Biology subjects

Siegel, J. B.

Publications and source records attributed to Siegel, J. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Computationally Guided Design of Two Novel Phosphodiesterase-4 Inhibitors for Treatment of Plaque Psoriasis

Plaque psoriasis is a chronic inflammatory skin disease characterized by itchy, scaly skin. Although there is currently no cure for this disease, it can be managed with medication, such as phosphodiesterase-4 (PDE4) inhibitors. Current PDE4 inhibitors are limited and show adverse effects. With the goal to develop better PDE4 inhibitors for the treatment of plaque psoriasis, two novel drug candidates were designed with computational studies, and observed to have better ADMET properties compared to Roflumilast. A homology analysis was performed to determine the best model organism for preclinical studies of the novel candidates.

pharmacology and toxicology↗

bioRxiv2026

De novo Rubisco design with protein language models

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fixes the majority of carbon dioxide globally but is challenged with low specificity for CO2 versus O2 and low catalytic efficiencies. Traditional engineering efforts have remained difficult because folding, assembly, specificity, and catalysis are tightly coupled, hampering efforts to explore sequence space. Therefore, we leveraged recent advances in protein large language models (PLMs) to generate sequences beyond those observed in nature, using both ProGen-2 that was fine-tuned on a limited dataset of non-Form I Rubiscos and an ESM-2 discriminator. With this approach, we generated 5.6 million novel Rubisco-like sequences and identified 21 highly diverse candidates predicted to be active that occupy regions of Rubisco phylogenetic space not previously observed in nature. Six designs were soluble in Escherichia coli, and five were shown to produce quantifiable 3PGA. One design produced an apparent CO2/O2 specificity estimate beyond the range of the natural representative Rubiscos assayed. We also solved the crystal structure of one de novo design that reproduced the predicted dimer and active-site geometry with sub-angstrom C agreement. Sequence-only generation followed by independent structural filtering therefore recovered soluble, active Rubiscos from regions of sequence space that are not represented in genomic databases. Together, these results establish a scalable strategy for accessing previously unexplored Rubisco sequence space, providing a broadly accessible path toward generating de novo Rubiscos that may have activity and specificity parameters needed to address longstanding limitations in biological carbon fixation.

biochemistry↗

bioRxiv2026

Computational Design of Two Novel BRAF V600E Inhibitors: Exploiting Sulfoximine Bioisosterism and Chiral Constraints to Evade Paradoxical Activation

Metastatic melanoma is an aggressive cutaneous malignancy frequently driven by the oncogenic V600E mutation within the BRAF kinase. While first-generation Type IS BRAF inhibitors, such as dabrafenib, are currently prescribed to target this specific molecular vulnerability, paradoxical MAPK pathway activation, and acquired drug resistance necessitate the continuous development of structurally optimized lead molecules. In this study, chemical intuition, bioisosteric replacement, and computational molecular docking were employed to propose two novel BRAFV600E drug candidates. The proposed therapeutics, engineered to incorporate constrained sp3-hybridized aliphatic rings and a sulfoximine bioisostere, demonstrated thermodynamically superior docking scores within the mutant catalytic cleft compared to dabrafenib. Lastly, a homology analysis determined that Mus musculus is a suitable model organism for future preclinical studies and confirmed crucial structural selectivity against microbial off-target kinases.

pharmacology and toxicology↗

bioRxiv2026

Computational Design of Two New 5-HT1B Serotonin Receptor Agonists Derived from Naratriptan

Migraine headaches affect over one billion people internationally and can be defined as episodes of acute severe pain wrapping around the head and are normally accompanied by nausea, blurry vision, and sensitivity to light and sound. While triggers that cause migraines may vary among patients, evidence shows they are involved with the trigeminovascular system (a network of blood vessels in the brain in conjunction with the trigeminal nerve). Activation of the trigeminal neurons triggers the release of vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and vasodilation of cranial blood vessels. The development of 5-HT1B serotonin agonist drugs, commonly known as triptans, have been an effective measure of migraine relief. The drugs created in this research were found to have improved docking scores within the 5-HT1B binding site compared to that of naratriptan. The two drugs proposed in this paper would need to undergo further investigation to determine the feasibility of laboratory synthesis and clinical trials.

pharmacology and toxicology↗

bioRxiv2026

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: Y333F, A88E, L219Q, A408H, Y173L, E340S, and Y422F

