@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-drug-target-validation/SKILL.md

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+---
+name: tooluniverse-drug-target-validation
+description: Comprehensive computational validation of drug targets for early-stage drug discovery. Evaluates targets across 10 dimensions (disambiguation, disease association, druggability, chemical matter, clinical precedent, safety, pathway context, validation evidence, structural insights, validation roadmap) using 60+ ToolUniverse tools. Produces a quantitative Target Validation Score (0-100) with GO/NO-GO recommendation. Use when users ask about target validation, druggability assessment, target prioritization, or "is X a good drug target for Y?"
+---
+
+# Drug Target Validation Pipeline
+
+Validate drug target hypotheses using multi-dimensional computational evidence before committing to wet-lab work. Produces a quantitative Target Validation Score (0-100) with priority tier classification and GO/NO-GO recommendation.
+
+**KEY PRINCIPLES**:
+1. **Report-first approach** - Create report file FIRST, then populate progressively
+2. **Target disambiguation FIRST** - Resolve all identifiers before analysis
+3. **Evidence grading** - Grade all evidence as T1 (experimental) to T4 (computational)
+4. **Disease-specific** - Tailor analysis to disease context when provided
+5. **Modality-aware** - Consider small molecule vs biologics tractability
+6. **Safety-first** - Prominently flag safety concerns early
+7. **Quantitative scoring** - Every dimension scored numerically (0-100 composite)
+8. **Negative results documented** - "No data" is data; empty sections are failures
+9. **Source references** - Every statement must cite tool/database
+10. **Completeness checklist** - Mandatory section showing analysis coverage
+11. **English-first queries** - Always use English terms in tool calls. Respond in user's language
+
+---
+
+## When to Use This Skill
+
+Apply when users:
+- Ask "Is [target] a good drug target for [disease]?"
+- Need target validation or druggability assessment
+- Want to compare targets for drug discovery prioritization
+- Ask about safety risks of modulating a target
+- Need chemical starting points for target validation
+- Ask about pathway context for a target
+- Need a GO/NO-GO recommendation for a target
+- Want a comprehensive target dossier for investment decisions
+
+**NOT for** (use other skills instead):
+- General target biology overview -> Use `tooluniverse-target-research`
+- Drug compound profiling -> Use `tooluniverse-drug-research`
+- Variant interpretation -> Use `tooluniverse-variant-interpretation`
+- Disease research -> Use `tooluniverse-disease-research`
+
+---
+
+## Input Parameters
+
+| Parameter | Required | Description | Example |
+|-----------|----------|-------------|---------|
+| **target** | Yes | Gene symbol, protein name, or UniProt ID | `EGFR`, `P00533`, `Epidermal growth factor receptor` |
+| **disease** | No | Disease/indication for context | `Non-small cell lung cancer`, `Pancreatic cancer` |
+| **modality** | No | Preferred therapeutic modality | `small molecule`, `antibody`, `protein therapeutic`, `PROTAC` |
+
+---
+
+## Target Validation Scoring System
+
+### Score Components (Total: 0-100)
+
+**Disease Association** (0-30 points):
+- Genetic evidence: 0-10 (GWAS, rare variants, somatic mutations)
+- Literature evidence: 0-10 (publications, clinical studies)
+- Pathway evidence: 0-10 (disease pathway involvement)
+
+**Druggability** (0-25 points):
+- Structural tractability: 0-10 (structure quality, binding pockets)
+- Chemical matter: 0-10 (known compounds, bioactivity data)
+- Target class: 0-5 (validated target family bonus)
+
+**Safety Profile** (0-20 points):
+- Tissue expression selectivity: 0-5 (expression in critical tissues)
+- Genetic validation: 0-10 (knockout phenotypes, human genetics)
+- Known adverse events: 0-5 (safety signals from modulators)
+
+**Clinical Precedent** (0-15 points):
+- Approved drugs: 15 (strong precedent, validated target)
+- Clinical trials: 10 (moderate precedent)
+- Preclinical compounds: 5 (weak precedent)
+- None: 0 (novel target)
+
+**Validation Evidence** (0-10 points):
+- Functional studies: 0-5 (CRISPR, siRNA, biochemical)
+- Disease models: 0-5 (animal models, patient data)
+
+### Priority Tiers
+
+| Score | Tier | Recommendation |
+|-------|------|----------------|
+| **80-100** | Tier 1 | Highly validated - proceed with confidence |
+| **60-79** | Tier 2 | Good target - needs focused validation |
+| **40-59** | Tier 3 | Moderate risk - significant validation needed |
+| **0-39** | Tier 4 | High risk - consider alternatives |
+
+### Evidence Grading System
+
+| Tier | Symbol | Criteria | Examples |
+|------|--------|----------|----------|
+| **T1** | [T1] | Direct mechanistic, human clinical proof | FDA-approved drug, crystal structure with mechanism, patient mutation |
+| **T2** | [T2] | Functional studies, model organism | siRNA phenotype, mouse KO, biochemical assay, CRISPR screen |
+| **T3** | [T3] | Association, screen hits, computational | GWAS hit, DepMap essentiality, expression correlation |
+| **T4** | [T4] | Mention, review, text-mined, predicted | Review article, database annotation, AlphaFold prediction |
+
+---
+
+## Phase 0: Target Disambiguation & ID Resolution (ALWAYS FIRST)
+
+**Objective**: Resolve target to ALL needed identifiers before any analysis.
