15-PGDH Target Analysis & Binder Design Strategy

🔬 Adaptyv × Nucleate Berlin · Binder Design Competition 2025

15-PGDH Target Analysis
& Binder Design Strategy

Sequence · Structure · Conservation · Inhibitors · Design Rationale

Target Protein

15-PGDH / HPGD (UniProt P15428)

Protein Size

266 amino acids · ~29 kDa/subunit

Family

SDR (Short-Chain Dehydrogenase/Reductase)

Cofactor

NAD⁺ (obligate)

2

15-PGDH — Protein Overview & Biology

Protein Identity

266

amino acids / subunit

~29

kDa per subunit

2

subunits (homodimer)

1.65

Å best resolution (2GDZ)

NAD⁺

obligate cofactor

Domain Architecture (266 aa)

Key Biological Facts

Property	Value
Full name	15-hydroxyprostaglandin dehydrogenase
Gene	HPGD (SDR36C1) · Chr 4q34.1
Reaction	PGE₂ + NAD⁺ → 15-keto-PGE₂ + NADH
Quaternary structure	Obligate homodimer (α9 helix packing)
Expression	Lung, colon, placenta, stomach
Disease role	Tumor suppressor — inactivated in CRC, lung, gastric cancers
Therapeutic interest	Inhibition → anti-inflammatory, bone marrow, DMD

⚡ Why This Target

15-PGDH degrades PGE₂, a pro-inflammatory eicosanoid that suppresses immunity and promotes tumor growth. Inhibiting 15-PGDH raises tissue PGE₂ → accelerates hematopoietic recovery after transplantation and promotes muscle regeneration in DMD.

🔑 Binder Opportunity

The substrate binding pocket (adjacent to NAD⁺) is large, well-defined, and validated — SW033291 achieves Ki 0.1 nM. The dynamic lid domain (F185/Y217 hinges) is unique to 15-PGDH and offers a selectivity handle over other SDR family members.

⚠️ Design Constraint

NAD⁺ is obligate cofactor at near-saturating cellular concentrations (~0.5 mM). Avoid NAD⁺-site competition. Best approach: substrate-competitive pocket OR allosteric lid domain targeting.

3

Structural Architecture — SDR Fold & Catalytic Tetrad

Structural Detail

Active Site — Rossmann Fold + Catalytic Tetrad (PDB 2GDZ)

Reaction Mechanism

Y151 (pKa raised by K155) abstracts proton from C15–OH of PGE₂ → hydride transferred to NAD⁺ → 15-keto-PGE₂ released

GxGxxG Motif (pos 12–18)

G·A·A·Q·G·I·G — Gly16 (0.856) and Gly18 (0.903) coordinate NAD⁺ pyrophosphate via backbone NH

Catalytic Tetrad — Residue Detail

Residue	Role	Scorecon	Notes
Asn107	Substrate anchor	0.8448	H-bonds C15–OH of PGE₂; tetrad entry point
Ser138	Proton relay	0.9396	H-bond network bridging Tyr151 to substrate
Tyr151	Catalytic base ★	1.0000	Perfectly conserved — all SDR orthologs
Lys155	Tyr151 activator	0.9756	Ion-dipole lowers Tyr pKa ~4 units

Homodimer Interface

α9 Antiparallel Helix Packing

F161 · A146 · Y206 · L167 · L171

Primary dimerization element. Disrupting dimerization abolishes activity — both subunits required for correct active site geometry.

Interface Area ~1200 Å²

Hydrophobic core; conserved F264

Large buried surface. Stable under physiological conditions. Good target for protein binder approach via surface complementarity.

🏆 Best Structure: PDB 2GDZ (1.65 Å)

Highest-resolution human 15-PGDH structure with NAD⁺ bound. Complete substrate-binding cleft visible. Use as primary template for all docking and binder design calculations.