This study explores computational design predictions related to experimental enzyme behavior by analyzing seven single-point mutants of {beta}-glucosidase B (BglB) from Paenibacillus polymyxa: Y333F, A88E, L219Q, A408H, Y173L, E340S, and Y422F. Each mutation was modeled using Foldit Standalone, and mutant selections were based on predicted thermodynamic stability changes of interest. Six of the seven mutants in this set yielded soluble, expressed protein. Most variants had similar catalytic efficiency compared to the wild type with one exception. The melting temperatures for most variants were also similar to the wild type. Correlation analysis revealed weak but potentially informative relationships between predicted {Delta}TSE and (a) thermal stability and (b) catalytic efficiency. These results further support known limitations of TSE score as a tool for single point mutation design and add to a growing dataset being generated to build the next generation of functionally predictive protein models.

biochemistry↗

bioRxiv2025

Reprogramming the Rossmann Fold Signature Motif Creates Orthogonal Redox Biocatalysts

Biological reducing power is carried by nicotinamide adenine dinucleotide (phosphate) (NAD(P)/H), which supports cellular functions and cannot be specifically directed to engineered metabolic pathways. Nicotinamide mononucleotide (NMN(H)) has emerged as an orthogonal redox cofactor to address this challenge, yet strategies remain elusive for creating NMN(H)-specific enzymes that no longer interact with the cellular NAD(P)/H pools. Previous designs avoided perturbing the most ancient and conserved GxGxxG motif in Rossmann fold enzymes, the root cause of persistent NAD(P)/H recognition. Herein, we demonstrated that this motif, though long considered essential, is in fact mutable to yield active NMN+-specific enzymes. This is implemented on two unrelated model enzymes chosen to garner orthogonal reducing power from either the cheap electron source phosphite or glycolysis. On phosphite dehydrogenase (PTDH), variants NRC-01 and NRC-02 eliminated electron leaking to numerous NAD(P)H-dependent side reactions in whole cells and crude cell lysates while driving NMNH-dependent biotransformation with [~]240-fold higher productivity than existing catalysts. On glyceraldehyde-3-phosphate dehydrogenase (GapA), variant RSQ featured a [~]2.9x104-fold cofactor specificity switch to NMN+ from NAD+. Combined Rosetta modeling, systematic structural alignment, and experimental results revealed that Rossmann fold reprogramming, paired with engineered structural reinforcement, may offer a general route to orthogonal redox biocatalysts.

molecular biology↗

bioRxiv2025

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: N160L, N160S, N160C, N160M, N160G

Protein design is advancing toward quantitative modeling of enzyme function and stability. However, progress remains limited by the scarcity of standardized experimental datasets for training and benchmarking computational models. The Design to Data (D2D) program addresses this need by generating harmonized measurements of catalytic and stability parameters across an extensive {beta}-glucosidase B (BglB) variant library. Here, we expand the D2D dataset with kinetic and thermal characterization of five single-point BglB variants and the wild-type (WT), including soluble expression, Michaelis-Menten constants (kcat, KM, and kcat/KM), and melting temperature (TM,). Foldit Standalone was used to model the structural effects of the mutations. In this study, a weak but consistent association between Foldit total system energy (TSE) and TM was observed, suggesting local energetic effects that may influence stability. Together with the broader D2D corpus, these data enhance the functional mapping of BglB and provide model-ready benchmarks for developing and evaluating data-driven predictors of enzyme activity and stability.

bioengineering↗

bioRxiv2025

Diversity-driven biochemical survey reveals dimeric structural origin of rubisco

Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural diversity of rubisco, we systematically sampled and synthesized a library of diverse rubisco sequences with an emphasis on clades that have previously not been characterized. Our updated phylogenetic analysis reveals that many deep-branching rubiscos assemble as dimers, supporting a dimeric origin for the superfamily -- in contrast to the ecologically dominant hexadecameric form I. Additionally, we discover and structurally characterize the largest rubisco described to date, originating from a cryptic, early-branching subclade with novel structural folds that have previously not been observed in the rubisco superfamily. By integrating biochemical data with an updated phylogenetic framework, we propose a revised nomenclature for the rubisco protein family that reflects current insights and will better accommodate future discoveries.

biochemistry↗

bioRxiv2025

A Sequence Motif Enables Widespread Use of Non-Canonical Redox Cofactors in Natural Enzymes

Non-canonical redox cofactors (NRCs) are promising alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) for biomanufacturing due to low cost and exquisite electron delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Here, we screened the aldehyde dehydrogenase (ALDH) protein family and identified a conserved RH/QxxR sequence motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 and Pseudanabaena biceps ALDH exhibit unprecedented turnover with nicotinamide mononucleotide (NMN+), with kcat values matching or exceeding that of NAD+ and surpassing most engineered NRC-active enzymes by 10 to 105-fold, based on the relative NRC to native activity. Structural and dynamic analyses reveal this motif reinforces cofactor positioning and pre-organizes the active site without dependence on the adenosine monophosphate moiety of NAD+. When introduced into diverse ALDH scaffolds, the RH/QxxR motif enhances NMN+ activity up to 60-fold. In addition to NMN+, this motif also supports activity across multiple non-nucleotide, simple synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide (AmNA+). These findings elucidate Natures solution to the engineering challenge of obtaining NRC-active enzymes and offers a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.

