Protein Chimeras Research Articles

Functional expression of the chimera proteins of Nav1.7 and NavAb in Escherichiacoli

Nav1.7 is a eukaryotic voltage-dependent Na channel (Nav) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Nav subtype are targeted in the development of Nav1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Nav1.7 inhibitors, the chimera protein (NavAb/1.7VSD) of Nav from prokaryotic Arcobacter butzleri (NavAb), in which extracellular loops of VSD were replaced with homologous loops from Nav1.7, serves as an effective model. This is because NavAb harbors only one VSD and the biological activity of NavAb/1.7VSD was previously confirmed. To date, NavAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of NavAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Nav1.7-specific peptide inhibitor. The results indicate that NavAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Nav1.7 inhibitors using the PERISS screening method.

Bioengineered Silk Fibroin Hydrogel Reinforced with Collagen-Like Protein Chimeras for Improved Wound Healing.

The study investigates the potentials of the rapid crosslinking hydrogel concoction comprising of natural silk fibroin (SF) and recombinant tailorable collagen-like protein with binding domains for wound repair. The formation of dityrosine crosslinks between the tyrosine moieties augments the formation of stable hydrogels, in the presence of the cytocompatible photo-initiator riboflavin and visible light. This uniquely engineered PASCH (Photo-activated silk fibroin and tailor-made collagen-like protein hydrogel) confers the key advantage of improved biological properties over the control hydrogels comprising only of SF. The physico-chemical characterization of the hydrogels with respect to crosslinking, modulus, and thermal stability delineates the ascendancy of PASCH 7:3 over other combinations. Furthermore, the hybrid protein hydrogel proves to be a favorable cellular matrix as it enhances cell adhesion, elongation, growth, and proliferation in vitro. Time-lapse microscopy studies reveal an enhanced wound closure in human endothelial cell monolayer (EA.hy926), while the gene expression studies portray the dynamic interplay of cytokines and growth factors in the wound milieu facilitating the repair and regeneration of cells, sculpted by the proteins. The results demonstrate the improved physical and biological properties of fabricated PASCH, depicting their synergism, and implying their competency for use in tissue engineering applications.

Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5’-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P<0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P<0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as prototypes of enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it a potential player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. Therefore, it constitutes a prototype of a microreactor with potential use in bioremediation.

Abstract 7524: Targeting siglec-7/9 glyco-immune checkpoints in prostate cancer for enhanced immune responses and tumor suppression

Abstract Prostate cancer is one of the leading causes of death in men worldwide. Despite significant advancements in the fight against cancer, immune checkpoint inhibitors have limited effectiveness in treating prostate cancer. Evidence shows that disrupting the interactions between Siglec-7/9 and sialic acids can enhance immune responses and effectively inhibit tumor growth in several cancers. However, the understanding of Siglec-7/9-sialic acid interactions in prostate cancer remains limited. Here, we found that tumor-infiltrating immune cells derived from prostate cancer patients exhibited high levels of Siglec-7 and Siglec-9. TCGA data analysis suggests that high levels of Siglec-7/9 are correlated to worse patient survival. Elevated levels of Siglec-7/9 ligands were found in prostate tumor tissues compared to adjacent normal tissues. Prostate cancer cell lines exhibited significant expression levels of Siglec-7 and Siglec-9 ligands, and cell surface sialic acids. Proteomic analysis suggests that increased sialylation was observed in tumor tissues in comparison to adjacent normal tissues. Disrupting interactions between Siglec-7/9 and sialoglycans enhanced T cell-mediated cytotoxicity against prostate cancer cells. Treatment with anti-Siglec-7/9 antibodies effectively suppressed the growth of PC3 and 22Rv1 tumors in a humanized mouse model. Immunohistochemistry analysis of tumor tissues showed that PC3 and 22Rv1 tumors treated with anti-Siglec-7/9 exhibited reduced proliferation, decreased vascularization, increased apoptosis, and enhanced immune cell infiltration. The CRISPR screen and mass spectrometry analysis suggested CD59 as a potential ligand for Siglec-9. Knockout of CD59 in prostate cancer cells decreased their binding activity of Siglec-9-Fc chimera proteins and promoted T cell-mediated killing ability. These findings provide valuable insights into the potential therapeutic strategy of targeting Siglec-7/9 and their ligands for prostate cancer treatment. Citation Format: Ru Wen, G Edward Marti, Jessica Stark, Fernando Jose Garcia Marques, Nick Riley, Abel Bermudez, Hongjuan Zhao, Carolyn Bertozzi, Sharon Pitteri, James Brooks. Targeting siglec-7/9 glyco-immune checkpoints in prostate cancer for enhanced immune responses and tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7524.