4

Key PDB Structures & Structural Resources

Structure Database

1.65

Å — best resolution (2GDZ)

8+

PDB entries available

2023

cryo-EM lid domain solved

AF2

AlphaFold model available

PDB ID	Complex / Ligand	Resolution	Method	Key Insight	Priority
2GDZ	Human 15-PGDH + NAD⁺ (homodimer)	1.65 Å	X-ray	Highest-resolution human structure; defines substrate cleft; Rossmann fold + catalytic tetrad in active conformation	★★★★★
2HHQ	NAD⁺ + substrate analog	2.1 Å	X-ray	Productive NAD⁺ + substrate co-binding; defines substrate vector and key pocket contacts	★★★★
2O8U	Indomethacin-bound	2.3 Å	X-ray	First small molecule co-crystal; substrate pocket accommodates larger scaffolds	★★★★
cryo-EM	SW033291-bound (Huang et al. 2023, Nat Commun)	3.2 Å	Cryo-EM	Reveals dynamic lid domain — F185/Y217 hinges; lid closes over inhibitor. First view of full conformational change	★★★★
AF-P15428	AlphaFold2 predicted model	in silico	ML	Full-length model including flexible N-term; useful for binder design vs. regions not in X-ray	★★★

Recommended Templates for Binder Design

2GDZ (1.65 Å, NAD⁺ complex) — primary template; highest-precision substrate pocket geometry.
Cryo-EM SW033291 structure (Huang 2023) — lid-closed conformation for allosteric strategies.
Combining open (2GDZ) + closed (cryo-EM) maximizes coverage of conformational states.

Lid Domain Uniqueness

Lid domain is disordered in apo structures, ordered only upon inhibitor/substrate binding. Binders that engage F185/Y217 achieve conformational selectivity not possible with rigid-site targeting.

5

Conservation Analysis — Valdar Scorecon (150 Orthologs)

Computational · MSA Analysis

Data Source

NCBI BLASTp vs SwissProt
E < 1×10⁻⁵ · ≥25% identity
150 homologs retained

MSA

MUSCLE v5.3
150 seqs × 732 columns
Henikoff sequence weighting

Scoring

Valdar (2002) Scorecon
BLOSUM62 normalized
Weighted pairwise scoring

Results

Score range: 0.0 – 1.0
13 positions > 0.80
Tyr151 = 1.0000 (perfect)

Per-Residue Scorecon Heatmap — All 266 Positions (hover for details)

Score: <0.35 0.35–0.50 0.50–0.65 0.65–0.75 0.75–0.85 0.85–0.95 >0.95 threshold 0.80

All 13 Highly Conserved Positions (Scorecon > 0.80)

Position	AA	Score	Module	Known Role
Tyr151	Y	1.0000	Catalytic Tetrad	General base; proton abstraction from C15–OH; perfectly conserved across all SDRs
Lys155	K	0.9756	Catalytic Tetrad	Activates Tyr151 by lowering pKa; essential for activity
Ser138	S	0.9396	Catalytic Tetrad	Proton relay; H-bond to Tyr151 and substrate
Pro183	P	0.9069	Lid Hinge Region	Structural constraint adjacent to F185 lid hinge
Ile133	I	0.9029	Substrate Pocket	Hydrophobic core; shapes substrate vector toward Y151
Gly18	G	0.9027	Rossmann GxGxxG	NAD⁺ pyrophosphate contact; critical for cofactor binding
Val176	V	0.8943	Lid Region	Hydrophobic core near lid base; positions lid for substrate entry
Gly131	G	0.8477	Substrate Pocket	Structural Gly enabling tight packing in catalytic domain
Asn107	N	0.8448	Catalytic Tetrad	Anchors substrate C15–OH; first member of tetrad
Gly16	G	0.8557	Rossmann GxGxxG	NAD⁺ phosphate binding via backbone NH
Ala92	A	0.8332	Cofactor Loop	Part of NAD⁺ binding loop; nicotinamide ring positioning
Asp64	D	0.8208	NAD⁺ Binding	Anchors adenine ribose 2'-OH; conserved SDR Asp
Met1	M	0.8487	N-terminus	High within SDR family; likely alignment boundary artifact

6

Conservation Insights — Key Findings for Binder Design

Interpretation

Critical Observations

★ Entire Catalytic Tetrad Is Highly Conserved

N107 (0.84), S138 (0.94), Y151 (1.00), K155 (0.98) — all 4 tetrad members rank in the top 13. Any binder contacting these positions will compete with the catalytic mechanism and show potent inhibition of enzymatic activity.