synthetic biology↗

bioRxiv2025

Activation-Free Upgrading of Carboxylic Acids to Aldehydes and Alcohols

Advances in organic and gas waste valorization have enabled high-yield production of carboxylic acids, positioning them as promising feedstocks for the bioeconomy. However, carboxylic acids must be activated before downstream use, typically requiring ATP, CoA, or reduced ferredoxin to overcome unfavorable thermodynamics. These activators are costly to generate and divert carboxylic acids into CO2-releasing pathways, reducing carbon efficiency. Here, we demonstrate that aldehyde dehydrogenases (ALDHs) can directly reduce carboxylic acids to aldehydes without prior activation, a process previously thought to be biologically inaccessible. Screening 133 ALDHs revealed that this activity is remarkably widespread within the protein family, enabling production of aliphatic aldehydes and alcohols, diols, and aromatic alcohols, at titers >1 g/L, in some cases, after optimization of thermodynamic driving forces. Additionally, we applied this system to upgrade waste-derived carboxylic acid effluent streams from wastewater sludge, food waste, and waste gas (CO2). This activation-free process, termed "reverse aldehyde oxidation" (rAOX), establishes a broadly applicable, energy-efficient platform for carboxylic acid valorization at 100% carbon yield. Analogous to the reverse tricarboxylic acid cycle (rTCA) and reverse {beta}-oxidation (rBOX), rAOX exemplifies that metabolic reactions classically defined as unidirectional may have unexpected plasticity to operate in reverse and open new avenues in biomanufacturing.

synthetic biology↗

bioRxiv2025

Engineering the key central metabolic enzyme to utilize a non-canonical redox cofactor

Redesigning central metabolism enzymes to utilize non-canonical cofactors that are orthogonal to NAD+ and NADP+ may allow partitioning of reducing power into a distinct redox cofactor pool that is preserved for user-desired biosynthetic tasks, without being dissipated by the complex natural metabolism in the cells. Here, we engineer Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GapA), the key enzyme in glycolysis, to utilize nicotinamide mononucleotide (NMN+) while greatly reducing its original activity with its native cofactor, NAD+. Through rational design, we identify the variant A180S with a [~]3-fold increase in NMN+ specific activity compared to that of the wildtype (WT). Next, we design a double mutant, G10R-A180S (RS), where G10R mutation hampers the catalytic efficiency for NAD+ but also decreased the activity toward NMN+. Importantly, based on the hypothesis that the role of NAD+ in coordinating oligomer interface must be recapitulated in NMN+-utilizing enzymes, we develop a triple mutant G10R-A180S-G187Q (RSQ). Rosetta modeling predicts that the new mutation G187Q bridges separate subunits across the cofactor binding pocket, which possibly affords stronger association of the GapA tetramer, the active form of this enzyme. This final GapA variant features a [~]2.8 x 104 switch of cofactor specificity from GapAs natural cofactor NAD+ to the non-canonical cofactor NMN+. Overall, this work demonstrates the engineering of a highly conserved, central metabolic enzyme for the utilization of a non-canonical redox cofactor NMN+. The design principle discovered here may be broadly applicable to many enzymes that bind cofactors using an intersubunit pocket.

molecular biology↗

bioRxiv2025

Expression and Characterization of SARS-CoV-2 Spike Protein in Thermothelomyces heterothallica C1

The COVID-19 pandemic demonstrated a pressing need for rapid, adaptive, and scalable manufacturing of vaccines and reagents. With the transition into an endemic disease and rising threats of other emerging pandemics, production of these biologicals requires a stable and sustainable supply chain and accessible distribution methods. In this study, we demonstrate the strength of an engineered filamentous fungal platform, Thermothelomyces heterothallica C1, for high volumetric productivity of the full-length spike glycoprotein. Spike protein produced in this system is highly thermostable and immunization of mice with spike made in C1 or mammalian platforms resulted in a similar humoral response. Additionally, it was shown that the native N-glycan profile can be redecorated with complex sialylated structures, if necessary, resulting in a more human-like glycan profile, without impacting binding characteristics as shown experimentally and in simulations. Through extensive physicochemical analysis, the C1 produced spike performs similarly to spike proteins produced in other commercially available systems. The data presented is evidence that C1 can be a strong platform for production of complex glycosylated recombinant proteins such as subunit antigen vaccines.