Molecular handcraft of a well-folded protein chimera.

Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous 'copy-paste' processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/β-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

Engineered Chimera Protein Constructs to Facilitate the Production of Heterologous Transmembrane Proteins in E. coli.

To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, E. coli has the lowest cost and fastest turnover. However, many of the TMPs expressed in E. coli are misfolded. Several strategies have been developed to either direct the foreign TMPs to E. coli's membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of Mycobacterium tuberculosis, HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs' expression in E. coli.

A protein chimera for dsRNA binding and delivery into oocytes for large-scale gene silencing in crustacean aquaculture

RNA interference (RNAi) is one of the most intensively used gene silencing techniques for gene function studies and biotechnology applications. However, the gonads, specifically the ovary, of arthropods appear to be intractable to efficient gene silencing. In this article, we report the development of a tool for specific binding and delivery of double-stranded (ds) RNA to the crustacean oocyte for gene silencing. The tool, an oocyte-specific silencing protein chimera, which we designated OSSCot, is composed of a dsRNA-binding domain retrieved from a specific Macrobrachium rosenbergii dsRNA binding protein and 24-amino acid peptide derived from the yolk protein, vitellogenin. The results of the in vitro and in vivo examinations carried out in this study indicate that an OSSCot-piggybacked dsRNA specifically enters the oocytes. The results also indicate that the OSSCot protein protects the dsRNA from degradation by endonucleases in the ovary. When OSSCot was linked to the specific PAX6-like dsRNA of M. rosenbergii (where PAX6 is the master gene for eye development) and injected into the hemolymph of vitellogenic females, the expression of the RNAi enzyme machinery was enhanced, and eye development was impaired in the embryos of the treated females. These findings validate the concept of exploiting OSSCot to piggyback a functional dsRNA into crustacean oocytes for gene silencing. The newly developed tool will find application for oocyte-specific gene silencing in other crustacean species and for research and biotechnology applications.

Porcine UL-16 Binding Protein 1 Is Not a Functional Ligand for the Human Natural Killer Cell Activating Receptor NKG2D.

Natural killer (NK) cells play a vital role in xenotransplantation rejection. One approach to induce NK cell immune tolerance is to prevent the NK cell-mediated direct killing of porcine cells by targeting the interaction of the activating receptor NKG2D and its ligands. However, the identity of porcine ligands for the human NKG2D receptor has remained elusive. Previous studies on porcine UL-16 binding protein 1 (pULBP-1) as a ligand for human NKG2D have yielded contradictory results. The goal of the present study was to clarify the role of pULBP-1 in the immune response and its interaction with human NKG2D receptor. To accomplish this, the CRISPR/Cas9 gene editing tool was employed to disrupt the porcine ULBP-1 gene in a 5-gene knockout porcine endothelial cell line (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin, 5GKO). A colony with two allele mutations in pULBP-1 was established as a 6-gene knockout pig cell line (6GKO). We found that pULBP-1-deficient pig cells exhibited a reduced binding capacity to human NKG2D-Fc, a recombinant chimera protein. However, the removal of ULBP-1 from porcine endothelial cells did not significantly impact human NK cell degranulation or cytotoxicity upon stimulation with the pig cells. These findings conclusively demonstrate that pULBP-1 is not a crucial ligand for initiating xenogeneic human NK cell activation.

