TRAP1 — MOA And Pipeline Page (Page 1124)

Bulk / Single Cell Transcriptomics Plan (continuation)

	Sonia Gandhi (UCL), Steven Lee (University of Cambridge), Michele Vendruscolo (University of Cambridge)	and human brain tissues	relevant regions and over different cell types, → 이 map에서 oligomer-containing cell을 골라→ LCM (laser capture microdissection)으로 single cell 빼내 → sc trnscriptomics. genomics (ie SNP발굴, 이것도 oligomer-containing인지 불언급) -validation: iPSC+oligomer→ readouts: ROS, mito, Ca, synapse, UPR, oligomer flux
Mice	(Zhong, 2020 #1309) 6-month old α-syn A53T		region-specific dysfunctions regarding proteostasis, channelosome (calcium지칭듯), extracellular environment, neuroinflammation → BM제안 (high fidelity BM)

Midbrain DA neurons

	Midbrain DA neurons	DA neurons in other brain regions
Developmental path	develop from radial 21 glial cells of the foor plate	Develop from neural stem cells
During development	exposed to high levels of the SHH transcription factor	Determined by Pax6 transcription factor
transcriptome	Different transcriptome

TRAP1

gene	protein	function	Pipeline
TRAP1 (TNF Receptor Associated Protein 1) Maiko: Peter Maycox is working in TRAP1.	Heat shock protein 75 kDa, =mitochondrial member of Hsp90 family, resides in mito matrix, phosphorylated by PINK1, TRAP1 protein comprises an N-terminal ATPase domain, a middle domain and a C-terminal dimerization domain.	https://www.uniprot.org/uniprot/Q12931 Chaperone that expresses an ATPase activity (ATP → ↑ADP). Involved in maintaining mitochondrial function and polarization, downstream of PINK1 and MC I. The impact of TRAP1 on mitochondrial respiration is probably mediated by modulation of mitochondrial SRC and inhibition of SDHA spare respiratory capacity (SRC). SRC is the extra capacity available in cells to produce energy in response to increased stress or work The SDHA gene provides instructions for making one of four parts (subunits) of the succinate dehydrogenase (SDH) enzyme succinate dehydrogenase (=mc2): succinate → ↑fumarate Fitzgerald, 2017 #1289), we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, the role of its ATPase domain is clearly associated with the binding of non-chaperone "client" proteins but not with oxidative phosphorylation. TRAP1 is also closely associated with the mitochondrial ATP synthase and many of its subunits are client proteins. hypoxic conditions induce the expression of TRAP1 TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction Case report) LOF TRAP1 mutation in LOPD, (Gaare, 2018 #1288) No evidence for rare TRAP1 mutations influencing the risk of idiopathic PD - fibroblasts derived from the patient reveal that oxygen consumption, ATP output and ROS are increased, but loss of MMP and sensitivity to mitochondrial removal and apoptosis. Hu ATPase, ADP-GLO : normally ATP → ADP by TRAP1: ADP production measured with ADP-Glo (Promega) Mt ETC mbe. potential, ETC Seahorse (OCR), Mito Stress Test kit	Amathus: TRAP1 positive allosteric modulator (PAM)
sharefoleder	https://onetakeda.box.com/s/atojegv302eix7lt6ln1lk97gwfddyva

MOA

	normal	PINK1 phosphorylates TRAP1	↑TRAP1 phosphorylation	Client proteins		(ATP → ↑ADP).		mt depolarization
				↑N-terminal ATPase domain,
				TRAP1 Inhibit cytochrome c oxidase
				TRAP1 binds and Inhibit succinate dehydrogenase (SDH, =mc2)	그럼 이론상으론, ↑succinate (근데 PD plasma에선 ↓), ↓fumarate	↓MC2 activity, ↓OCR (Sciacovelli, 2013 #2405)	↓ATP synthesis (Sciacovelli, 2013 #2405)
				TRAP1 interacts w F-ATP synthase		↑F-ATP synthase activity
			?↓TRAP1 phosphorylation	↓TRAP1 function (KD)				mt fragmentation
	PD		?
Tx Hypothesis	PD+ ↑TRAP1 (ie PAM)			↑ATPase activity (eg.Amathus compound 20231019	Client protein synergy	OCR: Seahorse	↓(감소시키려한다 게 맞을 듯) Mito ETC:	↓ROS
								↓aSyn ?

in vitro pharmacology

					meeting slide #28)
in vitro pharmacology	Target				↑(50%) ATPase activity - 100 nM in i)human ATPase ADP-Glo & ii)Rodent (rat & mouse)	↑ expression of at least two from; SDHB, SIRT3, NDUFS1 (additional client proteins - CDK4, PRDX3)	EC50<1uM	EC50<1uM
	Progress 20230914					Methods developed for three proteins (Jess) SHDB, SIRT3 and NDUFS1		CellROX staining (이게 아마 started developing)
	progress 20231005					Client protein assay ready for compound screening.		ROS assay is almost ready to be completely developed.
	Progress 20231018				3 ADP-Glo assays (hTRAP1, Rodent TRAP1, HSP90) successfully developed and used in hit finding activities → being used for routine screening	Assay developed 3 principal client proteins (SDHB, Sirt3, NDUFS1). → being used for routine screening	CDA in place → (next step) MSA	Provisional conditions established; initial test compounds provided including Amathus patent compounds. → next step: Optimisation of compound treatment times.
	Progress 20231130					Complete repeat of 2nd Jess client protein run • Results consistent with other compounds — N=1 dataset indicates increases in NDUFS1 observed but no clear Sirt3/SDHB		No activity observed in ROS assay for Amathus patent compounds when tested following longer compound preincubation. • Further optimisation appears to have had a positive impact on variability.

Uncertain Spans

location	transcription	uncertainty
TRAP1 transcriptomics row	이것도 oligomer-containing인지 불언급	The penultimate Hangul token is rendered ambiguously between 불언급 and 불인급 in the OCR; the preferred reading 불언급 (“not mentioned”) matches the surrounding clause.
Mice row	channelosome (calcium지칭듯)	The hand-typed Korean note reads as 지칭듯 (“seems to refer to”) but the small font near the column edge keeps the second syllable visually close to 징.
MOA grid, leftmost rows	row labels	The leftmost two columns of the MOA grid are largely blank in this capture; some state-row labels (between “Client proteins” header and “Tx Hypothesis”) remain unlabeled rather than cut off.
in vitro pharmacology grid, right edge	progress columns past EC50<1uM	The rightmost progress narrative cells extend off the photo edge near the binding column and continue in 20240722_184539; only the cells fully visible in this capture are transcribed here.