+
+### Resolution Strategy
+
+```python
+# Step 1: Determine input type and get initial identifiers
+# If gene symbol (e.g., "EGFR"):
+mygene = tu.tools.MyGene_query_genes(query="EGFR", species="human", fields="symbol,name,ensembl.gene,uniprot.Swiss-Prot,entrezgene")
+# Extract: ensembl_id, uniprot_id, entrez_id, symbol, name
+
+# If UniProt ID (e.g., "P00533"):
+uniprot = tu.tools.UniProt_get_entry_by_accession(accession="P00533")
+# Extract: gene names, Ensembl xrefs, function
+
+# Step 2: Resolve Ensembl ID and get versioned ID for GTEx
+ensembl = tu.tools.ensembl_lookup_gene(gene_id=ensembl_id, species="homo_sapiens")
+# CRITICAL: species parameter is REQUIRED
+# CRITICAL: Response is wrapped in {status, data, url, content_type} - access via ensembl['data']
+ensembl_data = ensembl.get('data', ensembl) if isinstance(ensembl, dict) else ensembl
+# Extract: version for versioned_id (e.g., "ENSG00000146648.18")
+
+# Step 3: Get Ensembl cross-references
+xrefs = tu.tools.ensembl_get_xrefs(id=ensembl_id)
+# Extract: HGNC, UniProt, EntrezGene mappings
+
+# Step 4: Get OpenTargets target info
+ot_target = tu.tools.OpenTargets_get_target_id_description_by_name(targetName="EGFR")
+# Verify ensemblId matches
+
+# Step 5: Get ChEMBL target ID
+chembl_targets = tu.tools.ChEMBL_search_targets(pref_name__contains="EGFR", organism="Homo sapiens", limit=5)
+# Extract: target_chembl_id for later use
+
+# Step 6: Get UniProt function summary
+function_info = tu.tools.UniProt_get_function_by_accession(accession=uniprot_id)
+# Returns list of strings (NOT dict)
+
+# Step 7: Get alternative names for collision detection
+alt_names = tu.tools.UniProt_get_alternative_names_by_accession(accession=uniprot_id)
+```
+
+### Identifier Resolution Output
+
+```markdown
+## 1. Target Identity
+
+| Database | Identifier | Verified |
+|----------|-----------|----------|
+| Gene Symbol | EGFR | Yes |
+| Full Name | Epidermal growth factor receptor | Yes |
+| Ensembl | ENSG00000146648 | Yes |
+| Ensembl (versioned) | ENSG00000146648.18 | Yes |
+| UniProt | P00533 | Yes |
+| Entrez Gene | 1956 | Yes |
+| ChEMBL | CHEMBL203 | Yes |
+| HGNC | HGNC:3236 | Yes |
+
+**Protein Function**: [from UniProt_get_function_by_accession]
+**Subcellular Location**: [from UniProt_get_subcellular_location_by_accession]
+**Target Class**: [from OpenTargets_get_target_classes_by_ensemblID]
+```
+
+### Known Parameter Corrections
+
+| Tool | WRONG Parameter | CORRECT Parameter |
+|------|-----------------|-------------------|
+| `ensembl_lookup_gene` | `id` | `gene_id` (+ `species="homo_sapiens"` REQUIRED) |
+| `Reactome_map_uniprot_to_pathways` | `uniprot_id` | `id` |
+| `ensembl_get_xrefs` | `gene_id` | `id` |
+| `GTEx_get_median_gene_expression` | `gencode_id` only | `gencode_id` + `operation="median"` |
+| `OpenTargets_*` | `ensemblID` (uppercase) | `ensemblId` (camelCase) |
+| `OpenTargets_get_publications_*` | `ensemblId` | `entityId` |
+| `OpenTargets_get_associated_drugs_by_target_ensemblID` | `ensemblId` only | `ensemblId` + `size` (REQUIRED) |
+| `MyGene_query_genes` | `q` | `query` |
+| `PubMed_search_articles` | returns `{articles: [...]}` | returns **plain list** of dicts |
+| `UniProt_get_function_by_accession` | returns dict | returns **list of strings** |
+| `HPA_get_rna_expression_by_source` | `ensembl_id` | `gene_name` + `source_type` + `source_name` (ALL required) |
+| `alphafold_get_prediction` | `uniprot_accession` | `qualifier` |
+| `drugbank_get_safety_*` | simple params | `query`, `case_sensitive`, `exact_match`, `limit` (ALL required) |
+
+---
+
+## Phase 1: Disease Association Evidence (0-30 points)
+
+**Objective**: Quantify the strength of target-disease association from genetic, literature, and pathway evidence.
+
+### 1A. OpenTargets Disease Associations (Primary)
+
+```python
+# Get ALL disease associations for target
+diseases = tu.tools.OpenTargets_get_diseases_phenotypes_by_target_ensembl(ensemblId=ensembl_id)
+
+# If specific disease provided, get detailed evidence
+if disease_name:
+    disease_info = tu.tools.OpenTargets_get_disease_id_description_by_name(diseaseName=disease_name)
+    efo_id = disease_info.get('id')  # e.g., "EFO_0003060"
+
+    evidence = tu.tools.OpenTargets_target_disease_evidence(
+        efoId=efo_id, ensemblId=ensembl_id
+    )
+
+    # Get evidence by data source for detailed breakdown
+    datasource_evidence = tu.tools.OpenTargets_get_evidence_by_datasource(
+        efoId=efo_id, ensemblId=ensembl_id,
+        datasourceIds=["ot_genetics_portal", "eva", "gene2phenotype", "genomics_england", "uniprot_literature"],
+        size=100
+    )
+```
+
+### 1B. GWAS Genetic Evidence
+
+```python
+# GWAS associations for target gene
+gwas_snps = tu.tools.gwas_get_snps_for_gene(mapped_gene=gene_symbol, size=50)
+
+# If specific disease, search for trait-specific associations
+if disease_name:
+    gwas_studies = tu.tools.gwas_search_studies(query=disease_name, size=20)
+```
+
+### 1C. Constraint Scores (gnomAD)
+
+```python
+# Genetic constraint - intolerance to loss of function
+constraints = tu.tools.gnomad_get_gene_constraints(gene_symbol=gene_symbol)
+# Extract: pLI, LOEUF, missense_z, pRec
+# High pLI (>0.9) = highly intolerant to LoF = likely essential
+```
+
+### 1D. Literature Evidence
+
+```python
+# PubMed for target-disease association
+articles = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND "{disease_name}" AND (target OR therapeutic OR inhibitor)',
+    limit=50
+)
+# PubMed_search_articles returns a plain list of dicts
+
+# OpenTargets publications
+pubs = tu.tools.OpenTargets_get_publications_by_target_ensemblID(entityId=ensembl_id)
+```
+
+### Scoring Logic - Disease Association
+
+```
+Genetic Evidence (0-10):
+  - GWAS hits for specific disease: +3 per significant locus (max 6)
+  - Rare variant evidence (ClinVar pathogenic): +2
+  - Somatic mutations in disease: +2
+  - pLI > 0.9 (essential gene): +2
+
+Literature Evidence (0-10):
+  - >100 publications on target+disease: 10
+  - 50-100 publications: 7
+  - 10-50 publications: 5
+  - 1-10 publications: 3
+  - 0 publications: 0
+
+Pathway Evidence (0-10):
+  - OpenTargets overall score > 0.8: 10
+  - Score 0.5-0.8: 7
+  - Score 0.2-0.5: 4
+  - Score < 0.2: 1
+```
+
+---
+
+## Phase 2: Druggability Assessment (0-25 points)
+
+**Objective**: Assess whether the target is amenable to therapeutic intervention.
