Protein-like Structure Research Articles

Abstract 5940: JAB-30355: A highly potent, orally bioavailable p53-Y220C reactivator

Abstract Background: As a key tumor suppressor, p53 precisely regulates cellular events such as cell cycle arrest, apoptosis, senescence, and DNA repair under physiological conditions. In 50-60% of human cancers, TP53 gene is mutated. The TP53 Y220C is a hotspot loss-of-function mutation occurring in around 1% of solid tumors. Application of p53-Y220C reactivator for restoration of p53 function represents a promising treatment strategy for patients with this mutation. Methods: The Induced Allosteric Drug Discovery Platform (IADDP), which integrates medicinal chemistry, biochemical and biophysical studies, and computational techniques, was applied for the development of JAB-30355. The proportion of p53-Y220C with wild-type protein-like structure after JAB-30355 treatment was determined by immunoprecipitation. Thermal shift assay was applied to assess the thermal stability of JAB-30355-reactivated p53-Y220C protein. DNA binding assay was developed to evaluate the in vitro DNA binding activity. p53-luciferase reporter system was developed to evaluate the cellular transcriptional activity. qPCR and Western blotting were performed to evaluate the transcriptional and translational levels of p53 downstream targets, respectively. CellTiter-Glo assay was performed to evaluate the inhibitory activity of JAB-30355 on the viability of tumor cell lines harboring wild-type or Y220C mutated TP53. In vivo PK-PD study was conducted to evaluate the relationship between JAB-30355 concentration and expression of p53 target genes. Multiple CDX and PDX mouse models harboring TP53 Y220C were used to evaluate the antitumor activity of JAB-30355. Results: Immunoprecipitation assay and thermal shift assay results demonstrated that JAB-30355 efficiently restored the wild-type p53-like structure and enhanced protein stability of p53-Y220C in a dose-dependent manner. In addition, JAB-30355 significantly enhanced the DNA binding activity of p53-Y220C and subsequently increased the expression of p53 target genes, such as CDKN1A, MDM2 and PUMA. JAB-30355 inhibited cell viability of multiple TP53 Y220C-mutated cancer cell lines with IC50s ranging from 0.2 to 0.7 μM, and exhibited good selectivity against TP53 wild-type cells. Overall, the in vitro data demonstrated that JAB-30355 is a potent and selective p53-Y220C reactivator. In vivo PK-PD study showed good correlation between JAB-30355 exposure and activation of p53 target genes. Furthermore, JAB-30355 exhibited dose-dependent anti-tumor activity, inducing tumor stasis or regression in multiple CDX and PDX models of ovarian cancer, pancreatic cancer, gastric cancer, and small cell lung cancer, with overall good tolerability. Conclusions: JAB-30355 is a highly potent, selective, and orally bioavailable p53-Y220C reactivator that will be tested in clinical space soon. Citation Format: Qian Zheng, Peng Wang, Chen Liang, Yao Li, Xin Sun, Amin Li, Wei Zhang, Wei Long, Yanping Wang. JAB-30355: A highly potent, orally bioavailable p53-Y220C reactivator [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 5940.

Enzyme-mimic catalytic activities and biomedical applications of noble metal nanoclusters.

Noble metal (e.g., Au and Ag) nanoclusters (NCs), which exhibit structural complexity and hierarchy comparable to those of natural proteins, have been increasingly pursued in artificial enzyme research. The protein-like structure of metal NCs not only ensures enzyme-mimic catalytic activity, including peroxidase-, catalase-, and superoxide dismutase-mimic activities, but also affords an unprecedented opportunity to correlate the catalytic performance with the cluster structure at the molecular or atomic levels. In this review, we aim to summarize the recent progress in programming and demystify the enzyme-mimic catalytic activity of metal NCs, presenting the state-of-the-art understandings of the structure-property relationship of metal NC-based artificial enzymes. By leveraging on a concise anatomy of the hierarchical structure of noble metal NCs, we manage to unravel the structural origin of the catalytic performance of metal NCs. Noteworthily, it has been proven that the surface ligands and metal-ligand interface of metal NCs are instrumental in influencing enzyme-mimic catalytic activities. In addition to the structure-property correlation, we also discuss the synthetic methodologies feasible to tailoring the cluster structure at the atomic level. Prior to the closure of this review with our perspectives in noble metal NC-based artificial enzymes, we also exemplify the biomedical applications based on the enzyme-mimic catalysis of metal NCs with the theranostics of kidney injury, brain inflammation, and tumors. The fundamental and methodological advancements delineated in this review would be conducive to further development of metal NCs as an alternative family of artificial enzymes.

