Systematic Evaluation of Protein-Small Molecule Hybrids on the Yeast Surface.

Protein-small molecule hybrids are structures that have the potential to combine the inhibitory properties of small molecules and the specificities of binding proteins. However, achieving such synergies is a substantial engineering challenge with fundamental principles yet to be elucidated. Recent work has demonstrated the power of the yeast display-based discovery of hybrids using a combination of fibronectin-binding domains and thiol-mediated conjugations to introduce small-molecule warheads. Here, we systematically study the effects of expanding the chemical diversity of these hybrids on the yeast surface by investigating a combinatorial set of fibronectins, noncanonical amino acid (ncAA) substitutions, and small-molecule pharmacophores. Our results show that previously discovered thiol-fibronectin hybrids are generally tolerant of a range of ncAA substitutions and retain binding functions to carbonic anhydrases following click chemistry-mediated assembly of hybrids with diverse linker structures. Most surprisingly, we identified several cases where replacement of a potent acetazolamide warhead with a substantially weaker benzenesulfonamide warhead still resulted in the assembly of multiple functional hybrids. In addition to these unexpected findings, we expanded the throughput of our system by validating a 96-well plate-based format to produce yeast-displayed hybrid conjugates in parallel. These efficient explorations of hybrid chemical diversity demonstrate that there are abundant opportunities to expand the functions of protein-small molecule hybrids and elucidate principles that dictate their efficient discovery and design.

Enhancing arsenic adsorptions by optimizing Fe-loaded biochar and preliminary application in paddy soil under different water management strategies.

Arsenic (As) is widely distributed in nature and is a highly toxic element impacting human health through drinking water and rice. In this study, an optimized approach was attempted to improve As adsorption capabilities by combining pre- and post-pyrolysis modification of Fe(oxy)hydroxides to rice husk biochar (FRB), of which the method is rarely addressed in previous studies. Maghemite and goethite were successfully loaded onto biochar, characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoemission spectroscopy (XPS) analyzer. The FRB had maximum As(III) and As(V) adsorption capabilities of 7908 and 11,268 mg/kg, respectively, which was significantly higher than that of Fe-modified biochar in the pre-pyrolysis and/or post-pyrolysis process. Adsorption mechanisms for As explored by Fourier-transform infrared spectroscopy (FTIR), XPS analysis mainly included electronic attraction and ligand exchange with hydroxyl groups on the FRB. It was noteworthy that more than half of the As(II) species loaded on FRB were converted into less toxic As(V) species, which could be mediated by the redox-active groups on the biochar. The preliminary application of FRB in soil indicated that it has an effective remediation potential for As-contaminated soil under flooded conditions, while promoted As release under dry conditions. Finding of this study highlighted that the loading of metal oxides onto biochar by combining pre- and post-pyrolysis modification could potentially increase As adsorption capabilities and further help in strategic water management.

Chemical Diversification of Simple Synthetic Antibodies.

Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support the isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the third complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled the utilization of additional CDRs for the installation of chemically reactive and photo-cross-linkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without the abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without the apparent disruption of binding. Finally, antibodies substituted with a photo-cross-linkable ncAA were evaluated for ultraviolet-mediated cross-linking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful cross-linking. Key findings regarding CuAAC reactions and photo-cross-linking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface open up new possibilities for discovering "drug-like" protein leads in high throughput.

Future prospects for noncanonical amino acids in biological therapeutics.

There is growing evidence that noncanonical amino acids (ncAAs) can be utilized in the creation of biological therapeutics ranging from protein conjugates to cell-based therapies. However, when does genetically encoding ncAAs yield biologics with unique properties compared to other approaches? In this review, we attempt to answer this question in the broader context of therapeutic development, emphasizing advances within the past two years. In several areas, ncAAs add valuable routes to therapeutically relevant entities, but application-specific needs ultimately determine whether ncAA-mediated or alternative solutions are preferred. Looking forward, using ncAAs to perform 'protein medicinal chemistry,' in which atomic-level changes to proteins dramatically enhance therapeutic properties, is a promising emerging area. Further upgrades to the performance of ncAA incorporation technologies will be essential to realizing the full potential of ncAAs in biological therapeutics.

Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins.

The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as 'enhancers' and at least one class of mitigator to be avoided.