Tc-43/RPIA_Inhibitor_Designs_GA-II

RPIA Inhibitor Designs — Technetium GA-II

21 de novo small-molecule designs against RPIA (ribose-5-phosphate isomerase A), a first-in-class longevity target, each docked into a rigid AlphaFold receptor and supplied as a full protein–ligand complex.

Produced by an end-to-end agentic pipeline: Biomni performed target discovery, novelty filtering, safety profiling and pocket analysis; the Technetium TC-43.ai engine (GA-II) performed pocket-conditioned, scaffold-constrained generative design and docking.

Target	RPIA — ribose-5-phosphate isomerase A
UniProt	P49247 · EC 5.3.1.6
Designs	21
Vina	−8.0 to −8.9 kcal/mol
Status	Testable computational hypothesis — nothing synthesised or assayed

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Summary

Aging is the largest unaddressed therapeutic area in medicine. Hundreds of genes are known to modulate lifespan in model organisms, yet almost none have entered drug development — most are considered undruggable, or already carry inhibitor programs that disqualify them as novel opportunities. This dataset is the output of a pipeline built to close that gap.

RPIA catalyses the reversible isomerisation of ribose-5-phosphate to ribulose-5-phosphate in the pentose phosphate pathway, which supplies NADPH for redox homeostasis and ribose-5-phosphate for nucleotide biosynthesis. Inhibition reduces nucleotide biosynthesis and activates cellular quality-control pathways.

Genetic validation

System	Intervention	Effect
C. elegans	rpia-1 RNAi knockdown	Lifespan extension + healthspan (oxidative stress tolerance, reduced polyQ aggregation)
C. elegans	Neuronal-specific knockdown	Sufficient for lifespan extension (glutamatergic or cholinergic)
Drosophila	Rpi knockdown	Healthspan improvement
Human MSCs	RPIA target identification	Senescence-reversal target (AUROC = 0.909)
Lung cancer	RPIA knockdown	ROS, autophagy, apoptosis, senescence induction

The pro-longevity mechanism requires autophagy and AMPK activation with reduced TOR signalling, placing RPIA inhibition on the canonical mTOR/AMPK/autophagy axis. That neuronal-specific knockdown alone suffices indicates tissue-targeted strategies are viable.

Novelty — first-in-class

Source	Finding
ChEMBL	6 records, all from one incidental chemoproteomics screen (IC50 ≈ 10 µM)
Literature	No medicinal chemistry campaigns against human RPIA; a 2024 review states selective RPI inhibitors remain a challenge
Patents	No patents claiming human RPIA inhibitors
Known analogs	Substrate analogs exist for spinach and bacterial RPI only — phosphate-containing fragments, not drug-like

Safety

Source	Metric	Interpretation
gnomAD	pLI = 2.54 × 10⁻⁸, oe_lof = 0.768	Loss-of-function tolerant in humans
DepMap	Mean gene effect −0.33, 28.5% dependent	Mild essentiality, comparable to known drug targets
HPA	Unprognostic across all TCGA cancer types	No cancer prognostic liability

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Workflow

1. Target identification — Biomni

Roughly 15 candidates were assembled from CRISPR-based lifespan screens in C. elegans, Drosophila genetic studies, AI-predicted longevity targets, and senescence CRISPR screens — among them HAAO, GLUD1, PITPNA, MAPK9, CNGA3, RPIA, XPO7, DIS3, BANF1, PRPF19, SLC25A1 and FMO2. These passed through a filtering cascade on five dimensions — novelty, genetic validation, structural availability, druggability and safety.

RPIA was the only candidate to clear all five. Competitors failed on prior art (MAPK9, 1034 ChEMBL entries; CNGA3, ~500; HAAO, 58 including the tool compound NCR-631) or on druggability (BANF1, DNA-binding; XPO7, no structure and hard to drug).

2. Structure and pocket modelling — Biomni

No experimental structure of human RPIA exists in the PDB, so the AlphaFold model AF-P49247-F1 (model_v6) was used. Active-site residues were assigned by mapping from the high-resolution E. coli RpiA crystal structure (PDB 1O8B, 1.25 Å, bound arabinose-5-phosphate) at 19.2% sequence identity. Pocket-specific pLDDT was 96–99, with 100% of pocket residues above 90.

The site is an elongated inter-domain cleft — 49 residues within 12 Å of the centroid — resolving into three sub-pockets:

Sub-pocket	Residues	Pharmacophore implication
Catalytic centre	Glu164, Asp168, Asn255	H-bond donor / acceptor
Phosphate binding	Lys183, Lys276, Lys173	Cation–π target (cationic)
Sugar-ring binding	Asp259, Asp283, Trp260 + hydrophobic wall	Hydrophobic / aromatic fill

Low identity to the bacterial template meant the initial homology mapping needed correction at four positions (His168→Asp168, Arg181→Gln181, His255→Asn255, Arg280→Gly280) before constraints were issued — a reminder that structure-based docking is more robust to imperfect homology mapping than residue-level annotation is.