bioengineering↗

bioRxiv2023

Protein Stability Prediction by Fine-tuning a Protein Language Model on a Mega-scale Dataset

The stability of a protein is crucial to its utility in industrial applications. While engineering campaigns can now be routinely used to enhance protein thermal stability to the level needed in an industrial setting, there is a significant desire to fast-track these efforts through predictive tools allowing one to jump in a minimal number of design iterations to a highly stabilized protein. In this work, we explore utilizing a mega-scale dataset for development of a protein language model tuned for stability. This model is trained on the folding stability of 528k sequences derived from 461 small protein domains and designs, and can accommodate deletions, insertions, and multiple-point mutations. We show that a protein language model can be fine-tuned to predict folding stability. The fine-tuned protein language model, named ESMtherm, performs reasonably on small protein domains and generalizes to sequences distal from the training set. Lastly, we discuss its limitations when compared to other state-of-the-art methods in generalizing to larger protein scaffolds and highlight the need of large-scale stability measurement on a diverse dataset that represents the distribution of sequence lengths commonly observed in nature.

biophysics↗

bioRxiv2023

Computationally Driven Design of Novel BACE-1 Inhibitors for Alzheimer's Disease

Alzheimers disease is the most common form of dementia affecting 35 million people globally. One of the major efforts in the development of a treatment for Alzheimers is to reduce the rate of plaque formation, the common hallmark of Alzheimers disease. The protease BACE-1 has been demonstrated to play a role in catalyzing plaque formation and is therefore a major drug target. Here we report potential new drug candidates building upon Verubecestat, developed through computationally driven molecular modeling methods. Both designed molecules have improved docking scores relative to Verubecestat when modeled in the BACE-1 active site, and therefore present potential new leads for more effective therapeutics to combat Alzheimers disease.

biochemistry↗

bioRxiv2023

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.

synthetic biology↗

bioRxiv2023

Computational Drug Design of Novel Agonists of the μ-Opioid Receptor to Inhibit Pain Signaling

Opioids such as Morphine, Codeine, Hydrocodone, and Oxycodone target the -opioid receptor, a G-protein-coupled receptor (GPCR), blocking the transmission of nociceptive signals. In this study, four opioids were analyzed for ADMET properties and molecular interactions with a GPCR crystal structure (PDB ID: 8EF6). This aided in the computational design of two novel drug candidates with improved docking scores and ADMET properties when compared to Hydrocodone. Homology analysis indicated that a Mus musculus (house mouse) animal model could be used in the preclinical studies of these drug candidates in the development of safer and more effective opioid drugs for pain management with reduced side effects.

molecular biology↗

bioRxiv2023

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: V311D, F248N, Y166H, Y166K, M221K

Engaging computational tools for protein design is gaining traction in the enzyme engineering community. However, current design and modeling algorithms have limited functionality predictive capacities for enzymes due to limitations of the dataset in terms of size and data quality. This study aims to expand training datasets for improved algorithm development with the addition of five rationally designed single-point enzyme variants. {beta}-glucosidase B variants were modeled in Foldit Standalone and then produced and assayed for thermal stability and kinetic parameters. Functional parameters: thermal stability (TM) and Michaelis-Menten constants (kcat, KM, and kcat/KM) of five variants, V311D, Y166H, M221K, F248N, and Y166K, were added into the Design2Data database. As a case study, evaluation of this small mutant set finds mutational effect trends that both corroborate and contradict findings from larger studies examining the entire dataset.

biochemistry↗

bioRxiv2023

Cradle to production gate life cycle assessment of cultured meat growth media: A comparison of Essential 8™ and Beefy-9

There is an increasing interest in use of biotechnology as a means of sustainable food manufacturing; however, biotechnology processing is considerably resource and energy intensive. Recent interest in animal cell-based meat (ACBM) has prompted scientific and engineering questions about the economic and environmental viability of these proposed ACBM products. This study provides an environmental assessment of two proposed growth mediums (Essential 8TM and Beefy-9) for ACBM production. The study found that the addition of antibiotics/antimycotics (10,000 g/mL) to the growth media increased the environmental metrics, such as the cumulative energy demand and global warming potential, by two orders of magnitude. To account for additional processing for animal cell culture, a scenario analysis was conducted to assess the potential environmental impacts of growth medium production with varying level of refinement required for the input components. The study indicates that the heavy refinement of the growth medium components is likely to undermine the potential sustainability of future ACBM products.

ecology↗