+
+### 2A. OpenTargets Tractability
+
+```python
+# Tractability assessment across modalities
+tractability = tu.tools.OpenTargets_get_target_tractability_by_ensemblID(ensemblId=ensembl_id)
+# Returns: label, modality (SM, AB, PR, OC), value (boolean/score)
+# Modalities: Small Molecule, Antibody, PROTAC, Other Clinical
+```
+
+### 2B. Target Class & Family
+
+```python
+# Target classification (kinase, GPCR, ion channel, etc.)
+target_classes = tu.tools.OpenTargets_get_target_classes_by_ensemblID(ensemblId=ensembl_id)
+
+# Pharos target development level
+pharos = tu.tools.Pharos_get_target(gene=gene_symbol)
+# TDL: Tclin (approved drug) > Tchem (compounds) > Tbio (biology) > Tdark (unknown)
+
+# DGIdb druggability categories
+druggability = tu.tools.DGIdb_get_gene_druggability(genes=[gene_symbol])
+```
+
+### 2C. Structural Tractability
+
+```python
+# PDB structures available
+if uniprot_id:
+    uniprot_entry = tu.tools.UniProt_get_entry_by_accession(accession=uniprot_id)
+    # Extract PDB cross-references from entry
+
+# AlphaFold prediction
+alphafold = tu.tools.alphafold_get_prediction(qualifier=uniprot_id)
+alphafold_summary = tu.tools.alphafold_get_summary(qualifier=uniprot_id)
+
+# For top PDB structures, analyze binding pockets
+# ProteinsPlus DoGSiteScorer for pocket detection
+for pdb_id in top_pdb_ids[:3]:
+    pockets = tu.tools.ProteinsPlus_predict_binding_sites(pdb_id=pdb_id)
+    # Returns predicted druggable pockets with scores
+```
+
+### 2D. Chemical Probes & Enabling Packages
+
+```python
+# Chemical probes (validated tool compounds)
+probes = tu.tools.OpenTargets_get_chemical_probes_by_target_ensemblID(ensemblId=ensembl_id)
+
+# Target Enabling Packages (TEPs)
+teps = tu.tools.OpenTargets_get_target_enabling_packages_by_ensemblID(ensemblId=ensembl_id)
+```
+
+### Scoring Logic - Druggability
+
+```
+Structural Tractability (0-10):
+  - High-res co-crystal structure with ligand: 10
+  - PDB structure available, pockets detected: 7
+  - AlphaFold only, confident pocket prediction: 5
+  - AlphaFold low confidence / no structure: 2
+  - No structural data: 0
+
+Chemical Matter (0-10):
+  - Known drug-like compounds (IC50 < 100nM): 10
+  - Tool compounds (IC50 < 1uM): 7
+  - HTS hits only (IC50 > 1uM): 4
+  - No known ligands: 0
+
+Target Class Bonus (0-5):
+  - Validated druggable family (kinase, GPCR, nuclear receptor): 5
+  - Enzyme, ion channel: 4
+  - Protein-protein interaction, transporter: 2
+  - Novel/unknown class: 0
+```
+
+---
+
+## Phase 3: Known Modulators & Chemical Matter (Feeds into Phase 2 scoring)
+
+**Objective**: Identify existing chemical starting points for target validation.
