Biology subjects

Kosuri, S.

Publications and source records attributed to Kosuri, S..

At least 19 recordsLinked to original sources

bioRxiv2025

Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

Tyrosine Kinase 2 (TYK2) is a genetically defined target for autoimmune disease, with first-generation inhibitors showing clinical success in some but not all associated indications. A deeper understanding of TYK2 structure-function, protein-ligand interactions, and the impact of human variants could inform next-generation therapeutics. Here, we applied Deep Mutational Scanning (DMS) to assess >23,000 amino acid substitutions across two TYK2 functions: IFN- signaling and protein abundance. This enabled high-resolution structure-function mapping and the identification of novel allosteric sites. By coupling DMS with inhibitor treatment, we uncovered variants that modulate compound potency. We also show that human variants - both common and rare - that are protective against autoimmune phenotypes reduce TYK2 protein abundance. Together, these findings demonstrate that DMS can prospectively reveal novel druggable sites, clarify structure-activity relationships (SAR), and highlight TYK2 degradation as a potential therapeutic strategy in autoimmunity.

genomics↗

bioRxiv2025

Covalent Degraders of Immune Regulatory Transcription Factors IRF8 and IRF5

Transcription factors are among the most challenging targets for drug discovery due to their lack of classical binding pockets and high degree of intrinsic disorder, despite the therapeutic importance of many of these proteins. IRF5 and IRF8 are key transcriptional regulators of innate immune signaling that orchestrate pro-inflammatory gene expression programs in response to stimuli such as toll-like receptor activation, making them central players in autoimmune and inflammatory diseases. Despite their therapeutic interest, direct targeting of IRF5 and IRF8 has remained challenging. Through a screen originally intended to identify cysteine-reactive electrophilic ligands that could directly and covalently engage and degrade IRF5, we identified an acrylamide hit, EN1033, that not only targeted and degraded IRF5 but also degraded a related inflammatory transcription factor, IRF8, more robustly and rapidly. EN1033 destabilized and degraded IRF5 and IRF8 by covalently targeting C28 and C223, respectively, as evidenced by the attenuation of their degradation through mutagenesis of these cysteines. We also found that IRF8 loss led to downregulation and inhibition of IRF5 activity, suggesting crosstalk between these two transcription factors, both of which are targeted by EN1033. Upon exploring structure-activity relationships, we identified an optimized compound, TH-B10, that more potently and selectively targeted and degraded IRF8 and, secondarily, downregulated IRF5 by targeting C223 on IRF8. Overall, we identify an early-stage pathfinder molecule, TH-B10, that directly covalently targets and degrades IRF8 and secondarily modulates IRF5 to shut down pro-inflammatory transcriptional activity.

biochemistry↗

bioRxiv2024

High resolution deep mutational scanning of the melanocortin-4 receptor enables target characterization for drug discovery

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6,600 single amino acid substitutions on MC4Rs function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gs- and Gq-signaling pathways, which could be used to design drugs that selectively bias MC4Rs activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.

synthetic biology↗

bioRxiv2023

Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this librarys utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.

synthetic biology↗

bioRxiv2021

Functional regulatory variants implicate distinct transcriptional networks in dementia

Predicting functionality of noncoding variation is one of the major challenges in modern genetics. We employed massively parallel reporter assays to screen 5,706 variants from genome-wide association studies for both Alzheimers disease (AD) and Progressive Supranuclear Palsy (PSP). We identified 320 functional regulatory polymorphisms (SigVars) comprising 27 of 34 unique tested loci, including multiple independent signals across the complex 17q21.31 region. We identify novel risk genes including PLEKHM1 in PSP and APOC1 in AD, and perform gene-editing to validate four distinct causal loci, confirming complement 4 (C4A) as a novel genetic risk factor for AD. Moreover, functional variants preferentially disrupt transcription factor binding sites that converge on enhancers with differential cell-type specific activity in PSP and AD, implicating a neuronal SP1-driven regulatory network in PSP pathogenesis. These analyses support a novel mechanism underlying noncoding genetic risk, whereby common genetic variants drive disease risk via their aggregate activity on specific transcriptional programs. One Sentence SummaryHigh-throughput functional analysis of GWAS loci reveals cell-type specific regulatory networks that mediate genetic risk for dementia.

