Mitochondrial Potentiation Screen in Aged Hematopoietic Stem Cells Identified Known and Novel Rejuvenation Drugs

Aging progressively impairs Hematopoietic stem cell (HSC) function to culminate in myeloid skewing, expanded frequency and diminished regenerative potential. Previously we found that Urolithin A (UroA) enhanced aged HSC fitness by rescuing impaired mitochondrial function. Here we report a first drug screening in HSCs to benchmark known mitochondrial boosting agents and identify new compounds improving mitochondrial fitness in aged cells.

Given the encouraging results on Urolithin A, we assembled 395 analogues using an advanced ligand-based virtual screen of a 40M compound library. Furthermore, we included 21 exploratory compounds with beneficial HSC modulating activity, including 2 natural analogues (Apigenin, Luteolin), 10 synthetic HSC activating compounds (Stemregenin 1, Mitoquinol, etc.), and 9 natural HSC boosting compounds (ATRA, Vitamin C, NMN, etc.). Multiparametric Mitoprofiling by Flow Cytometry (MMFlow) parallelized separate read-outs comprising cell viability, Lineage-/Sca-1+/Kit+ (LSK) immunophenotypic purity, mitochondrial mass (MT) and membrane potential (Ψ), CD38 and EPCR expression.

Screening data of 5 biological replicates was indexed, trimmed, and filtered for the Top50 compounds reducing median MT or Ψ, respectively, with reference to vehicle control (DMSO). The Top50[Ψ] contained UroA with 67% Ψ signal of control levels at rank 14, and 4 exploratory compounds Apigenin, Mitoquinol, Luteolin, 4-Octyl Itaconate at ranks 9, 12, 16, and 50. Clustering of Top50[Ψ] by maximum common substructure matching, minus exploratory compounds not analogues to UroA and plus Fisetin from a pilot screen experiment, yielded 7 clusters of structural subfamilies.

Next, we run dose response series by MMFlow, selecting 20 Top50[Ψ] candidates informed through structural similarities. Using the total number of live cells as an approximation for any effect on proliferation and viability, a fit for the LD50 was modeled. To separate desired effects associated with mitophagy from potential side effects on cell growth, ED50s for HSC Ψ and MT were modeled. For UroA we determined an LD50 of 5.9μM and an ED50[Ψ] of 2.7μM, whereas the natural compound analogue A3B5 had higher efficacy with an LD50 of 3.8μM and an ED50[Ψ] of 1.9μM. A3B5, ranked 6 in Top50[Ψ], co-clustered with the Flavonoid superfamily and is closely related to synthetic analogue A3E8, which demonstrated a very mild effect on cell growth even at high concentrations, while moderately decreasing Ψ and MT. Similarly strong response profiles with LD50 > ED50[Ψ] were obtained for Mitoquinol and the synthetic analogue A7C8, thus together 5 Hits from 3 structural subgroups were validated as showing non-toxic dose-dependent decrease in HSC Ψ.

The Top50[MT] comprised UroA with 52% MT signal of control levels at rank 19, and 4 exploratory compounds Luteolin, VitaminE, MitoTEMPO, VitaminB12 at ranks 1, 12, 15 and 39, clustering into 8 structural subfamilies. Fisetin, a close structural analog to Apigenin and Luteolin, had an efficacious response profile with 21.2μM LD50 vs 705nM ED50[MT], whereas polyphenolic Anthralin, known for its anti-proliferative effects and strong reducing properties, showed potent reduction of MT at 145nM ED50 with 684nM LD50.

By using the Seahorse assay to measure cellular respiration rates, UroA, as mitochondrial rejuvenation benchmark, significantly increased maximal respiration (MR) and spare respiratory capacity (SRC). A3B5 showed improved enhancement of MR and SRC compared to UroA. Surprisingly, A3E8 with moderate effects on Ψ and MT, strongly increased MR and SRC even above median levels of A3B5. Moreover, both Fisetin and Mitoquinol also showed a stronger improvement of MR and SRC than UroA.

Preliminary data from competitive bone marrow repopulation experiments reproduced the UroA effect of myeloid bias amelioration by increased lymphoid progeny and showed increased blood chimerism in HSCs treated with A3B5. Overall, we developed the first robust and reliable screening method to allow the identification of HSC rejuvenating molecules.