Effect of polyaspartic acid on the setting time and mechanical properties of α-hemihydrate gypsum

Polyaspartic acid (PASP) was an environment-friendly and biodegradable polymer, with a protein-like structure composed of L-aspartic acid residues. In this work, PASP with different molecular weights (2000, 4000–5000 and 6000–8000) was utilized as the retarder for α-hemihydrate gypsum (α-HH), and the effect of PASP on the coagulation, hydration and crystallization process of gypsum were investigated. Results showed that the addition of PASP with a low dosage of 0.01% efficiently delayed the hydration of α-HH and prolonged the setting process. Among three polyaspartic acids, PASP with molecular weight of 4000–5000 (PASP 4–5) exhibited the best performance not only in postponing the setting time to 95 min, but also in decreasing the negative impact of retarder on the strength of gypsum. Significantly, the slightest loss of 2.65% in compressive strength was obtained here when compared with other retarders previously reported, under the lowest dosage of 0.01% PASP 4–5. Consequently, the effect of PASP chain length on the retardation of α-HH was analyzed in detail. The conductivity, heat of hydration, X-ray diffraction (XRD), EDTA titration method and scanning electron microscopy (SEM) characterization found that PASP prevented the dissolution of α-HH. Total organic carbon analyzer (TOC) and X-ray photoelectron spectroscopy (XPS) measurements showed that the carboxyl groups of PASP adsorbed on the surface of α-HH to hinder the dissolution of α-HH. The molecular dynamic simulation of PASP further exhibited that three kinds of PASPs presented different conformations in supersaturated calcium sulfate solution due to the interaction between PASP and Ca2+ ions, and affected their solvent accessible surface area (SASA). In particular PASP 4–5 exhibited the smallest SASA and the strongest interaction with Ca2+ ions, causing the longest setting time of α-HH.

Thermosetting supramolecular polymerization of compartmentalized DNA fibers with stereo sequence and length control

Thermosetting supramolecular polymerization of compartmentalized DNA fibers with stereo sequence and length control

Poly(aspartic acid) in Biomedical Applications: From Polymerization, Modification, Properties, Degradation, and Biocompatibility to Applications.

Poly(aspartic acid) (PASP) is an anionic polypeptide that is a highly versatile, biocompatible, and biodegradable polymer that fulfils key requirements for use in a wide variety of biomedical applications. The derivatives of PASP can be readily tailored via the amine-reactive precursor, poly(succinimide) (PSI), which opens up a large window of opportunity for the design and development of novel biomaterials. PASP also has a strong affinity with calcium ions, resulting in complexation, which has been exploited for bone targeting and biomineralization. In addition, recent studies have further verified the biocompatibility and biodegradability of PASP-based polymers, which is attributed to their protein-like structure. In light of growing interest in PASP and its derivatives, this paper presents a comprehensive review on their synthesis, characterization, modification, biodegradation, biocompatibility, and applications in biomedical areas.

Polymers with controlled assembly and rigidity made with click-functional peptide bundles.

The engineering of biological molecules is a key concept in the design of highly functional, sophisticated soft materials. Biomolecules exhibit a wide range of functions and structures, including chemical recognition (of enzyme substrates or adhesive ligands1, for instance), exquisite nanostructures (composed of peptides2, proteins3 or nucleic acids4), and unusual mechanical properties (such as silk-like strength3, stiffness5, viscoelasticity6 and resiliency7). Here we combine the computational design of physical (noncovalent) interactions with pathway-dependent, hierarchical 'click' covalent assembly to produce hybrid synthetic peptide-based polymers. The nanometre-scale monomeric units of these polymers are homotetrameric, α-helical bundles of low-molecular-weight peptides. These bundled monomers, or 'bundlemers', can be designed to provide complete control of the stability, size and spatial display of chemical functionalities. The protein-like structure of the bundle allows precise positioning of covalent linkages between the ends of distinct bundlemers, resulting in polymers with interesting and controllable physical characteristics, such as rigid rods, semiflexible or kinked chains, and thermally responsive hydrogel networks.Chain stiffness can be controlled by varying only the linkage. Furthermore, by controlling the amino acid sequence along the bundlemer periphery, we use specific amino acid side chains, including non-natural 'click' chemistry functionalities, to conjugate moieties into a desired pattern, enabling the creation of a wide variety of hybrid nanomaterials.