+
+### 3A. ChEMBL Bioactivity
+
+```python
+# Search for ChEMBL target
+chembl_targets = tu.tools.ChEMBL_search_targets(
+    pref_name__contains=gene_symbol, organism="Homo sapiens", limit=10
+)
+
+# Get activities for best matching target
+target_chembl_id = chembl_targets[0]['target_chembl_id']
+activities = tu.tools.ChEMBL_get_target_activities(
+    target_chembl_id__exact=target_chembl_id, limit=100
+)
+# Parse: compound IDs, pChEMBL values, activity types (IC50, Ki, Kd)
+# Filter: potent compounds (pChEMBL >= 6.0 = IC50 <= 1uM)
+```
+
+### 3B. BindingDB Ligands
+
+```python
+# Experimental binding data
+ligands = tu.tools.BindingDB_get_ligands_by_uniprot(
+    uniprot=uniprot_id, affinity_cutoff=10000  # nM
+)
+# Returns: SMILES, affinity_type (Ki/IC50/Kd), affinity value, PMID
+```
+
+### 3C. PubChem Bioassays
+
+```python
+# HTS screening data
+assays = tu.tools.PubChem_search_assays_by_target_gene(gene_symbol=gene_symbol)
+# Get details for top assays
+for aid in assay_ids[:5]:
+    summary = tu.tools.PubChem_get_assay_summary(aid=str(aid))
+    targets = tu.tools.PubChem_get_assay_targets(aid=str(aid))
+    actives = tu.tools.PubChem_get_assay_active_compounds(aid=str(aid))
+```
+
+### 3D. Known Drugs Targeting This Protein
+
+```python
+# OpenTargets known drugs
+drugs = tu.tools.OpenTargets_get_associated_drugs_by_target_ensemblID(
+    ensemblId=ensembl_id, size=25
+)
+
+# ChEMBL drug mechanisms
+drug_mechanisms = tu.tools.ChEMBL_search_mechanisms(
+    target_chembl_id=target_chembl_id, limit=50
+)
+
+# Drug interaction databases
+dgidb = tu.tools.DGIdb_get_gene_info(genes=[gene_symbol])
+```
+
+### Report Format - Chemical Matter
+
+```markdown
+### 4. Known Modulators & Chemical Matter
+
+#### 4.1 Approved Drugs
+| Drug | ChEMBL ID | Mechanism | Phase | Indication | Source |
+|------|-----------|-----------|-------|------------|--------|
+| Erlotinib | CHEMBL553 | Inhibitor | 4 | NSCLC | [T1] OpenTargets |
+| Gefitinib | CHEMBL939 | Inhibitor | 4 | NSCLC | [T1] OpenTargets |
+
+#### 4.2 ChEMBL Bioactivity Summary
+**Total Activities**: 12,456 datapoints across 2,341 assays
+**Most Potent Compound**: CHEMBL413456 (IC50 = 0.3 nM) [T1]
+**Chemical Series**: 8 distinct scaffolds with pChEMBL >= 7.0
+**Selectivity Data**: Available for 45 compounds (kinase panel)
+
+#### 4.3 BindingDB Ligands
+**Total Ligands**: 856 with measured affinity
+**Best Affinity**: 0.1 nM (Ki)
+**Affinity Distribution**: <1nM: 23, 1-10nM: 89, 10-100nM: 234, 100nM-1uM: 510
+
+#### 4.4 Chemical Probes
+| Probe | Source | Potency | Selectivity | Use |
+|-------|--------|---------|-------------|-----|
+| SGC-1234 | SGC | IC50=5nM | >100x | In vitro |
+```
+
+---
+
+## Phase 4: Clinical Precedent (0-15 points)
+
+**Objective**: Assess clinical validation from approved drugs and clinical trials.
+
+### 4A. FDA-Approved Drugs
+
+```python
+# FDA label information
+fda_moa = tu.tools.FDA_get_mechanism_of_action_by_drug_name(drug_name=gene_symbol)
+fda_indications = tu.tools.FDA_get_indications_by_drug_name(drug_name=known_drug_name)
+
+# DrugBank pharmacology
+drugbank_targets = tu.tools.drugbank_get_targets_by_drug_name_or_drugbank_id(
+    query=known_drug_name, case_sensitive=False, exact_match=False, limit=10
+)
+
+# DrugBank safety info
+drugbank_safety = tu.tools.drugbank_get_safety_by_drug_name_or_drugbank_id(
+    query=known_drug_name, case_sensitive=False, exact_match=False, limit=10
+)
+```
+
+### 4B. Clinical Trials
+
+```python
+# Active clinical trials targeting this protein
+trials = tu.tools.search_clinical_trials(
+    query_term=gene_symbol,
+    intervention=gene_symbol,
+    pageSize=50
+)
+
+# If specific disease context
+if disease_name:
+    disease_trials = tu.tools.search_clinical_trials(
+        query_term=gene_symbol,
+        condition=disease_name,
+        pageSize=50
+    )
+```
+
+### 4C. Failed Programs (Learn from Failures)
+
+```python
+# Drug warnings and withdrawals
+for drug_chembl_id in known_drug_ids:
+    warnings = tu.tools.OpenTargets_get_drug_warnings_by_chemblId(chemblId=drug_chembl_id)
+    adverse = tu.tools.OpenTargets_get_drug_adverse_events_by_chemblId(chemblId=drug_chembl_id)
+```
+
+### Scoring Logic - Clinical Precedent
+
+```
+Clinical Precedent (0-15):
+  - FDA-approved drug for SAME disease: 15
+  - FDA-approved drug for DIFFERENT disease: 12
+  - Phase 3 clinical trial: 10
+  - Phase 2 clinical trial: 7
+  - Phase 1 clinical trial: 5
+  - Preclinical compounds only: 3
+  - No clinical development: 0
+
+Adjustment factors:
+  - Failed clinical program for safety: -3
+  - Drug withdrawal: -5
+  - Multiple approved drugs (validated class): +2
+```
+
+---
+
+## Phase 5: Safety & Toxicity Considerations (0-20 points)
+
+**Objective**: Identify safety risks from expression, genetics, and known adverse events.
