Phasing biology and disease
Literature Seminar

2024/8/5 (Mon)M2 Yuma TakeuchiTable of contents
 Introduction• Phasing biology (相分離生物学)• Life phenomena from the perspective of phasing biology• LLPS and disease• LLPS and small-molecule therapeutics

 New Tool
• Prediction of molecular localization in LLPS
with deep learning approaches

 Summary

2Phasing biology (相分離生物学)
 Four classes of protein structure3

 Quinary protein structure (五次構造)(= phase separation structure)

Structure

Main interaction

Primary

Covalent bond

SecondaryHydrogen bondTertiary

Side chain interaction

Quaternary Protein-Protein interaction(specific, strong)
Quinary

Protein-X interaction(X = Protein, RNA, DNA…)(multivalent, weak)

Kroschwald. S et al., Cell, 2017, 168, 947-948.

Phasing biology (相分離生物学)

4

 Phase separation
as a survival strategy

 Positioning ofphasing biology

Kroschwald. S et al., Cell, 2017, 168, 947-948.

Phasing biology (相分離生物学)

5 Liquid-Liquid Phase Separation (LLPS) in biology
condensed phase

dilute phase
Phase separation
Biomolecule

Liquid droplet

Wang. B et al., Sig. Transduct. Target Ther., 2021, 6, 290. Alberti. S et al. Cell, 2019, 176, 419-434.

Phasing biology (相分離生物学)

6

 Factors driving biological LLPS
IDP (Intrinsically Disordered Protein, 天然変性タンパク質)
IDR(天然変性領域)
Hydrophilic residues(for interaction)

Hydrophobic residues(for folding)Wang. B et al., Sig. Transduct. Target Ther., 2021, 6, 290.

Phasing biology (相分離生物学)

7

 History of the discovery and development of biological LLPS

Wang. B et al., Sig. Transduct. Target Ther., 2021, 6, 290.

8Life phenomena from the perspective of phasing biology
 Continuous enzyme reaction
Metabolic pathway map

Theoretical calculation
Continuous enzyme reaction cannot occur
if the active sites of two enzymes are >10 nm
M. Castellana et al., Nat. Biotechnol., 2014, 32(10), 1011-1018.

Scheme of continuous enzyme reaction
Substrates, Intermediates, Enzymes:
inside droplet
Metabolon: complex of enzymes
Purinosome: complex of purine synthases

LLPS = reaction field
P. A. Srere, Ann Rev. Biochem., 1987, 56, 89-124.

Purine:depletedrichHeLa cells
hFGAMS-GFP

re-depleted

S. An et al., Science, 2008, 320(5872), 103-106.

Life phenomena from the perspective of phasing biology

9 Post Translational Modification (PTM)

In vitro
25 mM Tris (pH 7.5),
150 mM KOAc, 1 mM MgOAc,
5% [w/v] glycerol

DNA: polyanion
Histone tail: polycation IDR

small PTM

change of protein properties(c.f. charge, hydrophilicity)large LLPS (field)
B. A. Gibson et al., Cell, 2019, 179, 470-484.

Life phenomena from the perspective of phasing biology

10 RNA sequence, structure and recognition
RNA recognition by ribosome
Some mRNAs are enriched in stress granules(>95%) ,while other mRNAs are NOT (<1%)
A. Khong et al., Mol. Cell, 2017, 68(4), 808-820.

Factors driving LLPS (Protein and RNA)・RNA sequence and structure
・RNA structural changeby RNA binding protein
E. M. Langdon et al., Science, 2018, 360(6391), 922-927.

Importance of RNA tertiary and quinary structure
The capacity of phase separation, as well as amino acid, are coded by mRNALLPS and diseaseLLPS

11LLPS=Life activity

Cascades of biochemical reactions
Disorder=Disease

LLPS and disease12 Amyloidosis

aggregation

Amyloidogenicprotein

Amyloid-β (Aβ)α-synuclein

Amyloid fibril

Tau
Transthyretin

Amyloidosis

Alzheimer disease
Parkinson disease

TTR amyloidosis

Amyloids are deposited in various organs and cause functional disorder.

LLPS and disease

13

 LLPS and amyloid function(normal)Phase separation

Monomer

Amyloid LLPS

Primary cortical mouse neurons(初代皮質神経) expressed with
GFP-tagged full-length tau(tau441)
Liquid-like

S. Wegmann et al., EMBO J., 2018, 37(7), e98049.

B. Solomiia et al., PNAS., 2020, 117(50), 1882-1890.

LLPS and disease

14

 LLPS in amyloid aggregation process(abnormal)

aggregation level

Aggregation
trigger

Phase separation

Monomer

Amyloid LLPS
aggregation

S. Wegmann et al., EMBO J., 2018, 37(7), e98049.

Fibril(cross-β-sheet rich)
In vitro
ThT aggregation
assay

B. Solomiia et al., PNAS., 2020, 117(50), 1882-1890.

LLPS and disease15

 Cancer
Dysregulation of LLPS in malignant cells
Transcriptional regulation by Super-enhancer(euchromatin)

B. R. Sabari et al., Science, 2018, 361, eaar3958.

A. Boija et al., Cancer Cell, 2021, 39, 174-192.

LLPS and small-molecule therapeutics16 Therapeutic small molecules concentrate in distinct intracellular environments
HCT-116 cells:
Human colorectal cancer 116 cells(ヒト結腸直腸がん細胞)

Sunitinib and Bosutinib:
anticancer receptor tyrosine kinase inhibitors
target: in the lipid bilayer andperhaps the cytoplasm
concentration: nucleoli (核小体)(membrane less organelle)

