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Concise appraisal

The paper reports that select sulfonamidecontaining TEAD lipid pocket binders promote a TEAD cofactor switch from oncogenic YAP to repressive VGLL4, reducing YAPdependent transcription and slowing tumor growth in vitro and in vivo — a mechanistic alternative to simple YAPTEAD blockade that requires VGLL4 presence for efficacy Preprint Main Paper
PNAS Published Version

Detailed critical review

What the paper claims

• Sulfonamidecontaining TEAD lipid pocket binders (Compound 2, VT103 and related LPB molecules) increase VGLL4 association with TEAD and promote VGLL4 chromatin recruitment, producing a cofactor switch from YAP to VGLL4 and transcriptional repression of progrowth and mechanosignaling genes Preprint Evidence Summary
• VGLL4 is necessary for the antiproliferative response: VGLL4 KO cells and VGLL4low lines are resistant unless VGLL4 is overexpressed; xenograft inhibition by Compound 2/VT103 is lost in VGLL4 KO tumors VGLL4 Functional Data
• Mechanistic hypothesis: sulfonamide moiety is key; compounds may act via mild allosteric change or behave as molecular glue to favor VGLL4 binding; crystallography shows little global TEAD conformational change so resolution requires insolution structural techniques Mechanistic Hypothesis Note

Strengths

1. Multiorthogonal evidence: APMS, endogenous coIP (multiple cell lines), biochemical peptide pulldown, ChIPseq, RNAseq, genetic KO and overexpression, and in vivo xenografts provide convergent support that VGLL4 engagement is functionally relevant Multimodal Methods
2. Translationally relevant insight: shows TEAD LPB compounds can work via cofactor modulation rather than only by YAP displacement, a conceptual advance for TEADdrug design and biomarker selection (VGLL4 status) PNAS Translation Note

Key limitations and blindspots

• Paralog coverage: APMS focused on TEAD1 and TEAD4; TEAD2/3 were not comprehensively profiled — paralogs can have distinct expression and genomic occupancy, affecting generality Paralog Limitation
• Structural mechanism unresolved: available cocrystal data show no large TEAD fold change with Compound 2, so proposed allostery or molecular glue behavior is plausible but unproven; reliance on crystallography alone is insufficient Structure Data Gap
• Cell line / model bias: many experiments are in engineered cell lines or NCIH226 with high endogenous VGLL4; tumor heterogeneity in patients may include VGLL4 loss or silencing making therapy less broadly applicable Model Limitations
• APMS stability bias: affinity purification selects for stable interactors and may miss transient cofactor dynamics; authors state transient interactors may be lost in washes APMS Bias
• Intellectual property and COI: all authors are/ were Genentech employees and patents are declared; industry affiliation is transparent but raises the need to replicate independently COI Statement

Mechanistic plausibility and outstanding mechanistic experiments

The chemical genetics and peptide pulldown data make a credible case that lipidgetting sulfonamide compounds alter TEAD behavior so it favors VGLL4. However, this leaves open whether the small molecules act as classical molecular glues (forming a ternary compoundTEADVGLL4 complex with direct ligandcontacts to both proteins) or as subtle allosteric modulators that change TEAD conformational ensemble to favor VGLL4 affinity — Xray data argue for small changes and thus for either mild allostery or ordered water/entropy effects. Definitive tests should include:

• Native mass spectrometry and HDXMS or NMR to detect ternary complex formation and map altered dynamics at residue resolution.
• Surface plasmon resonance or BLI measuring ternary assembly kinetics and whether the small molecule increases VGLL4 peptide kon or reduces koff.
• CryoEM or solution SAXS with full length TEADcofactors or crosslinked cryoEM assemblies to visualize any ligandmediated bridging.

Translational implications and biomarkers

Key actionable insight: VGLL4 protein (or RNA) expression in tumors is a plausible biomarker to predict responses to sulfonamidecontaining TEAD LPB inhibitors; conversely VGLL4 loss would predict resistance. The authors explicitly recommend evaluating VGLL4 status during clinical development Biomarker Recommendation

Concrete recommendations to strengthen the work

1. Perform in solution structural/dynamics experiments (HDXMS, NMR, nativeMS) and ternary binding kinetics to distinguish molecular glue from allostery.
2. Profile TEAD2 and TEAD3 for compounddependent interactions and validate VGLL4 cofactor effects across all four TEAD paralogs in representative tumor lines.
3. Survey VGLL4 expression/loss across patient tumor datasets (TCGA/DepMap) and correlate with predicted sensitivity to LPB sulfonamide chemotypes to assess clinical reach.
4. Assess off target binding and general toxicity in noncancer primary cells and organoids since promoting VGLL4TEAD in normal tissues could have unanticipated effects on tissue homeostasis.

Conclusion and confidence

The study presents a wellcontrolled, multipronged body of evidence that select sulfonamide TEAD LPB compounds induce a TEAD cofactor switch from YAP to VGLL4 and that this switch mediates antiproliferative effects in models that express VGLL4. The major unresolved element is the precise structural mechanism (molecular glue versus allostery) and the populationlevel generality given VGLL4 heterogeneity in tumors. Confidence is moderatehigh for the central biological claim within the tested models, but clinical generalizability requires VGLL4 biomarker validation and further mechanistic resolution PNAS Confidence Note