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments.

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8M urea (pH8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

Sequence Programmable Peptoid Polymers for Diverse Materials Applications.

Polymer sequence programmability is required for the diverse structures and complex properties that are achieved by native biological polymers, but efforts towards controlling the sequence of synthetic polymers are, by comparison, still in their infancy. Traditional polymers provide robust and chemically diverse materials, but synthetic control over their monomer sequences is limited. The modular and step-wise synthesis of peptoid polymers, on the other hand, allows for precise control over the monomer sequences, affording opportunities for these chains to fold into well-defined nanostructures. Hundreds of different side chains have been incorporated into peptoid polymers using efficient reaction chemistry, allowing for a seemingly infinite variety of possible synthetically accessible polymer sequences. Combinatorial discovery techniques have allowed the identification of functional polymers within large libraries of peptoids, and newly developed theoretical modeling tools specifically adapted for peptoids enable the future design of polymers with desired functions. Work towards controlling the three-dimensional structure of peptoids, from the conformation of the amide bond to the formation of protein-like tertiary structure, has and will continue to enable the construction of tunable and innovative nanomaterials that bridge the gap between natural and synthetic polymers.

Protein backbone engineering as a strategy to advance foldamers toward the frontier of protein-like tertiary structure.

A variety of non-biological structural motifs have been incorporated into the backbone of natural protein sequences. In parallel work, diverse unnatural oligomers of de novo design (termed "foldamers") have been developed that fold in defined ways. In this Perspective article, we survey foundational studies on protein backbone engineering, with a focus on alterations made in the context of complex tertiary folds. We go on to summarize recent work illustrating the potential promise of these methods to provide a general framework for the construction of foldamer mimics of protein tertiary structures.

On the role of physics and evolution in dictating protein structure and function.

How many of the structural and functional properties of proteins are inherent? Computer simulations provide a powerful tool to address this question. A series of studies on QS, quasi-spherical, compact polypeptides which lack any secondary structure; ART, artificial, proteins comprised of compact homopolypeptides with protein-like secondary structure; and PDB, native, single domain proteins shows that essentially all native global folds, pockets and protein-protein interfaces are in the ART library. This suggests that many protein properties are inherent and that evolution is involved in fine-tuning. The completeness of the space of ligand binding pockets and protein-protein interfaces suggests that promiscuous interactions are intrinsic to proteins and that the capacity to perform the biochemistry of life at low level does not require evolution. If so, this has profound consequences for the origin of life.

An injectable and biodegradable hydrogel based on poly(α,β‐aspartic acid) derivatives for localized drug delivery

An injectable hydrogel via hydrazone cross-linking was prepared under mild conditions without addition of cross-linker or catalyst. Hydrazine and aldehyde modified poly(aspartic acid)s were used as two gel precursors. Both of them are water-soluble and biodegradable polymers with a protein-like structure, and obtained by aminolysis reaction of polysuccinimide. The latter can be prepared by thermal polycondensation of aspartic acid. Hydrogels were prepared in PBS solution and characterized by different methods including gel content and swelling, Fourier transformed-infrared spectroscopy, and in vitro degradation experiment. A scanning electron microscope viewed the interior morphology of the obtained hydrogels, which showed porous three-dimensional structures. Different porous sizes were present, which could be well controlled by the degree of aldehyde substitution in precursor poly(aspartic acid) derivatives. The doxorubicin-loaded hydrogels were prepared and showed a pH-sensitive release profile. The release rate can be accelerated by decreasing the environmental pH from a physiological to a weak acidic condition. Moreover, the cell adhesion and growth behaviors on the hydrogel were studied and the polymeric hydrogel showed good biocompatibility.