⚡ Lid Domain Hinges Are Low Conservation

F185 (0.163) and Y217 (0.288) — the cryo-EM-defined lid hinges — show low scores. The lid diverged across SDR family members. Lid-targeting binders will be species- and isoform-selective, a significant advantage for therapeutic development.

Pro183 — Underappreciated Hotspot (0.9069)

4th highest-scoring residue. Adjacent to F185 lid hinge. Acts as a rigid structural constraint before the lid. Targeting Pro183 may lock the lid in closed state — a novel allosteric mechanism not yet exploited by any known inhibitor.

GxGxxG: G18 > G16 (Both High)

Gly18 (0.903) and Gly16 (0.856) confirm NAD⁺ as obligate across all 150 orthologs. These glycines are structurally irreplaceable — define the cofactor selectivity filter of the Rossmann fold.

Score Distribution Summary

1

perfect score (1.00)

12

high (> 0.80)

~28

moderate (> 0.65)

4

catalytic tetrad in top 13

Conservation vs. Known Function

Residue	Score	Literature Role	Design Use
Tyr151	1.000	Catalytic base	Primary anchor — potency
Lys155	0.976	pKa modulator	Co-anchor with Y151
Ser138	0.940	Proton relay	Pharmacophore hydrogen bond
Phe185	0.163	Lid hinge (cryo-EM)	Species-selective contact
Tyr217	0.288	Lid hinge (cryo-EM)	Species-selective contact
Asp64	0.821	NAD⁺ adenine anchor	Avoid — cofactor binding site
Phe161	0.410	Dimer interface	Alternative allosteric site

        Design verdict: Lead with substrate pocket (Y151/K155/S138) for potency. Layer in lid domain (F185/Y217) contacts for selectivity. Avoid NAD⁺ site competition.
      

7

NAD⁺ Cofactor — Four Critical Roles in 15-PGDH Function

Cofactor Biology

NAD⁺ is not merely an electron acceptor — it structurally organizes the active site, sets reaction geometry, and gates substrate binding via ordered kinetics.

① Hydride Acceptor — Direct Chemistry

C4 of nicotinamide ← hydride from C15 of PGE₂

The B-face of the NAD⁺ nicotinamide ring accepts the pro-S hydride from PGE₂ C15–OH, producing NADH + 15-keto-PGE₂. This is the direct chemistry that inactivates PGE₂ inflammatory signaling.

② Structural Organizer — Rossmann Fold

G12·G16·G18 (GxGxxG) + D64 + N91 + A92

NAD⁺ binding via the Rossmann fold stabilizes the entire cofactor domain. GxGxxG contacts pyrophosphate backbone; Asp64 anchors adenine ribose 2'-OH. Cofactor binding is obligate for correct tertiary structure.

③ Active Site Organizer — Substrate Positioning

Nicotinamide ring · T188 · V186 · G184

The bound nicotinamide ring forms the floor of the substrate pocket, positioning PGE₂ C15 within 3.5 Å for hydride transfer. Without NAD⁺ the substrate pocket collapses — explaining the strict ordered binding mechanism.

④ Kinetic Gate — Ordered Bi-Bi Mechanism

Order: NAD⁺ first → PGE₂ second → NADH last

15-PGDH follows ordered bi-bi kinetics: NAD⁺ must bind first to open the substrate portal. NADH releases last after oxidation. This gating means binders to the substrate site must compete with substrate, not cofactor — a more tractable competition.

NAD⁺ Km & Cellular Context

Km(NAD⁺) ≈ 20–80 µM; cellular NAD⁺ ~0.5 mM → enzyme is normally NAD⁺-saturated in vivo. Binders competing with NAD⁺ face a ~10–25× concentration disadvantage.

NADH Product Inhibition

NADH is a competitive inhibitor (Ki ~15 µM). NAD⁺/NADH ratio partially controls enzyme activity in vivo. Oxidative stress → increased NAD⁺ → more PGE₂ degradation.

Design Implication

Avoid displacing NAD⁺ — extremely high competition barrier. Best targets: substrate pocket (opens only after NAD⁺ binds) or lid domain (gates substrate entry). Both are accessible from the enzyme exterior.

