“BIOINFORMATICS UTILIZATION FOR DRUG

DISCOVERY AND DRUG DEVELOPMENT

FROM HERBAL: FOCUSING ON ANTI-AGING
AND CANCER CHEMOPREVENTION”
Prof. Dr. apt. Edy Meiyanto, M.Si.
Cancer Chemoprevention Research Center (CCRC)
Lab. Rekayasa Makromolekul, Kimia Farmasi
Faculty of Pharmacy, Universitas Gadjah Mada
edy_meiyanto@ugm.ac.id
http://ccrc.farmasi.ugm.ac.id
DRUG DISCOVERY
Penemuan obat atau drug discovery adalah suatu proses yang bertujuan untuk
mengidentifikasi suatu senyawa yang secara terapeutik berguna dalam
menyembuhkan dan mengobati penyakit. Proses ini berkaitan dengan
identifikasi kandidat, sintesis, karakterisasi, validasi, optimasi, penyaringan dan
pengujian untuk kemanjuran terapeutik (Deore et al., 2019).

1
TRL 1 - 6 TRL 7 - 9
DRUG DISCOVERY: TRL 1-6

2
DRUG DISCOVERY: dari yang sudah ada

3
Bioinfomatika dalam Drugs Discovery
Biologi molekuler
Genomic, proteomic, X-omics
CADD (computer aided drug design)
Computational resources
Information technology / information
management
Application, Algorithm, machine learning,
database
Manajeman dan interpretasi data
Bioinformatika, penemuan obat,
dan pengembangan obat
• Mengidentifikasi target penyakit
• Studi terkait senyawa obat
• Mendeteksi penanda molekuler suatu penyakit
• Tehnik desain obat secara rasional
• Penyempurnaan suatu senyawa
• Quantitative Structure Activity Relationships
(QSAR)
• Pengujian suatu obat
4
Bioinfomatika dalam Drugs Discovery (Concept)
Molecular pathways

What is bioinformatics? Application of techniques from computer science to problems from biology 5
PERKEMBANGAN BIOINFORMATIKA YANG SANGAT PESAT

6
Bioinformatics in Drugs Discovery Tujuan Bioinformatika dalam proses penemuan obat

7
(Gupta et al., 2021)
Bioinformatics Tools (Contoh)
UALCAN membantu dalam mengidentifikasi
kandidat biomarker dari subkelas kanker
tertentu, beserta dengan implikasi diagnostik,
prognostik, dan terapeutik
Swiss Institute of
GEPIA2 menyediakan data ekspresi dan
membandingkan data tersebut dengan koleksi
besar sampel TCGA dan GTEx
ChEMBL menyediakan informasi terkai target,
organisme, dan pengukuran bioaktivitas yang
dilaporkan dalam literatur ilmiah,
ChEMBL
KNIME memungkinkan para ilmuwan dan
pemrogram untuk memberikan ekstensi mereka
sendiri kepada komunitas ilmiah.
KEGG adalah ensiklopedia gen dan genom.
Menetapkan makna fungsional untuk gen dan
genom pada tingkat molekuler
(SIB, 2015; Sfran, 2010; Kaneshia, 2017; Chandrasheka,
2017)
SIB menyediakan database bioinformatika, perangkat
lunak, layanan dan pelatihan kepada komunitas ilmu
kehidupan internasional. SIB terintegrasi dengan lebih
dari 150 sumber daya bioinformatika seperti
UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt,
STRING, dll. Contoh platform,SwissTargetPrediction,
SwissDock, dll
Bioinformatics
CMAUP menyediakan informasi terkait target target (kelas
target ChEMBL) dan tingkat aktivitas (dalam peta panas
bahan target 2D), dan ontologi gen yang diatur, jalur
biologis, dan untuk lebih dari 5000 tanaman yang
dikumpulkan dari atau digunakan secara tradisional di 153
negara dan wilayah
Memberikan informasi yang komprehensif tentang gen
manusia, ditambang dan terintegrasi dari lebih dari 80
sumber data. Dengan menyatukan informasi gen dari
sumber publik yang besar seperti HGNC, NCBI, ENSEMBL
dan UniProtKB dll. GeneCards menyediakan genom,
proteome, transkriptom, data penyakit dan fungsi yang
ringkas pada semua gen manusia yang diketahui dan
diprediksi.
8
 TREND IN HERBAL
PRODUCTS AND RESEARCH
Anti Aging dan Chemoprevention
MENGAPA PENELITIAN PADA BAHAN ALAM?

➢ Bahan alam merupakan sumber struktur kimia yang paling kompleks dan
menarik
➢ Bahan alam menggambarkan keanekaragaman hayati
➢ Bahan alam adalah ekspresi dari genom
➢ Bahan alam memiliki aktivitas biologis baik dalam bentuk senyawa tunggal
atau sebagai campuran yang kompleks
➢ Bahan alam merupakan bagian dari kekayaan alam negara dan dapat
menjadi sumber kehidupan yang penting, mulai dari pertanian dan pangan,
FARMASI, serta industri kimia
➢ Bahan alam menjadi jembatan antara tradisional menuju perkembangan
ilmu pengetahuan modern, termasuk genetika, biologi molekuler,
bioteknologi, dan ilmu farmasi

10
THE PERSPECTIVE OF NATURAL PRODUCTS RESEARCH

CONTOH

11
Adapted from:
Li and Weng. Demystifying traditional herbal medicine with modern approaches. Nature Plants (2017).
 Trend Produk Herbal, Kosmetik dan Pangan:
preventive medicine
Nutrasetikal Kosmeseutikal

Nutrasetikal dapat didefinisikan

sebagai makanan atau bagian
dari makanan atau nutrisi yang
memberikan manfaat untuk
kesehatan, termasuk pencegahan
dan pengobatan suatu penyakit
Kosmesetikal adalah produk kosmetik
dengan bahan bioaktif yang diklaim
memiliki manfaat medis. Nama tersebut
merupakan kombinasi dari "cosmetics" dan
"pharmaceuticals".