CD58 Genetic Alterations and Its Contribution to Upregulation of PD-L1 and IDO Via LYN/CD22/SHP1 Axis in DLBCL

Abstract Background: Recurrent abnormalities of immune surveillance-related genes play a crucial role in DLBCL progression. Prior studies have shown that CD58, a key adhesion molecule that acts as a ligand for the T-cell costimulatory molecule CD2, is frequently mutated or deleted in certain hematological malignancies. Aberrant expression of CD58 may result in tumor cells evading immune surveillance mechanisms mediated by T/NK cells. Downregulation or loss of CD58 is linked to resistance to ICB therapy in melanoma and CAR-T therapy in B-cell malignancies. Nevertheless, the role of CD58 in cancer is not yet well understood. Thus, the aim of this study was to comprehensively characterize CD58 genetic alterations in DLBCL, unveil the multiple roles of CD58 in anti-tumor immunity, and provide insights for potential therapeutic strategies. Methods: Comprehensive analysis of the genetic characteristics of CD58 were performed through targeted deep sequencing (n=176), whole exome sequencing (n=38), and RNA-sequencing (n=162) in patients with de novo DLBCL. To investigate the mechanistic impacts of CD58 alterations on co-inhibitory molecules expression and immune cell function, we performed bulk and single-cell RNA-sequencing analysis of tumor samples and conducted co-IP, flow cytometry and co-culture assays in vitro. Results: We identified that CD58 mutagenesis rate was 9.1%, with 88.9% of the identified mutations resulting in truncated protein or impairment of its normal function. 61.1% mutations occurred in exon 2, which may disrupt CD58-CD2 binding. Co-mutation analysis revealed higher frequencies of KMT2D, CD79B and MYD88 mutations in patients with CD58 mutations(p &amp;lt; 0.05) (Fig 1A). The CD58 copy number loss rate was 44.7% (Fig 1B). Both mutations and copy number loss led to a mild decrease in mRNA expression of CD58. Patients with CD58 mutation, copy number loss or low expression exhibited lower complete response rates following first-line R-CHOP therapy, as well as significantly poorer PFS and OS. To reveal the profound impact of CD58 on the immune microenvironment of DLBCL, we performed bulk and single-cell RNA sequencing and revealed that increased NK cells, activated CD4 +/CD8 + T cells, as well as the weaker exhaustion status of CD8 + T cells, correlated with CD58 high expression. By co-culturing assays, we identified that CD58 downregulation or mutation in DLBCL cells suppressed T cell proliferation and CAR-T cell-mediated killing. Interestingly, blocking CD58-CD2 signaling with a CD2 antibody did not fully eliminate the inhibitory effect, indicating that CD58 alterations could trigger additional immunosuppressive mechanisms. We further revealed that CD58 downregulation or mutation in DLBCL cells resulted in elevated expression of PD-L1 and IDO. This suggested that CD58 may have a dual effect on T cells, whereby its downregulation or mutation may lead to a lack of CD2 co-stimulatory signaling, as well as an increase in PD-L1 and IDO inhibitory signaling. We found that CD58 downregulation or mutation caused JAK2/STAT1 phosphorylation, which in turn promoted PD-L1 and IDO expression. Co-IP identified that CD58 can interact with Lyn, which phosphorylated ITIM of the inhibitory receptor CD22, leading to recruitment and activation of the inhibitory phosphatase SHP1. CD58 downregulation suppressed the negative regulation of the LYN/CD22/SHP1 axis, thereby promoting the activation of JAK2/STAT1 and upregulating PD-L1 and IDO expression. To overcome the dual effects induced by CD58 alterations, anti-PD-L1 antibody or IDO inhibitor and recombinant CD58-Fc chimera protein were added alone or in combination to the co-culture cells. We showed that the combination regimens reversed the inhibitory effect of CD58-downregulated or mutated DLBCL cells on T cell proliferation and restored sensitivity to CAR-T cell-mediated killing (Fig 1C, D). Conclusions: Our study comprehensively characterized CD58 genetic alterations in DLBCL. We demonstrated that CD58 downregulation or mutation led to upregulation of PD-L1 and IDO expression mainly by regulating the LYN/CD22/SHP1 axis. Moreover, we explored strategies to directly activate CD2 co-stimulatory signaling or in combination with immune checkpoint inhibitors to address the diverse effects of CD58 alterations. Our findings provide novel insights for individualized therapy for DLBCL patients with CD58 mutation or deletion.

Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Innovative Breakthroughs for the Treatment of Advanced and Metastatic Synovial Sarcoma.

Synovial sarcoma (SyS) is a rare aggressive soft tissue sarcoma carrying the chromosomal translocation t(X;18), encoding the fusion transcript SS18::SSX. The fusion oncoprotein interacts with both BAF enhancer complexes and polycomb repressor complexes, resulting in genome-wide epigenetic perturbations and a unique altered genetic signature. Over 80% of the patients are initially diagnosed with localized disease and have a 5-year survival rate of 70-80%, but metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease has a 5-year survival rate below 10%, representing a critical issue. This review summarizes the molecular mechanisms behind SyS and illustrates current treatments in front line, second line, and beyond settings. We analyze the use of immune check point inhibitors (ICI) in SyS that do not behave as an ICI-sensitive tumor, claiming the need for predictive genetic signatures and tumor immune microenvironment biomarkers. We highlight the clinical translation of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells. Adoptive cell transfer of engineered T-cell receptor cells targeting selected cancer/testis antigens has shown promising results against metastatic SyS in early clinical trials and further improvements are awaited from refinements involving immune cell engineering and tumor immune microenvironment enhancement.

Administration of soluble gp130Fc disrupts M-1 macrophage polarization, dendritic cell activation, MDSC expansion and Th-17 induction during experimental cerebral malaria

Regulatory effect of IL-6 on various immune cells plays a crucial role during experimental cerebral malaria pathogenesis. IL-6 neutralization can restore distorted ratios of myeloid dendritic cells and plasmacytoid dendritic cells as well as the balance between Th-17 and T-regulatory cells. IL-6 can also influence immune cells through classical and trans IL-6 signalling pathways. As trans IL-6 signalling is reportedly involved during malaria pathogenesis, we focused on studying the effects of trans IL-6 signalling blockade on various immune cell populations and how they regulate ECM progression. Results show that administration of sgp130Fc recombinant chimera protein lowers the parasitemia, increases the survivability of Plasmodium berghei ANKA infected mice, and restores the distorted ratios of M1/M2 macrophage, mDC/pDC, and Th-17/Treg. IL-6 trans signalling blockade has been found to affect both expansion of myeloid derived suppressor cells (MDSCs) and expression of inflammatory markers on them during Plasmodium berghei ANKA infection indicating that trans IL-6 signalling might regulate various immune cells and their function during ECM. In this work for the first time, we delineate the effect of sgp130Fc administration on influencing the immunological changes within the host secondary lymphoid organ during ECM induced by Plasmodium berghei ANKA infection.

A ubiquitination-mediated degradation system to target 14-3-3-binding phosphoproteins

The phosphorylation of 14-3-3 binding motif is involved in many cellular processes. A strategy that enables targeted degradation of 14-3-3-binding phosphoproteins (14-3-3-BPPs) for studying their functions is highly desirable for basic research. Here, we report a phosphorylation-induced, ubiquitin-proteasome-system-mediated targeted protein degradation (TPD) strategy that allows specific degradation of 14-3-3-BPPs. Specifically, by ligating a modified von Hippel-Lindau E3-ligase with an engineered 14-3-3 bait, we generated a protein chimera referred to as Targeted Degradation of 14-3-3-binding PhosphoProtein (TDPP). TDPP can serve as a universal degrader for 14-3-3-BPPs based on the specific recognition of the phosphorylation in 14-3-3 binding motifs. TDPP shows high efficiency and specificity to a difopein-EGFP reporter, general and specific 14-3-3-BPPs. TDPP can also be applied for the validation of 14-3-3-BPPs. These results strongly support TDPP as a powerful tool for 14-3-3 related research.