+
+### 5A. OpenTargets Safety Profile
+
+```python
+safety = tu.tools.OpenTargets_get_target_safety_profile_by_ensemblID(ensemblId=ensembl_id)
+# Returns: safety liabilities, adverse effects, experimental toxicity
+```
+
+### 5B. Expression in Critical Tissues
+
+```python
+# GTEx tissue expression (identifies essential organ expression)
+gtex = tu.tools.GTEx_get_median_gene_expression(
+    operation="median", gencode_id=ensembl_versioned_id
+)
+# If empty, try unversioned ID
+
+# HPA expression
+# NOTE: HPA_get_rna_expression_by_source requires gene_name, source_type, source_name
+hpa = tu.tools.HPA_search_genes_by_query(search_query=gene_symbol)
+hpa_details = tu.tools.HPA_get_comprehensive_gene_details_by_ensembl_id(ensembl_id=ensembl_id)
+
+# Check expression in safety-critical tissues
+# Heart, liver, kidney, brain, bone marrow = high risk if target is expressed
+```
+
+### 5C. Knockout Phenotypes
+
+```python
+# Mouse model phenotypes
+mouse_models = tu.tools.OpenTargets_get_biological_mouse_models_by_ensemblID(ensemblId=ensembl_id)
+
+# Genetic constraint (proxy for essentiality)
+constraints = tu.tools.gnomad_get_gene_constraints(gene_symbol=gene_symbol)
+# High pLI = essential gene = potential safety concern
+```
+
+### 5D. Known Adverse Events from Target Modulation
+
+```python
+# For known drugs targeting this protein
+for drug_name in known_drug_names:
+    fda_adr = tu.tools.FDA_get_adverse_reactions_by_drug_name(drug_name=drug_name)
+    fda_warnings = tu.tools.FDA_get_warnings_and_cautions_by_drug_name(drug_name=drug_name)
+    fda_boxed = tu.tools.FDA_get_boxed_warning_info_by_drug_name(drug_name=drug_name)
+    fda_contraindications = tu.tools.FDA_get_contraindications_by_drug_name(drug_name=drug_name)
+```
+
+### 5E. Homologs & Off-Target Risks
+
+```python
+# Paralogs (close family members that might be hit)
+homologs = tu.tools.OpenTargets_get_target_homologues_by_ensemblID(ensemblId=ensembl_id)
+# Paralogs with high sequence identity = selectivity challenge
+```
+
+### Scoring Logic - Safety
+
+```
+Tissue Expression Selectivity (0-5):
+  - Target restricted to disease tissue: 5
+  - Low expression in heart/liver/kidney/brain: 4
+  - Moderate expression in 1-2 critical tissues: 2
+  - High expression in multiple critical tissues: 0
+
+Genetic Validation (0-10):
+  - Mouse KO viable, no severe phenotype: 10
+  - Mouse KO viable with mild phenotype: 7
+  - Mouse KO has concerning phenotype: 3
+  - Mouse KO lethal: 0
+  - No KO data, low pLI (<0.5): 5
+  - No KO data, high pLI (>0.9): 2
+
+Known Adverse Events (0-5):
+  - No known safety signals: 5
+  - Mild, manageable ADRs: 3
+  - Serious ADRs reported: 1
+  - Black box warning or drug withdrawal: 0
+```
+
+---
+
+## Phase 6: Pathway Context & Network Analysis
+
+**Objective**: Understand the target's role in biological networks and disease pathways.
+
+### 6A. Reactome Pathways
+
+```python
+# Map target to pathways
+pathways = tu.tools.Reactome_map_uniprot_to_pathways(id=uniprot_id)
+
+# Get pathway details for top pathways
+for pathway in top_pathways[:5]:
+    detail = tu.tools.Reactome_get_pathway(id=pathway['stId'])
+    reactions = tu.tools.Reactome_get_pathway_reactions(id=pathway['stId'])
+```
+
+### 6B. Protein-Protein Interactions
+
+```python
+# STRING network
+string_ppi = tu.tools.STRING_get_protein_interactions(
+    protein_ids=[gene_symbol], species=9606, confidence_score=0.7
+)
+# Higher confidence = more reliable
+
+# IntAct interactions (experimental)
+intact_ppi = tu.tools.intact_get_interactions(identifier=uniprot_id)
+
+# OpenTargets interactions
+ot_ppi = tu.tools.OpenTargets_get_target_interactions_by_ensemblID(ensemblId=ensembl_id)
+```
+
+### 6C. Functional Enrichment
+
+```python
+# GO annotations
+go_terms = tu.tools.OpenTargets_get_target_gene_ontology_by_ensemblID(ensemblId=ensembl_id)
+
+# Direct GO query
+go_annotations = tu.tools.GO_get_annotations_for_gene(gene_id=gene_symbol)
+
+# STRING functional enrichment of interaction partners
+enrichment = tu.tools.STRING_functional_enrichment(
+    protein_ids=[gene_symbol], species=9606
+)
+```
+
+### Report Format - Pathway Context
+
+```markdown
+### 7. Pathway Context & Network Analysis
+
+#### 7.1 Key Pathways
+| Pathway | Reactome ID | Relevance to Disease | Evidence |
+|---------|-------------|---------------------|----------|
+| EGFR signaling | R-HSA-177929 | Driver pathway in NSCLC | [T1] |
+| RAS-RAF-MEK-ERK | R-HSA-5673001 | Downstream effector | [T1] |
+| PI3K-AKT signaling | R-HSA-2219528 | Resistance mechanism | [T2] |
+
+#### 7.2 Protein-Protein Interactions
+**Total Interactors**: 45 (STRING confidence > 0.7)
+**Key Interactors**: GRB2, SHC1, PLCG1, PIK3CA, STAT3
+
+#### 7.3 Pathway Redundancy Assessment
+**Compensation Risk**: MODERATE
+- Parallel pathways: HER2, HER3 can compensate
+- Feedback loops: RAS activation bypasses EGFR
+- Downstream convergence: MEK/ERK shared with other RTKs
+```
+
+---
+
+## Phase 7: Validation Evidence (0-10 points)
+
+**Objective**: Assess existing functional validation data.
+
+### 7A. DepMap Essentiality (CRISPR/RNAi)
+
+```python
+# Gene essentiality in cancer cell lines
+deps = tu.tools.DepMap_get_gene_dependencies(gene_symbol=gene_symbol)
+# Negative scores = essential (cells die upon KO)
+# Score < -0.5: moderately essential
+# Score < -1.0: strongly essential
+```
+
+### 7B. Literature Validation Evidence
+
+```python
+# Search for functional studies
+validation_papers = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND (CRISPR OR siRNA OR knockdown OR knockout OR "loss of function") AND "{disease_name}"',
+    limit=30
+)
+
+# Search for biomarker studies
+biomarker_papers = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND (biomarker OR "target engagement" OR "pharmacodynamic")',
+    limit=20
+)
+```
+
+### 7C. Animal Model Evidence
+
+```python
+# Mouse phenotypes from OpenTargets (already retrieved in Phase 5)
+# Reuse mouse_models data
+
+# CTD gene-disease associations (complementary)
+ctd_diseases = tu.tools.CTD_get_gene_diseases(input_terms=gene_symbol)
+```
+
+### Scoring Logic - Validation Evidence
+
+```
+Functional Studies (0-5):
+  - CRISPR KO shows disease-relevant phenotype: 5
+  - siRNA knockdown shows phenotype: 4
+  - Biochemical assay validates mechanism: 3
+  - Overexpression study only: 2
+  - No functional data: 0
+
+Disease Models (0-5):
+  - Patient-derived xenograft (PDX) response: 5
+  - Genetically engineered mouse model: 4
+  - Cell line model: 3
+  - In silico model only: 1
+  - No model data: 0
+```
+
+---
+
+## Phase 8: Structural Insights
+
+**Objective**: Leverage structural biology for druggability and mechanism understanding.