Topotecan:
Topoisomerase inhibitor
target: nucleus (核)concentration: nucleus (核)H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

LLPS and small-molecule therapeutics

17

 Drug resistance mechanism by LLPS
In breast cancer cells

In vitro

Tamoxifen: antineoplastic drug(binding ER (Estrogen Receptor))
MED1: Subunit of Mediator(transcriptional condensate-forming protein)

I. A. Klein et al., Science, 2020, 368, 1386-1392.

LLPS and small-molecule therapeutics
 Undesired therapeutics

Cell
Target protein (LLPS)

Other LLPS

Need of high dose↓
cytotoxity and side effect(c.f. Sunitinib, Bosutinib)

 Desired therapeutics

Cell
Target protein (LLPS)

Other LLPS

Possibility of low dose↓less cytotoxity and side effect(c.f. Tamoxifen): target protein: small-molecule therapeutics

18

Table of contents
 Introduction• Phasing biology (相分離生物学)• Life phenomena from the perspective of phasing biology• LLPS and disease• LLPS and small-molecule therapeutics

 New Tool
• Prediction of molecular localization in LLPS
with deep learning approaches

 Summary19

Chemical features and subcellular localization 20
 Therapeutic small molecules concentrate in distinct intracellular environments
HCT-116 cells:
Human colorectal cancer 116 cells(ヒト結腸直腸がん細胞)

Actinomycin D, Mitoxantrone,Proflavine, Psoralen:
nucleic acid binding compounds
Different chemical features
Different subcellular localization

H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

In vitro condensate reconstitution

21

H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

Selective partitioning of small molecules in simple condensates

22

Many probes were enriched in one or more condensates.
Partition ratio of each top 50 partitioning probes

The partition ratios of high-partitioning probes in these condensates
were generally greater than the partition ratios in the other condensates.
H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

Chemical similarity and partition ratio

23

Morgan fingerprint: presence (1) or absence (0) of a chemical feature
Tanimoto similarity: chemical similarity (molecular similarity)(=5/9)

H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

Success of deep learning prediction

24

Chemical feature

DL: Deep Learning
RS: Random SelectProbe data
Partition data

Train data

Compare to RS,
DL predicted probeswith high partitioning ratio.
Test data

H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

Importance of cationic and aromatic motifs

25

The probes predicted to partition into MED1, HP1α and NPM1 condensates
were identified to be primarily xanthene and BODIPY
that possess electron donating R-groups and electron rich π-system.

MED1 rationales: 1. aromatic rings functionalized with electron donating, withdrawing and neutral motifs
2. cationic amines and their N-acetyl propylamine derivatives
NPM1 rationales: 1. aromatic and amine rich moieties, which compose the scaffold of BODIPY
HP1α rationales: 1. aromatic ring structures and building blocks of BODIPY and xanthene
H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

26

Live cell partitioning predicted by deep learning

Heterochromatin
region

NPM1: 10 drugs predicted to concentrate in nucleoli (DL with vitro data)
5 were observed to do so and31 drugs predicted not to concentrate in nucleoli,
11 appeared to concentrate in nucleoli.
HP1α: 5 drugs predicted to concentrate in chromocenters (DL with vitro data)
4 were observed to do so and36 drugs predicted not to concentrate in chromocenters,
1 appeared to concentrate in chromocenters.

DL, trained on simple in vitro condensates,
could predict that some drugs will selectivelyconcentrate in the more complex environment of
the relevant condensates in cells,
albeit with limited accuracy.
H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

Table of contents
 Introduction• Phasing biology (相分離生物学)• Life phenomena from the perspective of phasing biology• LLPS and disease• LLPS and small-molecule therapeutics

 New Tool
• Prediction of molecular localization in LLPS
with deep learning approaches

 Summary27

Summary28

New era・Prediction of molecular localization
in LLPS with deep learning
・LLPS and disease, therapeutics

Table of contents
 Introduction• Phasing biology (相分離生物学)• Life phenomena from the perspective of phasing biology• LLPS and disease• LLPS and small-molecule therapeutics

 New Tool
• Prediction of molecular localization in LLPS
with deep learning approaches

 Summary29

Life phenomena from the perspective of phasing biology

30 Post Translational Modification (PTM)

P-TEFb (Positive Transcription Elongation Factor b):
consisting of CDK9 and cyclin T1 (CYCT1)

In vitro
20 mM Tris-HCl (pH7.5), 1 mM DTT, 37.5 mM NaCl,
6 mg/mL protein solutions
T1-IDR (Histidine rich):
IDR (491-686) of transcription-related cyclin T1 (CYCT1)
CTD (phos. type: use of CDK7):
C-terminal domain (YSPTSPS x52) of RPB1 subunitof human RNA polymerase II(RNAPII)
H. Kimura and Y. Sato, Current Opinion in Cell Biology. 2022, 74, 71-79. H. Lu et al., Nature. 2018, 558(7709), 318-323.

LLPS and small-molecule therapeutics

31 Cisplatin

I. A. Klein et al., Science, 2020, 368, 1386-1392.

LLPS and small-molecule therapeutics

32

 Therapeutic small molecules concentrate in distinct intracellular environments

H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

LLPS and small-molecule therapeutics

33

 Therapeutic small molecules concentrate in distinct intracellular environments(two-photon imaging for ultraviolet region excitation)

H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

Small molecule-protein interactions in condensates

34H. R. Kilgore et al., Nature Chemical Biology, 2024, 20, 291-301.

ActuAtor

35

H. Nakamura et al., Cell Reports, 2023, 42, 113089.