Protein Drug Development Research Articles

LM-GVP: an extensible sequence and structure informed deep learning framework for protein property prediction

Proteins perform many essential functions in biological systems and can be successfully developed as bio-therapeutics. It is invaluable to be able to predict their properties based on a proposed sequence and structure. In this study, we developed a novel generalizable deep learning framework, LM-GVP, composed of a protein Language Model (LM) and Graph Neural Network (GNN) to leverage information from both 1D amino acid sequences and 3D structures of proteins. Our approach outperformed the state-of-the-art protein LMs on a variety of property prediction tasks including fluorescence, protease stability, and protein functions from Gene Ontology (GO). We also illustrated insights into how a GNN prediction head can inform the fine-tuning of protein LMs to better leverage structural information. We envision that our deep learning framework will be generalizable to many protein property prediction problems to greatly accelerate protein engineering and drug development.

Ion-Mediated Morphological Diversity in Protein Amyloid Systems.

Salt ions are considered among the major determinants ruling protein folding, stability, and self-assembly in the context of amyloid-related diseases, protein drug development, and functional biomaterials. Here, we report that Hofmeister ions not only determine the rate constants of the aggregation reaction for human insulin and hen egg white lysozyme but also control the generation of a plethora of amyloid-like morphologies ranging from the nanoscale to the microscale. We anticipate that the latter is a result of a balance between colloidal and conformational stability combined with an ion-specific effect and highlight the importance of salt ions in controlling the biological functions of protein aggregates.

The role of PI3Kγ in the immune system: new insights and translational implications.

Over the past two decades, new insights have positioned phosphoinositide 3-kinase-γ (PI3Kγ) as a context-dependent modulator of immunity and inflammation. Recent advances in protein structure determination and drug development have allowed for generation of highly specific PI3Kγ inhibitors, with the first now in clinical trials for several oncology indications. Recently, a monogenic immune disorder caused by PI3Kγ deficiency was discovered in humans and modelled in mice. Human inactivated PI3Kγ syndrome confirms the immunomodulatory roles of PI3Kγ and strengthens newly defined roles of this molecule in modulating inflammatory cytokine release in macrophages. Here, we review the functions of PI3Kγ in the immune system and discuss how our understanding of its potential as a therapeutic target has evolved.

Subvisible Particles Derived by Dropping Stress Enhance Anti-PEG Antibody Production and Clearance of PEGylated Proteins in Mice

Bioconjugation with polyethylene glycol (PEG) is important for protein drug development as it has improved biological stability. In contrast, proteins including PEGylated ones are susceptible to physicochemical stresses. Particularly, protein drugs in solution may form aggregates or subvisible particles if they are exposed to dropping stress during transportation. However, many PEGylation studies have focused on its usefulness, such as the extension of half-life in blood, and changes in the physical properties or biological responses of PEGylated proteins under dropping stress remain unexplored. Here, we prepared four PEGylated ovalbumin (PEG-OVA) molecules conjugated with different lengths (5 or 20 kDa) and numbers (large [L] or small [S]) of PEG, analyzed the formation of subvisible particles under dropping stress, and examined their impact on antibody production and clearance. Under dropping stress, the aggregated particle concentration of 20 kDa PEG-OVA (S) and (L) solutions was approximately 3-fold that of the OVA solution. Moreover, administration of 20 kDa PEG-OVA with dropping stress induced anti-PEG antibody production and clearance of PEG-OVA. As a mechanism, dropping stress could enhance the uptake of 20 kDa PEG-OVA (L) by macrophages. These findings could provide insights into proper transportation conditions to ensure the quality of PEGylated protein drugs.

Fully automated virtual screening pipeline of FDA-approved drugs using Caver Web

Protein tunnels are essential in transporting small molecules into the active sites of enzymes. Tunnels' geometrical and physico-chemical properties influence the transport process. The tunnels are attractive hot spots for protein engineering and drug development. However, studying the ligand binding and unbinding using experimental techniques is challenging, while in silico methods come with their limitations, especially in the case of resource-demanding virtual screening pipelines. Caver Web 1.2 is a new version of the web server combining the capabilities for the detection of protein tunnels with the calculation of the ligand trajectories. The new version of the Caver Web server was expanded with the ability to fetch novel ligands from the Integrated Database of Small Molecules and with the fully automated virtual screening pipeline allowing for the fast evaluation of the predefined set of over 4,300 currently approved drugs. The virtual screening pipeline is accompanied by a comprehensive user interface, making it a viable service for the broader spectrum of companies and the academic user community. The web server is freely available for academic use at https://loschmidt.chemi.muni.cz/caverweb.