+
+### 8A. PDB Structures
+
+```python
+# Get PDB entries from UniProt cross-references
+uniprot_entry = tu.tools.UniProt_get_entry_by_accession(accession=uniprot_id)
+# Parse: uniProtKBCrossReferences where database == "PDB"
+
+# Get details for each PDB
+for pdb_id in pdb_ids[:10]:
+    metadata = tu.tools.get_protein_metadata_by_pdb_id(pdb_id=pdb_id)
+    quality = tu.tools.pdbe_get_entry_quality(pdb_id=pdb_id)
+    summary = tu.tools.pdbe_get_entry_summary(pdb_id=pdb_id)
+    experiment = tu.tools.pdbe_get_entry_experiment(pdb_id=pdb_id)
+    molecules = tu.tools.pdbe_get_entry_molecules(pdb_id=pdb_id)
+```
+
+### 8B. AlphaFold Prediction
+
+```python
+alphafold = tu.tools.alphafold_get_prediction(qualifier=uniprot_id)
+alphafold_info = tu.tools.alphafold_get_summary(qualifier=uniprot_id)
+# Check pLDDT scores for confidence
+```
+
+### 8C. Binding Pocket Analysis
+
+```python
+# ProteinsPlus DoGSiteScorer for best PDB structure
+pockets = tu.tools.ProteinsPlus_predict_binding_sites(pdb_id=best_pdb_id)
+# Returns: pocket locations, druggability scores, volume, surface
+
+# Interaction diagram for co-crystal structures
+if has_ligand:
+    diagram = tu.tools.ProteinsPlus_generate_interaction_diagram(pdb_id=pdb_id)
+```
+
+### 8D. Domain Architecture
+
+```python
+# InterPro domains
+domains = tu.tools.InterPro_get_protein_domains(uniprot_accession=uniprot_id)
+
+# Domain details for key domains
+for domain in domains[:5]:
+    detail = tu.tools.InterPro_get_domain_details(entry_id=domain['accession'])
+```
+
+---
+
+## Phase 9: Literature Deep Dive
+
+**Objective**: Comprehensive literature analysis with collision-aware search.
+
+### 9A. Collision Detection
+
+```python
+# Detect naming collisions before literature search
+test_results = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}"[Title]', limit=20
+)
+# PubMed returns plain list of dicts
+# Check if >20% of results are off-topic (no biology terms)
+# If collision detected, add filters: AND (protein OR gene OR receptor OR kinase)
+```
+
+### 9B. Publication Metrics
+
+```python
+# Total publications
+total = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND (protein OR gene)', limit=1
+)
+# Check total_count field
+
+# Recent publications (5-year trend)
+recent = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND (protein OR gene) AND ("2021"[PDAT] : "2026"[PDAT])',
+    limit=50
+)
+
+# Drug-focused publications
+drug_pubs = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND (drug OR therapeutic OR inhibitor OR antibody)',
+    limit=30
+)
+
+# EuropePMC for broader coverage
+epmc = tu.tools.EuropePMC_search_articles(
+    query=f'"{gene_symbol}" AND drug target',
+    limit=30
+)
+```
+
+### 9C. Key Reviews and Landmark Papers
+
+```python
+# Reviews for target overview
+reviews = tu.tools.PubMed_search_articles(
+    query=f'"{gene_symbol}" AND drug target AND review[pt]',
+    limit=10
+)
+
+# OpenAlex for citation metrics
+openalex_works = tu.tools.openalex_search_works(
+    query=f'{gene_symbol} drug target', limit=20
+)
+```
+
+---
+
+## Phase 10: Validation Roadmap (Synthesis)
+
+**Objective**: Generate actionable recommendations based on all evidence.
+
+This phase synthesizes all previous phases into:
+1. **Target Validation Score** (0-100)
+2. **Priority Tier** (1-4)
+3. **GO/NO-GO Recommendation**
+4. **Recommended Experiments**
+5. **Tool Compounds for Testing**
+6. **Biomarker Strategy**
+7. **Key Risks & Mitigations**
+
+### Score Calculation
+
+```python
+def calculate_validation_score(phase_results):
+    """
+    Calculate Target Validation Score (0-100).
+
+    Components:
+    - Disease Association: 0-30
+    - Druggability: 0-25
+    - Safety: 0-20
+    - Clinical Precedent: 0-15
+    - Validation Evidence: 0-10
+    """
+    score = {
+        'disease_genetic': 0,      # 0-10
+        'disease_literature': 0,   # 0-10
+        'disease_pathway': 0,      # 0-10
+        'drug_structural': 0,      # 0-10
+        'drug_chemical': 0,        # 0-10
+        'drug_class': 0,           # 0-5
+        'safety_expression': 0,    # 0-5
+        'safety_genetic': 0,       # 0-10
+        'safety_adverse': 0,       # 0-5
+        'clinical': 0,             # 0-15
+        'validation_functional': 0, # 0-5
+        'validation_models': 0,    # 0-5
+    }
+
+    # ... scoring logic from each phase ...