3. Design constraints issued to GA-II

#	Constraint	Specification
1	H-bond donor/acceptor	Match the catalytic-centre H-bond pattern
2	Cation–π capable aromatic	Aromatic rings to engage the cationic Lys pocket
3	Hydrophobic/aromatic group	Fill the sugar-ring sub-pocket
4	Elongated geometry	Span the cleft, ≈ 15 Å end-to-end
5	MW 300–500 Da	Balance potency and drug-likeness
6	No phosphate group	Neutral interactions, not phosphate mimics

4. Ligand generation — Technetium GA-II

The TC-43.ai engine ran pocket-conditioned, scaffold-constrained generative design against the receptor and constraints above, docking each candidate with AutoDock Vina and filtering to 21 complexes. The series converged on a naphthalene–imidazolidinone core: the fused aromatic system supplies the cation–π surface, the cyclic urea the H-bond donor/acceptor pair, and a single variable exit substituent carries the diversity.

A deliberate design choice was to pursue cation–π rather than ionic engagement of the cationic pocket. Cation–π interactions are worth roughly 5–15 kcal/mol in biological contexts — comparable to a salt bridge — but require no formal charge on the ligand, so they cost nothing in permeability. For a longevity target where broad tissue exposure and possible CNS penetration are desirable, avoiding permanently charged carboxylates or tetrazoles is the correct trade. Four ligands carry weakly basic amines (pKa ≈ 9–10) that equilibrate with a membrane-permeable free base, the same mechanism that lets memantine and donepezil reach the CNS.

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Contents

File	Description
designs.csv	One row per design — scores, physicochemical properties, measured contacts
ligands.sdf	21 docked ligand poses, 3D, bond orders and formal charges assigned
receptor_RPIA.pdb	Docking receptor, 311 residues, chain A
structures/	21 complex PDBs with REMARK SMILES and REMARK VINA RESULT

Receptor

AlphaFold model AF-P49247-F1 (UniProt P49247, human RPIA, 311 aa). Docking used a rigid receptor — chain A is byte-identical across all 21 complexes, so poses are directly superposable without alignment.

The designs

Designs	21
Vina score	−8.0 to −8.9 kcal/mol
Ligand efficiency	0.38 – 0.44
Molecular weight	348 – 452 Da
QED	0.35 – 0.82
Lipinski violations	0 in 16/21; 5 fail on cLogP > 5
Veber	21/21 pass
Aromatic rings	2 – 4
Murcko scaffolds	17 distinct across 21 designs

Binding mode

Recomputed from the deposited complexes. Contact = any ligand heavy atom within 4.5 Å.

Residue	Poses in contact
Lys183, Ile184, Asn272, Asp283, Leu286, Phe287, Ile288, Met290	21/21
Lys276	20/21
Thr180, Val282	19/21

Hydrogen bonds (ligand N/O to receptor N/O, < 3.5 Å): Ile288 backbone in 21/21 poses, Asn272 15/21, Leu286 9/21, Asp283 7/21.

The 21 poses form one tight cluster — maximum pairwise centroid spread 3.0 Å — in a hydrophobic sub-pocket walled by Leu286 / Phe287 / Ile288 / Met290 / Val282, with the cationic residues Lys183 and Lys276 at one rim.

Cation–π geometry

Aromatic-ring-centroid to Lys N^ζ distances:

Residue	Best	Mean	< 6.0 Å
Lys183	5.29 Å	5.44 Å	21/21
Lys276	4.82 Å	6.24 Å	5/21

Lys183 is engaged by every ligand in the series within the conventional 6 Å cation–π cutoff, making it the anchor of the binding mode; the ammonium sits 27–43° off the ring axis, so these are edge-on rather than axial contacts. Lys276 provides a secondary contact across part of the series.

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The testable hypothesis

Small-molecule RPIA inhibitors from this GA-II naphthalene–urea series will (1) inhibit recombinant human RPIA enzyme activity with measurable IC50, and (2) phenocopy the lifespan extension observed with rpia-1 genetic knockdown in C. elegans — validating RPIA as a druggable longevity target.

Validation roadmap

Step	Method	Go criterion	Timeline
1. Enzyme inhibition	Express and purify human RPIA; IC50 for top 5 compounds by coupled spectrophotometric assay (R5P → Ru5P)	≥ 1 compound with IC50 < 50 µM	2–3 months
2. Cellular activity	PPP flux (NADPH), autophagy markers (LC3-II, p62), AMPK phosphorylation	Reduced PPP flux + autophagy/AMPK activation at non-toxic concentrations	2–3 months
3. C. elegans lifespan	Compound in liquid culture or NGM plates vs vehicle; rpia-1 RNAi as positive control	Significant lifespan extension (p < 0.05, log-rank) comparable to RNAi	3–4 months

Total ≈ 7–10 months from synthesis to lifespan result.

Status and limitations

These are computational designs, not validated compounds. Nothing here has been synthesised or assayed.

Gap	Issue	Resolution
Docking ≠ binding	Vina scores rank poses; they do not measure affinity	Enzyme assay required
Genetic knockdown ≠ pharmacological inhibition	RNAi reduces protein levels; small molecules inhibit activity	C. elegans compound testing
AlphaFold ≠ experimental structure	Pocket geometry may differ from the real protein	Crystallography or cryo-EM
No selectivity data	The naphthalene-urea scaffold may bind other targets	Counter-screening

Docking was rigid-receptor throughout — no side-chain relaxation.

Citation

Technetium Therapeutics (2026). RPIA Inhibitor Designs — Technetium GA-II.
Target discovery, pocket analysis: Biomni. Generative design: Technetium TC-43.ai engine.