Catalytically active holo Homo sapiens adenosine deaminase I adopts a closed conformation.

Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells

BackgroundThe various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli.ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity.ConclusionsReengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Multifractal and cross-correlation analysis on mitochondrial genome sequences using chaos game representation

We characterized the multifractality and power-law cross-correlation of mitochondrial genomes of various species through the recently developed method which combines the chaos game representation theory and 2D-multifractal detrended cross-correlation analysis. In the present paper, we analyzed 32 mitochondrial genomes of different species and the obtained results show that all the analyzed data exhibit multifractal nature and power-law cross-correlation behaviour. Further, we performed a cluster analysis from the calculated scaling exponents to identify the class affiliation and its outcome is represented as a dendrogram. We suggest that this integrative approach may help the researchers to understand the phylogeny of any kingdom with their varying genome lengths and also this approach may find applications in characterizing the protein sequences, mRNA sequences, next-generation sequencing, and drug development, etc.

Abstract 199: Blossom AI: A novel drug discovery app for the prediction of hotspots on multiplex protein protein interaction complexes using random forest algorithms

Abstract Protein protein interactions (PPIs) form the backbone of signal transduction pathways in diverse physiological processes, mediating the transmission and regulation of oncogenic signals essential to cellular proliferation and survival, thus representing a potential new class of drug targets for anticancer therapeutic discovery. However, several challenges face the targeting of PPIs, including large PPI interface areas, a lack of deep pockets, the presence of noncontiguous binding sites, and a general lack of natural ligands. The presence of hot spots (small subsets of amino acid residues that contribute significantly to free binding energy) makes PPIs amenable to small molecule perturbations, playing essential roles in the stability of protein binding. Effectively identifying which specific interface residues of protein protein complexes form the hot spots is critical for understanding the principles of protein interactions and has broad application prospects in protein design and drug development. This project presents Blossom AI, a novel, user friendly mobile app developed in XCode and CoreML that uses random forest decision tree algorithms (RF) to computationally predict the presence of hotspots on protein complexes within seconds, aiding the design of small molecule and peptide drugs that target protein-protein interactions, particularly for anticancer therapy. Leveraging features such as solvent accessible surface area (ASA), blocks substitution matrix, physicochemical properties (hydrophobicity, polarity, polarizability, propensities), position specific scoring matrix (PSSM) and solvent exposure, the RF is trained through a dataset of 313 mutated interface residues (133 hotspot residues and 180 non hotspot residues) from over 60 protein complexes to produce a training accuracy of 88.75%, validation accuracy of 92.86%, specificity of 87.18%, sensitivity of 75.38%, PPV 94.23%, NPV 86.61%. Blossom is high speed, low cost, and user friendly with significantly improved accuracy over the standard of alanine scanning mutagenesis. Citation Format: Stephanie Zhang, Minsoo Kang. Blossom AI: A novel drug discovery app for the prediction of hotspots on multiplex protein protein interaction complexes using random forest algorithms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 199.

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.

Molecular Elucidation of a Urate Oxidase from Deinococcus radiodurans for Hyperuricemia and Gout Therapy.

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg2+ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.

Design of Polymers for Intracellular Protein and Peptide Delivery

AbstractCytosolic protein delivery techniques are of great importance for cell biology, biotechnology and protein drug development. The design of carriers with robust efficiency in cytosolic protein delivery is challenging. This account provides a progress report of polymeric carriers for this purpose in our group. During the past years, we have developed several types of functionalized polymers for cytosolic protein and peptide delivery by engineering polymers with ligands such as guanidinium, boronate, coordination ligands and fluoroalkyls. The designed polymers showed improved protein/peptide binding affinities, and successfully delivered various cargo proteins into the cytosol of living cells, while maintaining their bioactivity. In addition, the polymers showed potent efficiencies in the delivery of tumor antigens, therapeutic peptides, toxins and antioxidant proteins in vivo. We hope these polymers could be translated for protein delivery in the treatment of various diseases in the future. What is the most favorite and original chemistry developed in your research group?Fluorinated polymers for cytosolic biomolecule delivery.How do you get into this specific field? Could you please share some experiences with our readers?I get into this field by unexpected discoveries when designing polymeric gene delivery systems. Usually, modification of polymers with hydrophobic ligands yields more efficient gene carriers. Fluoroalkyl ligands wereproposed as one type of hydrophobic candidates in our experiments. Interestingly, the fluorous ligand modified polymers showed unprecedented efficiency in gene delivery. More importantly, the polymers exhibited excellent serum resistance and could achieve high efficiency at extremely low polymer dose. These unexpected discoveries motivated us to investigate the fluorine effect of fluorinated materials in gene, protein and peptide delivery.