genetics↗

bioRxiv2020

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

Myriad biological functions require protein-protein interactions (PPIs), and engineered PPIs are crucial for applications ranging from drug design to synthetic cell circuits. Understanding and engineering specificity in PPIs is particularly challenging as subtle sequence changes can drastically alter specificity. Coiled-coils are small protein domains that have long served as a simple model for studying the sequence-determinants of specificity and have been used as modular building blocks to build large protein nanostructures and synthetic circuits. Despite their simple rules and long-time use, building large sets of well-behaved orthogonal pairs that can be used together is still challenging because predictions are often inaccurate, and, as the library size increases, it becomes difficult to test predictions at scale. To address these problems, we first developed a method called the Next-Generation Bacterial Two-Hybrid (NGB2H), which combines gene synthesis, a bacterial two-hybrid assay, and a high-throughput next-generation sequencing readout, allowing rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way. After validating the NGB2H system on previously characterized libraries, we designed, built, and tested large sets of orthogonal synthetic coiled-coils. In an iterative set of experiments, we assayed more than 8,000 PPIs, used the dataset to train a novel linear model-based coiled-coil scoring algorithm, and then characterized nearly 18,000 interactions to identify the largest set of orthogonal PPIs to date with twenty-two on-target interactions.

molecular biology↗

bioRxiv2020

Massively parallel identification of causal variants underlying gene expression differences in a yeast cross

Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5,832 natural DNA variants in the promoters of 2,503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, which is consistent with the action of negative selection. Causal variants were also enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

genetics↗

bioRxiv2020

Rapid cost-effective viral genome sequencing by V-seq

Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3 end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.

genomics↗

bioRxiv2020

Preliminary support for a 'dry swab, extraction free' protocol for SARS-CoV-2 testing via RT-qPCR

Structured AbstractO_ST_ABSBackgroundC_ST_ABSThe urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. MethodsWe evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral activation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. ResultsAfter optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. ConclusionSwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

molecular biology↗

bioRxiv2020

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8,269 rationally designed IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. Using these data, we fit a statistical mechanics model that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three novel promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

synthetic biology↗

bioRxiv2020

Genome-wide Functional Characterization of Escherichia coli Promoters and Regulatory Elements Responsible for their Function.

Despite decades of intense genetic, biochemical, and evolutionary characterizations of bacterial promoters, we lack the ability to identify or predict transcriptional activities of promoters using primary sequence. Even in simple, well-characterized organisms such as E. coli there is little agreement on the number, location, and strength of promoters. We use a genomically-encoded massively parallel reporter assay to perform the first full characterization of autonomous promoter activity across the E. coli genome. We measure promoter activity of >300,000 sequences spanning the entire genome and map 2,228 promoters active in rich media. Surprisingly, 944 of these promoters were found within intragenic sequences and are associated with conciliatory sequence adaptations by both the protein-coding regions and overlapping RNAP binding sites. Furthermore, we perform a scanning mutagenesis of 2,057 promoters to uncover sequence elements regulating promoter activity, revealing 3,317 novel regulatory elements. Finally, we show that despite these large datasets and modern machine learning algorithms, predicting endogenous promoter activity from primary sequence is still challenging.

microbiology↗

bioRxiv2019

DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here we improve DropSynth, a low-cost, multiplexed method which builds gene libraries by compartmentalizing and assembling microarray-derived oligos in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction, and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity.

synthetic biology↗

bioRxiv2019

Multiplexed dissection of a model human transcription factor binding site architecture