Abstract GS3-03: GS3-03 ARV-471, a PROTAC® estrogen receptor (ER) degrader in advanced ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer: phase 2 expansion (VERITAC) of a phase 1/2 study

Abstract Background: ARV-471 is a selective, orally administered PROteolysis TArgeting Chimera (PROTAC®) protein degrader that targets wild-type and mutant ER. ARV-471 is being evaluated in patients with ER+/HER2- locally advanced or metastatic breast cancer in a first-in-human phase 1/2 study (NCT04072952). In the phase 1 dose escalation, ARV-471 monotherapy (dose range: 30–700 mg total daily dose) showed a manageable safety profile in patients who had previously received endocrine therapy and a cyclin-dependent kinase (CDK) 4/6 inhibitor. The clinical benefit rate (CBR; rate of confirmed complete or partial response or stable disease ≥24 weeks) was 40% (95% CI: 26–56) in 47 evaluable patients. The phase 2 expansion portion of the study (VERITAC) evaluated 2 doses of ARV-471.Methods: In VERITAC, ARV-471 monotherapy was administered at doses of 200 mg once daily (QD) or 500 mg QD to patients with ER+/HER2- locally advanced/metastatic breast cancer who had received ≥1 prior endocrine therapy for ≥6 months, ≥1 CDK4/6 inhibitor, and ≤1 chemotherapy regimen. The primary endpoint of CBR was evaluated in patients enrolled ≥24 weeks prior to the data cutoff. Results: As of June 6, 2022, 71 patients received ARV-471 (200 mg QD [n=35]; 500 mg QD [n=36]) in VERITAC. Across all treated patients, 69 (97.2%) were female and median age was 60 y (range: 41–86). Patients had received a median of 4 prior regimens in all settings (range: 1–10); 100% had prior CDK4/6 inhibitors, 78.9% had prior fulvestrant, and 73.2% had prior chemotherapy. ARV-471 was well tolerated at both doses, with most treatment-related adverse events (TRAEs) grade 1/2; the most common TRAEs were fatigue and nausea (Table). In all, 3 patients (1 in the 200 mg QD cohort and 2 in the 500 mg QD cohort) discontinued ARV-471 due to treatment-emergent adverse events (TEAEs); 3 patients had ARV-471 dose reductions due to TEAEs (all from 500 mg QD to 400 mg QD). CBR was 37.1% (95% CI: 21–55) in 35 evaluable patients treated at 200 mg QD and 38.9% (95% CI: 23–57) in 36 evaluable patients treated at 500 mg QD. CBR in evaluable patients with mutant ESR1 in the 200 mg QD (n=19) and 500 mg QD (n=22) cohorts was 47.4% (95% CI: 24–71) and 54.5% (95% CI: 32–76), respectively. Conclusions: In the phase 2 VERITAC expansion cohorts of patients with ER+/HER2- locally advanced/metastatic breast cancer and prior CDK4/6 inhibitor treatment, ARV-471 monotherapy showed evidence of clinical activity based on CBR, which was further enhanced in the subgroup with ESR1 mutations. The manageable AE profile observed in the phase 1 portion of the study was maintained during cohort expansion at doses of 200 mg QD and 500 mg QD. Additional analyses are ongoing.Table. TRAEs reported in ≥10% of patients overall aNo grade 3/4 TRAE occurred in &amp;gt;1 patient. AST=aspartate aminotransferase Citation Format: Anne F. Schott, Sara Hurvitz, Cynthia Ma, Erika Hamilton, Rita Nanda, George Zahrah, Natasha Hunter, Antoinette R. Tan, Melinda Telli, Jesus Anampa Mesias, Rinath Jeselsohn, Pamela Munster, Haolan Lu, Richard Gedrich, Cecile Mather, Janaki Parameswaran, Hyo S. Han. GS3-03 ARV-471, a PROTAC® estrogen receptor (ER) degrader in advanced ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer: phase 2 expansion (VERITAC) of a phase 1/2 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS3-03.

A phase 2 expansion study of ARV-766, a PROTACandrogen receptor (AR) degrader, in metastatic castration-resistant prostate cancer (mCRPC).