+
+    total = sum(score.values())
+
+    if total >= 80:
+        tier = "Tier 1"
+        recommendation = "GO - Highly validated target"
+    elif total >= 60:
+        tier = "Tier 2"
+        recommendation = "CONDITIONAL GO - Needs focused validation"
+    elif total >= 40:
+        tier = "Tier 3"
+        recommendation = "CAUTION - Significant validation needed"
+    else:
+        tier = "Tier 4"
+        recommendation = "NO-GO - Consider alternatives"
+
+    return total, tier, recommendation, score
+```
+
+---
+
+## Report Template
+
+**File**: `[TARGET]_[DISEASE]_validation_report.md`
+
+```markdown
+# Drug Target Validation Report: [TARGET]
+
+**Target**: [Gene Symbol] ([Full Name])
+**Disease Context**: [Disease Name] (if provided)
+**Modality**: [Small molecule / Antibody / etc.] (if specified)
+**Generated**: [Date]
+**Status**: In Progress
+
+---
+
+## Executive Summary
+
+**Target Validation Score**: [XX/100]
+**Priority Tier**: [Tier X] - [Description]
+**Recommendation**: [GO / CONDITIONAL GO / CAUTION / NO-GO]
+
+**Key Findings**:
+- [1-sentence disease association strength with evidence grade]
+- [1-sentence druggability assessment]
+- [1-sentence safety profile]
+- [1-sentence clinical precedent]
+
+**Critical Risks**:
+- [Top risk 1]
+- [Top risk 2]
+
+---
+
+## Validation Scorecard
+
+| Dimension | Score | Max | Assessment | Key Evidence |
+|-----------|-------|-----|------------|--------------|
+| **Disease Association** | | 30 | | |
+| - Genetic evidence | | 10 | | |
+| - Literature evidence | | 10 | | |
+| - Pathway evidence | | 10 | | |
+| **Druggability** | | 25 | | |
+| - Structural tractability | | 10 | | |
+| - Chemical matter | | 10 | | |
+| - Target class | | 5 | | |
+| **Safety Profile** | | 20 | | |
+| - Expression selectivity | | 5 | | |
+| - Genetic validation | | 10 | | |
+| - Known ADRs | | 5 | | |
+| **Clinical Precedent** | | 15 | | |
+| **Validation Evidence** | | 10 | | |
+| - Functional studies | | 5 | | |
+| - Disease models | | 5 | | |
+| **TOTAL** | **XX** | **100** | **[Tier]** | |
+
+---
+
+## 1. Target Identity
+[Researching...]
+
+## 2. Disease Association Evidence
+### 2.1 OpenTargets Disease Associations
+[Researching...]
+### 2.2 GWAS Genetic Evidence
+[Researching...]
+### 2.3 Constraint Scores (gnomAD)
+[Researching...]
+### 2.4 Literature Evidence
+[Researching...]
+
+## 3. Druggability Assessment
+### 3.1 Tractability (OpenTargets)
+[Researching...]
+### 3.2 Target Classification
+[Researching...]
+### 3.3 Structural Tractability
+[Researching...]
+### 3.4 Chemical Probes & Enabling Packages
+[Researching...]
+
+## 4. Known Modulators & Chemical Matter
+### 4.1 Approved/Clinical Drugs
+[Researching...]
+### 4.2 ChEMBL Bioactivity
+[Researching...]
+### 4.3 BindingDB Ligands
+[Researching...]
+### 4.4 PubChem Bioassays
+[Researching...]
+### 4.5 Chemical Probes
+[Researching...]
+
+## 5. Clinical Precedent
+### 5.1 FDA-Approved Drugs
+[Researching...]
+### 5.2 Clinical Trial Landscape
+[Researching...]
+### 5.3 Failed Programs & Lessons
+[Researching...]
+
+## 6. Safety & Toxicity Profile
+### 6.1 OpenTargets Safety Liabilities
+[Researching...]
+### 6.2 Expression in Critical Tissues
+[Researching...]
+### 6.3 Knockout Phenotypes
+[Researching...]
+### 6.4 Known Adverse Events
+[Researching...]
+### 6.5 Paralog & Off-Target Risks
+[Researching...]
+
+## 7. Pathway Context & Network Analysis
+### 7.1 Biological Pathways
+[Researching...]
+### 7.2 Protein-Protein Interactions
+[Researching...]
+### 7.3 Functional Enrichment
+[Researching...]
+### 7.4 Pathway Redundancy Assessment
+[Researching...]
+
+## 8. Validation Evidence
+### 8.1 Target Essentiality (DepMap)
+[Researching...]
+### 8.2 Functional Studies
+[Researching...]
+### 8.3 Animal Models
+[Researching...]
+### 8.4 Biomarker Potential
+[Researching...]
+
+## 9. Structural Insights
+### 9.1 Experimental Structures (PDB)
+[Researching...]
+### 9.2 AlphaFold Prediction
+[Researching...]
+### 9.3 Binding Pocket Analysis
+[Researching...]
+### 9.4 Domain Architecture
+[Researching...]
+
+## 10. Literature Landscape
+### 10.1 Publication Metrics
+[Researching...]
+### 10.2 Key Publications
+[Researching...]
+### 10.3 Research Trend
+[Researching...]
+
+## 11. Validation Roadmap
+### 11.1 Recommended Validation Experiments
+[Researching...]
+### 11.2 Tool Compounds for Testing
+[Researching...]
+### 11.3 Biomarker Strategy
+[Researching...]
+### 11.4 Clinical Biomarker Candidates
+[Researching...]
+### 11.5 Disease Models to Test
+[Researching...]
+
+## 12. Risk Assessment
+### 12.1 Key Risks
+[Researching...]
+### 12.2 Mitigation Strategies
+[Researching...]
+### 12.3 Competitive Landscape
+[Researching...]
+
+## 13. Completeness Checklist
+[To be populated post-audit...]
+
+## 14. Data Sources & Methodology
+[Will be populated as research progresses...]