8

Known Small Molecule Inhibitors — Structural Templates

Ligand Chemistry

Compound	Affinity	Mode	Key Contacts	Clinical Status	Template Value
SW033291 Best-in-class	Ki ≈ 0.1 nM	Noncompetitive vs. PGE₂	S138 · Y151 · F185 · Y217	Preclinical (bone marrow, hematopoiesis)	★★★★★ — cryo-EM co-structure; spans pocket + lid
SW209415	Ki ≈ 2 nM	Noncompetitive	S138 · Y151 · L139 · I190	Preclinical	★★★★ — expanded pocket contact map
SW222746	Ki ≈ 5 nM	Competitive vs. NAD⁺	G16 · G18 · D64 · A92	Preclinical	★★ — NAD⁺ site; hard to outcompete in vivo
ML148	IC₅₀ ≈ 25 nM	Mixed	Q148 · V145 · T246	Probe compound (MLPCN)	★★★ — outer substrate pocket contacts
MF-300 (Epirium Bio) Phase 1 ✓	IC₅₀ ≈ 0.8 nM	Substrate-competitive	S138 · Y151 · K155 · L139 · I194	Phase 1 complete Sep 2025 (oral, DMD)	★★★★★ — first clinical; validates target in humans
Nimbus compound	IC₅₀ < 10 nM	Undisclosed	Not published	Early discovery	★★ — confirms active drugging competition

★ SW033291 — Pharmacophore Template for Protein Binder

SW033291 (Ki 0.1 nM) contacts S138, Y151, F185, Y217 — spanning both conserved catalytic tetrad and variable lid domain. The cryo-EM structure shows lid closing over the compound to form a ~900 Å² buried surface. A protein binder mimicking this pharmacophore but adding 2–3× more surface area should achieve sub-pM affinity. This is the primary template for Strategy A+B hybrid binder design.

Clinical Validation: MF-300

MF-300 (Epirium Bio) entered Phase 1 for DMD in 2024 and completed dosing September 2025. This is the strongest possible target validation — 15-PGDH inhibition is safe and mechanistically active in humans at drug doses.

9

Binder Design Strategy — Three Complementary Approaches

Design Strategy

Strategy A — Substrate-Competitive Pocket Binder

Target: Y151 · K155 · S138 · N107 · L139 · V145 · Q148 · I190 · I194

Design a protein that occupies the PGE₂ substrate binding site. Anchored by perfectly conserved Y151 (1.000) and K155 (0.976). Pocket opens only after NAD⁺ binds → design against 2GDZ (NAD⁺-bound form).

Rationale: SW033291 (Ki 0.1 nM) and MF-300 (IC₅₀ 0.8 nM) validate this pocket. A protein binder with 2–3× more buried surface area should reach sub-nM Kd by entropy-enthalpy compensation on a larger interface.

Strategy B — Lid Domain Conformational Capture

Target: F185 · Y217 · P183 · V176 · I190 · I194 · T188

Stabilize the "closed" lid conformation seen in cryo-EM. F185/Y217 are low conservation (0.16/0.29) → species-selective. Binder locks lid closed over substrate site — allosteric inhibition without touching NAD⁺.

Rationale: Higher selectivity vs. other SDR family members due to lid sequence divergence. Cryo-EM provides a precise structural model of the closed state for design template.

Strategy C — Dimer Interface Disruption

Target: F161 · Y206 · L167 · L171 · A146 · F264

Target the α9 antiparallel dimer interface (~1200 Å² buried). Disrupting dimerization abolishes activity — both subunits are required. Interface is hydrophobic; flat but sizeable.

Rationale: Isoform-selective — only homodimeric 15-PGDH is disrupted. Low risk of cross-reacting with SDR monomers. Lower conservation (0.4–0.5) means lower selectivity pressure but a novel epitope.