TPS290 Background: Patients with mCRPC inevitably develop resistance to available therapies and lack curative options. In patients treated with novel hormonal agents (NHAs), mutations can develop in the ligand-binding domain (LBD) of the AR gene, some of which are associated with resistance to current therapies and disease progression. ARV-766 is a novel, potent, orally bioavailable proteolysis targeting chimera (PROTAC) protein degrader that degrades not only wild-type AR but also clinically relevant AR LBD mutants, including the most prevalent AR L702H, H875Y, and T878A mutations. Here we describe a phase 2 expansion study to evaluate the clinical activity and safety of ARV-766 in men with mCRPC who have experienced disease progression on prior NHA therapy. Methods: This phase 2 cohort expansion is part of an open-label, first-in-human, phase 1/2 clinical trial of ARV-766 in men (aged ≥18 years) with histologically, pathologically, or cytologically confirmed mCRPC and Eastern Cooperative Oncology Group performance status score of 0 or 1. Ongoing androgen deprivation therapy with a gonadotropin-releasing hormone analog or inhibitor or orchiectomy is required. Patients enrolled in the cohort expansion must have received 1–3 prior NHAs (eg, abiraterone or enzalutamide) and ≤2 prior chemotherapy regimens. Following completion of dose escalation in the phase 1 portion of the study, which is evaluating the safety and tolerability of ARV-766, 2 doses (100 mg and 300 mg administered orally once daily in 28-day cycles) were selected for the phase 2 cohort expansion. The primary objectives of the cohort expansion study are to evaluate the antitumor activity of ARV-766 based on the overall response rate (per Response Evaluation Criteria in Solid Tumors) and the rates of prostate-specific antigen (PSA) declines of 30% (PSA30) and 50% (PSA50). Enrollment in the phase 2 expansion study is ongoing. Clinical trial information: NCT05067140 .

Insights into the oligomeric structure of the HIV-1 Vpu protein

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in β-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

Protein degraders enter the clinic - a new approach to cancer therapy.

Heterobifunctional protein degraders, such as PROteolysis TArgeting Chimera (PROTAC) protein degraders, constitute a novel therapeutic modality that harnesses the cell's natural protein-degradation machinery-that is, the ubiquitin-proteasome system - to selectively target proteins involved in disease pathogenesis for elimination. Protein degraders have several potential advantages over small-molecule inhibitors that have traditionally been used for cancer treatment, including their event-driven (rather than occupancy-driven) pharmacology, which permits sub-stoichiometric drug concentrations for activity, their capacity to act iteratively and target multiple copies of a protein of interest, and their potential to target nonenzymatic proteins that were previously considered 'undruggable'. Following numerous innovations in protein degrader design and rigorous evaluation in preclinical models, protein degraders entered clinical testing in 2019. Currently, 18 protein degraders are in phase I or phase I/II clinical trials that involve patients with various tumour types, with a phase III trial of one initiated in 2022. The first safety, efficacy and pharmacokinetic data from these studies are now materializing and, although considerably more evidence is needed, protein degraders are showing promising activity as cancer therapies. Herein, we review advances in protein degrader development, the preclinical research that supported their entry into clinical studies, the available data for protein degraders in patients and future directions for this new class of drugs.

Increased ionic strength triggers multiple conformations in both apo and holo forms of bacterial ferric binding protein.

This study combines molecular dynamics (MD) simulations with solution X-ray scattering measurements to investigate the range of conformations that can be achieved by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations. To explore how the conformational multiplicity of proteins might be modified in the environmental niches of biological media, we focus on the periplasmic protein FbpA from H. influenzae which is a part of the mechanism developed by bacteria to capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its unique biological environment. The collected SAXS data complemented by SEC analyses and binding assays point to multiple conformations at physiological IS while they are well-explained by single x-ray structures in a 15 mM buffer. Moreover, by fitting the SAXS data with unique conformations sampled by a series of MD simulations carried out under conditions mimicking the buffers, we quantify with high accuracy the populations of the occupied substates. Furthermore, we find the D52A mutant that we predicted by coarse-grained computational approaches to allosterically control the iron binding site in FbpA responds to the environmental changes in our experiments with varied conformational selection scenarios. We show an application of the environment dependence of the conformers by designing a genetically encoded iron biosensor as a chimera of FbpA and green fluorescent protein using the D52 region as the insertion point. This work exemplifies how unifying a range of experimental and computational techniques provides platforms for achieving the next generation of applications that put protein dynamics rather than the static structure at the centerstage. Support by Turkish Atomic Energy Authority, TUBITAK grant no 121Z329 and a scholarship to GL through 2214-A program. Measurements conducted at EMBL-Hamburg.