrogel capacity for controlled release, quantified by enzyme-linked immunosorbent assays (ELISA). ResultsSynthetic and recombinant affibodies were determined to be -helical by circular dichroism. The synthetic high- and low-affinity BMP-2-specific affibodies demonstrated comparable BMP-2-binding dissociation constants to their recombinant counterparts. Recombinant affibodies retained some endotoxins after purification, while endotoxins were not detected in the synthetic affibodies. High-affinity affibody-conjugated hydrogels reduced cumulative BMP-2 release compared to the low-affinity affibody-conjugated hydrogels and hydrogels without affibodies. ConclusionsSynthetic affibodies demonstrate comparable structure and function to recombinant affibodies, while reducing endotoxin contamination and increasing product yield, indicating that solid-phase peptide synthesis is a viable method of producing affibodies for controlled protein release and other applications.

bioengineering↗

bioRxiv2023

Statistical Optimization of Hydrazone-Crosslinked Hyaluronic Acid Hydrogels for Protein Delivery

Hydrazone-crosslinked hydrogels are attractive protein delivery vehicles for regenerative medicine. However, each regenerative medicine application requires unique hydrogel properties to achieve an ideal outcome. The properties of a hydrogel can be impacted by numerous factors involved in its fabrication. We used design of experiments (DoE) statistical modeling to efficiently optimize the physicochemical properties of a hyaluronic acid (HA) hydrazone-crosslinked hydrogel for protein delivery for bone regeneration. We modified HA with either adipic acid dihydrazide (HA-ADH) or aldehyde (HA-Ox) functional groups and used DoE to evaluate the interactions of three input variables, the molecular weight of HA (40 or 100 kDa), the concentration of HA-ADH (1-3% w/v), and the concentration of HA-Ox (1-3% w/v), on three output responses, gelation time, compressive modulus, and hydrogel stability over time. We identified 100 kDa HA-ADH3.0HA-Ox2.33 as an optimal hydrogel that met all of our design criteria, including displaying a gelation time of 3.7 minutes, compressive modulus of 62.1 Pa, and minimal mass change over 28 days. For protein delivery, we conjugated affinity proteins called affibodies that were specific to the osteogenic protein bone morphogenetic protein-2 (BMP-2) to HA hydrogels and demonstrated that our platform could control the release of BMP-2 over 28 days. Ultimately, our approach demonstrates the utility of DoE for optimizing hydrazone-crosslinked HA hydrogels for protein delivery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/549125v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@bd1a4org.highwire.dtl.DTLVardef@1f0766borg.highwire.dtl.DTLVardef@1eea01corg.highwire.dtl.DTLVardef@151bd6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