In eukaryotes, transcription factors orchestrate gene expression by binding to TF-Binding Sites (TFBSs) and localizing transcriptional co-regulators and RNA Polymerase II to cis-regulatory elements. The strength and regulation of transcription can be modulated by a variety of factors including TFBS composition, TFBS affinity and number, distance between TFBSs, distance of TFBSs to transcription start sites, and epigenetic modifications. We still lack a basic comprehension of how such variables shaping cis-regulatory architecture culminate in quantitative transcriptional responses. Here we explored how such factors determine the transcriptional activity of a model transcription factor, the c-AMP Response Element (CRE) binding protein. We measured expression driven by 4,602 synthetic regulatory elements in a massively parallel reporter assay (MPRA) exploring the impact of CRE number, affinity, distance to the promoter, and spacing between multiple CREs. We found the number and affinity of CREs within regulatory elements largely determines overall expression, and this relationship is shaped by the proximity of each CRE to the downstream promoter. In addition, while we observed expression periodicity as the CRE distance to the promoter varied, the spacing between multiple CREs altered this periodicity. Finally, we compare library expression between an episomal MPRA and a new, genomically-integrated MPRA in which a single synthetic regulatory element is present per cell at a defined locus. We observe that these largely recapitulate each other although weaker, non-canonical CREs exhibited greater activity in the genomic context.

genomics↗

bioRxiv2019

Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning

In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor ({beta}2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for {beta}2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

molecular biology↗

bioRxiv2018

A Scalable, Multiplexed Assay for Decoding Receptor-Ligand Interactions

Chemicals such as drugs, hormones, and odorants can have many potential interactions with endogenous targets, and uncovering these relationships is critical for understanding and modulating function. Mammalian olfactory receptors (ORs), a large family of G protein-coupled receptors, mediate olfaction through activation by small molecules. Each OR can respond to many odorants, and vice versa, making exploring this space one interaction at a time difficult. We developed a high-throughput receptor screening platform in human cell lines to screen libraries of chemicals against a multiplexed library of receptors using next-generation sequencing of barcoded genetic reporters. We screened three concentrations of 181 odorants, where in each well we record the activity of 39 ORs simultaneously, and identified 79 novel associations, including ligands for 15 orphan receptors. This platform allows the cost-effective mapping of large chemical libraries to receptor repertoires at scale.

biochemistry↗

bioRxiv2017

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically-Encoded Multiplexed Reporter Assay in E. coli

Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In E. coli, decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange (RMCE) system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10,898 {sigma}70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our dataset using a simple log-linear statistical model. Simple neural network models explain greater than 95% of the variance in our dataset by capturing nonlinear interactions with the spacer, background, and UP elements.

molecular biology↗

bioRxiv2017

Large-scale screening of rare genetic variants in humans reveals frequent splicing disruptions

Any individuals genome contains [~]4-5 million genetic variants that differ from reference, and understanding how these variants give rise to trait diversity and disease susceptibility is a central goal of human genetics1. A vast majority (96-99%) of an individuals variants are common, though at a population level the overwhelming majority of variants are rare2-5. Because of their scarcity in an individuals genome, rare variants that play important roles in complex traits are likely to have large functional effects6,7. Mutations that cause an exon to be skipped can have severe functional consequences on gene function, and many known disease-causing mutations reduce or eliminate exon recognition8. Here we explore the extent to which rare genetic variation in humans results in near complete loss of exon recognition. We developed a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS) that allows us to measure exon inclusion in thousands of human exons and surrounding intronic sequence simultaneously. We assayed 27,733 extant variants in the Exome Aggregation Consortium (ExAC)9 within or adjacent to 2,339 human exons, and found that 3.8% (1,050) of the variants, almost all of which were extremely rare, led to large-effect defects in exon recognition. Importantly, we find that 83% of these splice-disrupting variants (SDVs) are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate that loss of exon recognition is an important and underappreciated means by which rare variants exert large functional effects, and that MFASS enables their empirical assessment for splicing defects at scale.

genetics↗

bioRxiv2017

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a genes synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized ~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.

biochemistry↗