Layer-by-layer assembled polyaspartamide nanocapsules for pH-responsive protein delivery

Biodegradable shell cross-linked nanocapsules were prepared via layer-by-layer assembly of PADH (tertiary amine and hydrazide grafted polyaspartamide) and PACA (carboxyl and aldehyde grafted polyaspartamide) on silica spheres. Both of the polyaspartamide derivatives are water-soluble and biodegradable polymers with a protein-like structure, and obtained by aminolysis reaction of polysuccinimide. The latter is prepared by thermal polycondensation of aspartic acid. Dynamic light scattering and zeta potential measurements were used to analyze the layer-by-layer assembly process. Bovine serum albumin (BSA), as a model protein, was entrapped in the nanocapsules via electrostatic adsorption. Nanocapsules encapsulating BSA were prepared via layer-by-layer assembly on protein-entrapping amino-functionalized silica spheres, hydrazone cross-linking and silica core removal. The BSA release profiles exhibited a pH-dependent behavior. BSA release rate increased significantly as the ambient pH dropped from the physiological pH to acidic. Cell viability study suggests that the obtained polymeric nanocapsules have good biocompatibility. These kinds of novel composite nanocapsules may offer a promising delivery system for proteins.

Fullerene Derivative as Anti-HIV Protease Inhibitor: Molecular Modeling and QSAR Approaches

A Fullerene based system is modified in order to increase its solubility and enhance its ability to carry a protein-like structure. The modified structure, which is proposed to act as HIV-1 protease inhibitor, is [C₆₀-C2H4N-(2,4- XCOCH₂OH)C₆H₄], where the X atom is either O, S or Se. The geometry optimization, vibrational spectra and thermodynamics were performed using semiempirical quantum mechanical PM3 method in order to study the proposed compounds. Furthermore, the quantitative structure activity relationship (QSAR) properties of the compounds are calculated at the same level of theory. Results indicate a possible use of the investigated structures as HIV-1 protease inhibitors. The compounds containing oxygen is more stable as compared to the other two compounds.

Complexion between mercury and humic substances from different landfill stabilization processes and its implication for the environment

Three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy was employed to investigate the structural properties and Hg(II)-binding behavior of humic substances (HS) extracted from different landfill stabilization processes. The EEM fluorescence properties of humic acid (HA) are characterized by intense fluorescence at Ex/Em=440/500nm and Ex/Em=380/460nm. Two relatively strong fluorescence peaks appeared in the region of Ex/Em=260–290/350–370nm with the landfill time extended, which represented a protein-like or soluble microbial byproduct structure. The fluorescence EEM spectrum of fulvic acid (FA) featured a prominent peak of strong relative fluorescence intensity (FI=1598) at Ex/Em=330/440nm (peak C) accompanied by a weak fluorophore (FI=594) located at Ex/Em=275/445nm (peak D). There were strong interactions between HA and Hg, and the overall stability constant of Hg(II)–HA was mainly determined by the abundant O-ligands existing in HA. FA had a much higher Hg(II)-complexing capacity compared to HA samples, which may be ascribed to its relatively high content of carboxylic groups. The Hg(II)-complexing capacity of HA tended to decrease with stabilization process extension. The much higher Hg(II)-complexing capacity of FA than that of HA implied that FA played an important role in binding Hg(II) in early landfill stabilization process.

Mammalian cell protein expression for biopharmaceutical production

Mammalian cell expression has become the dominant recombinant protein production system for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly. While expression and production have been fully developed and Chinese hamster ovary cells are used for the majority of products both on the market and in clinical development, significant progresses in developing and engineering new cell lines, introducing novel genetic mechanisms in expression, gene silencing, and gene targeting, have been reported in the last several years. With the latest analytical methods development, more attention is being devoted towards product quality including glycol profiling, which leads to better understanding the impact of culture condition during production. Additionally, transient gene expression technology platform plays more important role in biopharmaceutical early development stages. This review focused on the latest advancements in the field, especially in active areas such as expression systems, glycosylation impact factors, and transient gene expression.