Prioritized Hotspot Residues

Residue	Score	Strategy	Contact Type	Priority
Tyr151	1.000	A	H-bond acceptor (OH), π-stacking	Must include — primary anchor
Lys155	0.976	A	Salt bridge / H-bond (ε-NH₃⁺)	Must include — co-anchor
Ser138	0.940	A	H-bond donor/acceptor (OH)	High priority pharmacophore
Pro183	0.907	B	Hydrophobic / structural constraint	High priority — lid anchor point
Phe185	0.163	B	Hydrophobic / π-stacking	Selectivity — use for isoform specificity
Tyr217	0.288	B	H-bond / aromatic stacking	Selectivity — use for isoform specificity
Asn107	0.845	A	H-bond donor/acceptor (amide)	High priority — tetrad entry
Phe161	0.410	C	Hydrophobic / π-stacking (dimer)	Alternative strategy

🏆 Recommended Primary Strategy: A + B Hybrid

Design a binder that anchors to the substrate pocket (Y151/K155/S138) AND reaches into the lid domain (F185/Y217) when lid is in the closed conformation. This mirrors SW033291's mechanism but with 2–3× greater buried surface area. Use 2GDZ + cryo-EM lid-closed model as dual templates for de novo backbone generation.

10

Computational Pipeline & Competition Summary

Pipeline · Summary

Recommended Computational Design Pipeline

Step 1

Structure Prep

2GDZ + cryo-EM

Step 2

Pocket Analysis

fpocket / SiteMap

Step 3

Hotspot Map

FTMap / Rosetta

Step 4

Backbone Design

RFdiffusion

Step 5

Sequence Design

ProteinMPNN

Step 6

Filter + Rank

AF2 · ESMFold · ΔG

Step 7

Submit 100 Best

Adaptyv BLI assay

Master Residue Reference

Category	Residues	Design Role
Catalytic Tetrad	N107 · S138 · Y151 · K155	Primary anchor — potency driver
Rossmann / NAD⁺	G12 · G16 · G18 · D64 · A92	Avoid — cofactor competition
Substrate Pocket	L139 · A140 · V145 · Q148 · I190 · I194	Shape complementarity
Lid Domain	P183 · F185 · V186 · T188 · Y217	Selectivity over other SDRs
Dimer Interface	F161 · L167 · L171 · Y206 · F264	Alternative allosteric site

Key References

• Cho et al. (2006) Proc Natl Acad Sci — PDB 2GDZ, 1.65 Å human structure
• Huang et al. (2023) Nat Commun — cryo-EM lid domain, SW033291 mechanism
• Seo et al. (2003) J Biol Chem — SDR catalytic mechanism
• Epirium Bio (2025) — MF-300 Phase 1 completion press release

Top 5 Insights for Competition Entry

Tyr151 is perfectly conserved (1.000) — computational and experimental data agree; use it as the primary anchor in every design
SW033291 (Ki 0.1 nM) proves the pocket — a protein binder with 2–3× more buried surface should reach pM range by design
Lid domain (F185/Y217) enables selectivity — low conservation makes these residues ideal for isoform-specific designs
MF-300 Phase 1 validates in-vivo safety — 15-PGDH inhibition is tolerated in humans at therapeutic doses
Ordered bi-bi kinetics: design against NAD⁺-bound form — use 2GDZ (NAD⁺ co-crystal) for maximal pocket fidelity

          Competition target: 100 designs, Strategy A+B hybrid. Pharmacophore: Y151·K155·S138·F185·Y217. Pipeline: RFdiffusion → ProteinMPNN → AF2 validation → BLI screening.
        

11

3D Structure — 15-PGDH Monomer (PDB 2GDZ, X-ray 1.65 Å)

Interactive · 3D Viewer

          Loading PDB 2GDZ...
Chain A · NAD⁺ complex
        

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Color Legend

White — Protein backbone

All other residues, chain A monomer

Red — Catalytic Tetrad

N107 · S138 · Y151 · K155

Scorecon: 0.84–1.00 (Y151 perfect)

Orange — Dimer Interface

56 residues <5 Å from partner chain

Computed from biological assembly

Purple — Lid Domain

Residues 181–220 · F185 · Y217 hinges

Disordered in apo; open in 2GDZ

Yellow — NAD⁺ cofactor

Obligate cofactor (residue 300, chain A)

2GDZ — X-ray Crystal Structure

1.65 Å resolution. NAD⁺-bound, lid domain open. Use as primary template for substrate pocket docking and binder design.