+```
+
+---
+
+## Completeness Checklist (MANDATORY)
+
+Before finalizing, verify:
+
+```markdown
+## 13. Completeness Checklist
+
+### Phase Coverage
+- [ ] Phase 0: Target disambiguation (all IDs resolved)
+- [ ] Phase 1: Disease association (OT + GWAS + gnomAD + literature)
+- [ ] Phase 2: Druggability (tractability + class + structure + probes)
+- [ ] Phase 3: Chemical matter (ChEMBL + BindingDB + PubChem + drugs)
+- [ ] Phase 4: Clinical precedent (FDA + trials + failures)
+- [ ] Phase 5: Safety (OT safety + expression + KO + ADRs + paralogs)
+- [ ] Phase 6: Pathway context (Reactome + STRING + GO)
+- [ ] Phase 7: Validation evidence (DepMap + literature + models)
+- [ ] Phase 8: Structural insights (PDB + AlphaFold + pockets + domains)
+- [ ] Phase 9: Literature (collision-aware + metrics + key papers)
+- [ ] Phase 10: Validation roadmap (score + recommendations)
+
+### Data Quality
+- [ ] All scores justified with specific data
+- [ ] Evidence grades (T1-T4) assigned to key claims
+- [ ] Negative results documented (not left blank)
+- [ ] Failed tools with fallbacks documented
+- [ ] Source citations for all data points
+
+### Scoring
+- [ ] All 12 score components calculated
+- [ ] Total score summed correctly
+- [ ] Priority tier assigned
+- [ ] GO/NO-GO recommendation justified
+```
+
+---
+
+## Fallback Chains
+
+| Primary Tool | Fallback 1 | Fallback 2 | If All Fail |
+|--------------|------------|------------|-------------|
+| `OpenTargets_get_diseases_phenotypes_*` | `CTD_get_gene_diseases` | PubMed search | Note in report |
+| `GTEx_get_median_gene_expression` (versioned) | GTEx (unversioned) | `HPA_search_genes_by_query` | Document gap |
+| `ChEMBL_get_target_activities` | `BindingDB_get_ligands_by_uniprot` | `DGIdb_get_gene_info` | Note in report |
+| `gnomad_get_gene_constraints` | `OpenTargets_get_target_constraint_info_*` | - | Note as unavailable |
+| `Reactome_map_uniprot_to_pathways` | `OpenTargets_get_target_gene_ontology_*` | - | Use GO only |
+| `STRING_get_protein_interactions` | `intact_get_interactions` | `OpenTargets interactions` | Note in report |
+| `ProteinsPlus_predict_binding_sites` | `alphafold_get_prediction` | Literature pockets | Note as limited |
+
+---
+
+## Modality-Specific Considerations
+
+### Small Molecule Focus
+- Emphasize: binding pockets, ChEMBL compounds, Lipinski compliance
+- Key tractability: OpenTargets SM tractability bucket
+- Structure: co-crystal structures with small molecule ligands
+- Chemical matter: IC50/Ki/Kd data from ChEMBL/BindingDB
+
+### Antibody Focus
+- Emphasize: extracellular domains, cell surface expression, glycosylation
+- Key tractability: OpenTargets AB tractability bucket
+- Structure: ectodomain structures, epitope mapping
+- Expression: surface expression in disease vs normal tissue
+
+### PROTAC Focus
+- Emphasize: intracellular targets, surface lysines, E3 ligase proximity
+- Key tractability: OpenTargets PROTAC tractability
+- Structure: full-length structures for linker design
+- Chemical matter: known binders + E3 ligase binders
+
+---
+
+## Quick Reference: Verified Tool Parameters
+
+| Tool | Parameters | Notes |
+|------|-----------|-------|
+| `ensembl_lookup_gene` | `gene_id`, `species` | species="homo_sapiens" REQUIRED; response wrapped in `{status, data, url, content_type}` |
+| `OpenTargets_get_*_by_ensemblID` | `ensemblId` | camelCase, NOT ensemblID |
+| `OpenTargets_get_publications_by_target_ensemblID` | `entityId` | NOT ensemblId |
+| `OpenTargets_get_associated_drugs_by_target_ensemblID` | `ensemblId`, `size` | size is REQUIRED |
+| `OpenTargets_target_disease_evidence` | `efoId`, `ensemblId` | Both REQUIRED |
+| `GTEx_get_median_gene_expression` | `operation`, `gencode_id` | operation="median" REQUIRED |
+| `HPA_get_rna_expression_by_source` | `gene_name`, `source_type`, `source_name` | ALL 3 required |
+| `PubMed_search_articles` | `query`, `limit` | Returns plain list, NOT {articles:[]} |
+| `UniProt_get_function_by_accession` | `accession` | Returns list of strings |
+| `alphafold_get_prediction` | `qualifier` | NOT uniprot_accession |
+| `drugbank_get_safety_*` | `query`, `case_sensitive`, `exact_match`, `limit` | ALL required |
+| `STRING_get_protein_interactions` | `protein_ids`, `species` | protein_ids is array; species=9606 |
+| `Reactome_map_uniprot_to_pathways` | `id` | NOT uniprot_id |
+| `ChEMBL_get_target_activities` | `target_chembl_id__exact` | Note double underscore |
+| `search_clinical_trials` | `query_term` | REQUIRED parameter |
+| `gnomad_get_gene_constraints` | `gene_symbol` | NOT gene_id |
+| `DepMap_get_gene_dependencies` | `gene_symbol` | NOT gene_id |
+| `BindingDB_get_ligands_by_uniprot` | `uniprot`, `affinity_cutoff` | affinity in nM |
+| `Pharos_get_target` | `gene` or `uniprot` | Both optional but need one |
+
+---
+
+## Example Execution: EGFR for NSCLC
+
+### Phase 0 Result
+- Symbol: EGFR, Ensembl: ENSG00000146648, UniProt: P00533, ChEMBL: CHEMBL203
+
+### Expected Scores (EGFR for NSCLC)
+- Disease Association: ~28/30 (strong genetic + pathway + literature)
+- Druggability: ~24/25 (kinase, many structures, abundant compounds)
+- Safety: ~14/20 (widely expressed but manageable toxicity)
+- Clinical Precedent: 15/15 (multiple approved drugs)
+- Validation Evidence: ~9/10 (extensive functional data)
+- **Total: ~90/100 = Tier 1**
+
+### Example for Novel Target (e.g., understudied kinase)
+- Disease Association: ~8/30 (limited GWAS, few publications)
+- Druggability: ~15/25 (kinase family bonus, AlphaFold structure)
+- Safety: ~12/20 (limited data, unknown KO phenotype)
+- Clinical Precedent: 0/15 (no clinical development)
+- Validation Evidence: ~2/10 (minimal functional data)
+- **Total: ~37/100 = Tier 4**