Macromolecular Prodrugs Based on Synthetic Polyaminoacids: Drug Delivery and Drug Targeting in Antitumor Therapy

In the last twenty years a depth study on potential pharmaceutical applications of synthetic polymers at proteinlike structure as carrier for macromolecular prodrug production has been performed in academia and in industry. In particular α,β-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA), α,β-polyaspartylhydrazide (PAHy), poly(glutamic acid) (PGA), poly(aspartic acid) (PAA) and polylysine (PLL) have been extensively studied in this field. In the present review, the use of PHEA, PAHy, PGA as starting materials to prepare macromolecular prodrugs is reported and drug delivery and targeting aspects have been considered.

Nonclinical Safety Assessment of a Synthetic Peptide Thrombopoietin Agonist

RWJ-800088 is a novel, potent polyethylene glycol (PEG)-conjugated thrombopoietin (TPO) mimetic that increases platelet levels and protects against thrombocytopenia. A nonclinical safety program was customized for this peptide that takes into account its protein-like structure, synthetic chemical nature, agonist pharmacologic activity, and mode of administration. In repeat-dose toxicity studies, the salient findings were dose-related increases in circulating platelet counts, mean platelet volume, and megakaryocytes in the bone marrow with no antibody formation. Reversible myelofibrosis and hyperostosis were observed in rats, but not dogs, when the circulating platelet levels exceeded 3× those of vehicle controls. The bone effects were due to the exaggerated pharmacologic effect and excessive stimulation and elevation of megakaryocytes by TPO, which results in intramedullary proliferation of fibroblasts and mesenchymal cells followed by osseous metaplasia. These findings support the use of platelet elevations of >3× as a stopping criterion to prevent potential adverse bone-related effects in humans.

Protein Mimetic Materials from the Self Assembly of Bioinspired Polymers at the Air Water Interface

Peptoids are a class of non-natural biopolymer based on an N-substituted glycine backbone that are ideally suited for protein mimetic research. This bio-inspired material has many unique properties that bridge the gap between proteins and bulk polymers. Like proteins, they are a sequence-specific heteropolymer, capable of folding into specific shapes and exhibiting potent biological activities; and like bulk polymers they are chemically and biologically stable and relatively cheap to make. Peptoids are efficiently assembled via automated solid-phase synthesis from hundreds of chemically diverse building blocks allowing the rapid generation of huge combinatorial libraries. This provides an ideal platform to discover nanostructured materials capable of protein-like structure and function. We have designed a series of amphiphilic peptoid 36mers of specific sequence that self assemble into extremely thin (3 nm) crystalline sheets in aqueous solution. A key intermediate in their formation is an ordered monolayer at the air-water interface. These peptoid nanosheets are one of the thinnest two-dimensional organic crystalline materials known. Because of their atomically defined polar surface, the nanosheets serve as an excellent platform to design chem/bio sensors capable of specific molecular recognition.

Peptoid origins

Peptoid oligomers were initially developed as part of a larger basic research effort to accelerate the drug-discovery process in the biotech/biopharma industry. Their ease of synthesis, stability, and structural similarity to polypeptides made them ideal candidates for the combinatorial discovery of novel peptidomimetic drug candidates. Diverse libraries of short peptoid oligomers provided one of the first demonstrations in the mid-1990s that high-affinity ligands to pharmaceutically relevant receptors could be discovered from combinatorial libraries of synthetic compounds. The solid-phase submonomer method of peptoid synthesis was so efficient and general that it soon became possible to explore the properties of longer polypeptoid chains in a variety of areas beyond drug discovery (e.g., diagnostics, drug delivery, and materials science). Exploration into protein-mimetic materials soon followed, with the fundamental goal of folding a non-natural sequence-specific heteropolymer into defined secondary or tertiary structures. This effort first yielded the peptoid helix and much later the peptoid sheet, both of which are secondary-structure mimetics that are close relatives to their natural counterparts. These crucial discoveries have brought us closer to building proteinlike structure and function from a non-natural polymer and have provided great insight into the rules governing polymer and protein folding. The accessibility of peptoid synthesis to chemists and nonchemists alike, along with a lack of information-rich non-natural polymers available to study, has led to a rapid growth in the field of peptoid science by many new investigators. This work provides an overview of the initial discovery and early developments in the peptoid field.