Biology subjects

Shah, R. N.

Publications and source records attributed to Shah, R. N..

5 recordsLinked to original sources

bioRxiv2026

Folding scFv--Antigen Complexes at Scale

AO_SCPLOWBSTRACTC_SCPLOWAccurate modeling of antibody-antigen (Ab-Ag) complexes is central to biologic development, yet the reliability and failures of modern Ab-Ag folding pipelines remain poorly characterized. Single-chain variable fragments (scFvs) are thera-peutically important antibodies, but large-scale evaluations of structure prediction models on scFv-Ag complexes are largely lacking. We introduce a scalable bench-marking pipeline that generates large ensembles of scFv-Ag structure predictions by cofolding a curated subset of 3,800 Ab-Ag complexes from SAbDab using multiple state-of-the-art models under diverse inference-time settings. The resulting dataset, SCALE (scFv-Ag CompLex Ensembles), includes standardized scFv-Ag sequences and around 200,000 predicted complexes spanning different models, sampling strategies, and auxiliary inputs. Using SCALE, we evaluate model performance in recovering correct scFv-Ag interfaces and assess the ability of existing confidence metrics to select the best structure from prediction ensembles. We find that while confidence scores effectively distinguish easy from hard scFv-Ag complexes, they often fail to identify the highest-quality interface for a given target. Further analysis shows that near-correct interfaces typically appear in ensembles but at low frequency, and inference-time choices such as sampling, recycling, and using evolutionary or structural information are crucial for accurate scFv-Ag complex predictions. Dataset and analysis code are available at https://huggingface.co/datasets/ravishah1/SCALE

bioinformatics↗

bioRxiv2023

Specificity Guides Interpretation: On H3K4 Methylation at Enhancers and Broad Promoters

In 2018, we used internally calibrated chromatin immunoprecipitation (ICeChIP) to find that many of the most commonly used antibodies against H3K4 methylforms had significant off-target binding, which compromised the findings of at least eight literature paradigms that used these antibodies for ChIP-seq (Shah et al., 2018). In many cases, we were able to recapitulate the prior findings in K562 cells with the original, low-quality antibody, only to find that the models did not hold up to scrutiny with highly specific reagents and quantitative calibration. In a recent preprint originally prepared as a Letter to the Editor of Molecular Cell, though they agree with our overarching conclusions, Pekowska and colleagues take issue with analyses presented for two relatively minor points of the paper (Pekowska et al., 2023). We are puzzled by the assertion that these two points constitute the "bulk" of our findings, nor is it clear which components of our "analytical design" they find problematic. We feel their critique, however mild, is misguided.

molecular biology↗

bioRxiv2021

Re-evaluating the role of nucleosomal bivalency in early development

Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2-4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5-10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11-15. The apparent juxtaposition of these opposing marks, termed "bivalent domains"16-18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19-22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naive mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

molecular biology↗

bioRxiv2020

Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

MLL-rearranged leukemia depends on H3K79 methylation. Depletion of this transcriptionally-activating mark by DOT1L deletion or high concentrations of the inhibitor pinometostat downregulates HOXA9 and MEIS1, and consequently reduces leukemia survival. Yet some MLL-rearranged leukemias are inexplicably susceptible to low-dose pinometostat, far below concentrations that downregulate this canonical proliferation pathway. In this context, we define alternative proliferation pathways that more directly derive from H3K79me2 loss. By ICeChIP-seq, H3K79me2 is markedly depleted at pinometostat-downregulated and MLL-fusion targets, with paradoxical increases of H3K4me3 and loss of H3K27me3. Although downregulation of polycomb components accounts for some of the proliferation defect, transcriptional downregulation of FLT3 is the major pathway. Loss-of-FLT3-function recapitulates the cytotoxicity and gene expression consequences of low-dose pinometostat, whereas overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, largely rescues these defects. This pathway also depends on MLL1, indicating combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating FLT3-mutant leukemia.

cancer biology↗

bioRxiv2020

Multimodal small-molecule screening for human prion protein binders

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mM), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. While orthogonal binder discovery methods could yield high affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

biochemistry↗