Facile synthesis of mesoporous copper silicate aggregates for highly selective enrichment of hemoglobin

The purification of histidine-rich proteins in complex biological samples has been an important topic for proteomics research and protein drug development. In order to achieve high selectivity and binding capacity for histidine-rich protein, novel mesoporous material of copper silicate have been synthesized via in-situ growth method with dendritic mesoporous silica as the template in a hydrothermal treatment process. The resulted materials maintain large pore size with ample adsorption sites. The maximum adsorption capacity for bovine hemoglobin is 4150 mg g−1. In addition, the formed mesoporous copper silicate aggregates showed excellent performance for the removal of highly abundant hemoglobin in human blood, permitting their potential applications in the pretreatment of complex biological samples.

Barcode signal amplifying strategy for sensitive and accurate protein detection on LC-MS/MS.

Protein drugs showing strong pharmaceutical activity, high specificity, and low toxicity and side effects have drawn extensive attention in the field of life sciences and medicine. Precise evaluation of the function of these drugs requires accurate and sensitive detection methods. Here, we report a novel chromatography-tandem mass spectrometry (LC-MS/MS) method for sensitive and selective detection of protein drugs. Magnetic nanoparticles (Apt29@MNPs) were functionalized by thrombin aptamers, and quantum dots (Apt15@ss@QDs) were dual-functionalized with quantitative thrombin aptamers and small molecules with high ionization efficiency as the mass barcode. After Apt29@MNPs specifically purify and enrich thrombin from biological samples, they can form a nano "sandwich structure" when Apt15@ss@QDs are added, resulting in the release of the mass barcode for LC-MS/MS analysis via the cutting of the disulfide bond. Since there is a higher quantitative molecular ratio of mass barcode to thrombin in the nano-"sandwich structure", quantitative detection of thrombin with high sensitivity and selectivity can be achieved via the LC-MS/MS detection of the mass barcode with high ionization efficiency rather than thrombin, which effectively avoids the disadvantages of direct protein detection by mass spectrometry. The established method for thrombin detection shows a good linear relationship in a concentration range of 0.00115-1.15 nM with a limit of detection (LOD) of 0.0007 nM. The present work provides a new approach for the effective and sensitive quantitative analysis of protein drugs and would be of great significance in promoting the development of protein drugs and clinical applications.

Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target the E-box DNA Site.

Protein-based therapeutics are part of the next-generation arsenal of drugs being developed against proto-oncoprotein Myc. We designed protein MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of Max and Myc, which bind to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. We used phage-assisted continuous evolution (PACE), which uncovered mutations at Arg12 that contact the DNA phosphodiester backbone. The Arg12 mutations improved ME47's stability. We replaced Cys29 with Ala to eliminate potential undesired disulfide formation and fused the designed FosW leucine zipper to mutated ME47 to increase the dimerization interface and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper. Compared with ME47, MEF gives 2-fold stronger binding to E-box and 4-fold increased specificity for E-box over nonspecific DNA. The synergistic combination of rational design and PACE allowed us to make MEF and demonstrates the power and utility of our two-pronged approach toward development of promising protein drugs with robust structure and DNA-binding function.

A Review of Bioactive Peptides: Chemical Modification, Structural Characterization and Therapeutic Applications.

In recent years, the development and applications of protein drugs have attracted extensive attention from researchers. However, the shortcomings of protein drugs also limit their further development. Therefore, bioactive peptides isolated or simulated from protein polymers have broad application prospects in food, medicine, biotechnology, and other industries. Such peptides have a molecular weight distribution between 180 and 1000 Da. As a small molecule substance, bioactive peptide is usually degraded by various enzymes in the organism and have a short half-life. At the same time, such substances have poor stability and are difficult to produce and store. Therefore, these active peptides may be modified through phosphorylation, glycosylation, and acylation. Compared with other protein drugs, the modified active peptides are more easily absorbed by the body, have longer half-life, stronger targeting, and fewer side effects in addition to higher bioavailability. In the light of their functions, bioactive peptide can be divided into antimicrobial, anti-tumour, anti-angiogenic, antioxidant, anti-fatigue, and anti-hypertensive peptides. This article mainly focuses on the introduction of several promising biologically active peptides functioning as antimicrobial, anti-tumour, antiangiogenic, and antioxidant peptides from the three aspects modification, structural characteristics and mechanism of action.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

Transdermal peptide conjugated to human connective tissue growth factor with enhanced cell proliferation and hyaluronic acid synthesis activities produced by a silkworm silk gland bioreactor.

Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide that stimulates cell proliferation, migration, and extracellular matrix production during tissue development, differentiation, angiogenesis, implantation, wound healing, and fibrosis processes, with broad application in the medical and cosmetic medical fields. However, the production of CTGF is currently limited by its low yield and purity in current bioreactors. In this study, two genetically modified silkworm strains were generated harboring artificially designed CTGF-8ht and pepCTGF-8ht genes, respectively, that contain an enhanced His-tag with eight histidine residues with or without a transdermal peptide (pep). Both recombinant CTGF-8ht and pepCTGF-8ht proteins were successfully expressed in the silkworm silk gland and cocoon, and could be easily extracted and purified from the cocoon by single-affinity immunoprecipitation column chromatography, achieving a purity of more than 95%. Moreover, compared with CTGF-8ht protein, pepCTGF-8ht protein exhibited better cell proliferation activity by activating the extracellular signal-regulated kinase (ERK) pathway and enhanced hyaluronic acid synthesis activity by upregulating hyaluronan synthase 3 expression; moreover, the addition of pep significantly improved the transmembrane ability of CTGF-8ht protein. These results should help to promote the application prospects of CTGF and further guide the design and development of protein drugs from silkworm and other bioreactor systems. KEY POINTS : A silkworm bioreactor was optimized to produce connective tissue growth factor (CTGF) The transgene contained an enhanced 8-His-tag and transmembrane peptide (pep) Recombinant CTGF was easily purified with maintained or higher biological activity.

Improved prediction of HLA antigen presentation hotspots: Applications for immunogenicity risk assessment of therapeutic proteins.

Immunogenicity risk assessment is a critical element in protein drug development. Currently, the risk assessment is most often performed using MHC-associated peptide proteomics (MAPPs) and/or T-cell activation assays. However, this is a highly costly procedure that encompasses limited sensitivity imposed by sample sizes, the MHC repertoire of the tested donor cohort and the experimental procedures applied. Recent work has suggested that these techniques could be complemented by accurate, high-throughput and cost-effective prediction of in silico models. However, this work covered a very limited set of therapeutic proteins and eluted ligand (EL) data. Here, we resolved these limitations by showcasing, in a broader setting, the versatility of in silico models for assessment of protein drug immunogenicity. A method for prediction of MHC class II antigen presentation was developed on the hereto largest available mass spectrometry (MS) HLA-DR EL data set. Using independent test sets, the performance of the method for prediction of HLA-DR antigen presentation hotspots was benchmarked. In particular, the method was showcased on a set of protein sequences including four therapeutic proteins and demonstrated to accurately predict the experimental MS hotspot regions at a significantly lower false-positive rate compared with other methods. This gain in performance was particularly pronounced when compared to the NetMHCIIpan-3.2 method trained on binding affinity data. These results suggest that in silico methods trained on MS HLA EL data can effectively and accurately be used to complement MAPPs assays for the risk assessment of protein drugs.

Albumin displacement at the air\u2013water interface by Tween (Polysorbate) surfactants

Tween (polysorbate) 20 and 80 are surfactants used for the development of parenteral protein drugs, due to their beneficial safety profile and stabilisation properties. To elucidate the mechanism by which Tween 20 and 80 stabilise proteins in aqueous solutions, either by a “direct” protein to surfactant interaction and/or by an interaction with the protein film at the air–water interface, we used spectroscopic (Infrared Reflection Absorption Spectroscopy, IRRAS) and microscopic techniques (Brewster Angle Microscopy, BAM) in combination with surface pressure measurements. To this end, the impact of both types of Tweens with regard to the displacement of the protein from the air–water interface was studied. As a model protein, human serum albumin (HSA) was used. The results for the displacement of the adsorbed HSA films by Tweens 20 and 80 can partially be understood on the basis of an orogenic displacement mechanism, which depends on the critical surface pressure of the adsorbed protein film. With increasing concentration of Tween in the sub-phase, BAM images showed the formation of different domain morphologies. IRRA-spectra supported the finding that at high protein concentration in the sub-phase, the protein film could not be completely displaced by the surfactants. Comparing the impact of both surfactants, we found that Tween 20 adsorbed faster to the protein film than Tween 80. The adsorption kinetics of both Tweens and the speed of protein displacement increased with rising surfactant concentration. Tween 80 reached significant lower surface pressures than Tween 20, which led to an incomplete displacement of the observed HSA film.