12
FAKTA 3

Research on Market volume

Nutraceuticals and Cosmeceuticals Nutraceuticals
800 16000

700 14000

600 12000

500 10000
Herbal dietary suplement
Herbal food suplement
Cosmeceutical
400 8000
Herbal anti-wrinckle
Herbal anti-aging
300 6000 Natural antioxidant
Nutraceutical

200 4000

100 2000

0 0
2015 2016 2017 2018 2019 2020 2021 (April)

Source : PubMed
Cosmeceuticals
15
Rempah-rempah dari Indonesia

Rice brand
Turmeric Black pepper

Galangal Sappan wood Citrus

15
Citrus Peels Product
Produk Dasar Kulit Jeruk
•Minyak
•Powder
Penggunaan atau Aplikasi Kulit Jeruk
• Pharmaceutical
• Industri makanan
• Perawatan
• Dietary Supplements
• dll

17
CITRUS
100

Combination Index
PGV-1 (µM)
Cytotoxic effects of PGV-1 and diosmin on
Cell viability (%)

1 2 4
50
4T1 breast cancer cells.
48 0.1 0.2 0.3
Diosmin
96 0.2 0.3 0.4
(µM)
193 0.4 0.5 0.5

0
0 100 200 300 400 500 600
Diosmin (μM) ✱✱

30 ns

% mitotic catastrophe cell

ns
20

10

µM
ed

D PG 96 M +

µM +
µM

µM

µM

D GV 3 µ +

19 µ M
P 19 M
at

sm -1 M

sm V-1 µM
3
96

in 4 µ
in 2 µ
re

19

in 4
nt

in

V-

V-

3
in

sm 1
U

sm

PG

PG

V-
sm

D PG
io

io
D

io
io

io
Mitotic catastrophe effects of PGV-1 and 18
diosmin on 4T1 breast cancer cells.
Tumeric Product

19
Sumber: Report and Data
Tumeric

Curcumin increased cell senescence which related to

ROS generation in K562 leukemia cells (Larasati et al.,
2018)

SENESCENCE APOPTOSIS
ROS

TUMOR CELL

Curcumin increased intracellular ROS levels and inhibited

the growth of CML-derived leukemic cells (Larasati et al., 20
2018)
Kunyit
• Curcumin inhibits ROS metabolic enzymes
• Increasing intracellular ROS level over the limit
• Inducing cell death

Curcumin only kill the cancer cells,

and save for normal cells
due to the differences in
intracellular ROS level

(Larasati et al., 2018)

21
ROS METABOLIC ENZYMES
 Penelitian dan Produk lengkuas
Galangal related research in PubMed over last 30 years.

Keywords: Alpinia galanga

Keywords: Alpinia galanga and cancer

Keywords: Alpinia galanga and anti-aging

Keywords: Alpinia galanga and bioinformatic

Galangal related products

Further exploration utilizing bioinformatic is promising
22
GALANGAL
4T1 cancer cells
NIH-3T3 non-cancerous cells

GE has synergistic effect with Doxorubicin on 4T1 cells

GE at the concentration of 100 µg/mL induces G2/M

phase arrest, this effect also increased when
combined with Doxorubicin

GE decreases ROS level and inhibits

senescence formation on NIH3T3 cells

Protein target & mechanism?

GE Inhibits cancer cells migration also inhibits MMP9
GE increases ROS level and senescence activity that responsible for cell migration
formation on 4T1 cells Galangal potential metabolite? 23
G
GALANGAL

Galangin PGV-1

C
A 100

Viabilitas Sel (%)

Viabilitas Sel (%)

80

60 Untreated PGV-1 4 μM
40

20

0
0 500 1000 1500

Galangin (μM)

B 100
Viabilitas Sel (%)

80

60

40
Combination Index of Gal 60 μM PGV-1 4 μM + Gal 60 μM
20
Galangin and PGV-1 in 4T1 Cells 30 ✱✱✱
0
Combination of PGV-1 and

Persentase Sel (%)

0 5 10 15 20

PGV-1 (µM)

Galangin
PGV-1 (μM) 20 Galangal enhances mitotic
✱✱✱, p<0,001
(μM) catastrophe induction
IC50 of Galangin and PGV-1 in 4T1 Cells 1 2 4
10
Compound IC50 (μM) 15 -0,13 0,03 -0,24

Galangin 120 30 0,12 0,10 0,04 24

0
PGV-1 8 60 0,56 0,20 0,06 Gal 60 uM - +
PGV-1 4 uM + +
SAPPAN WOODS
1
IC50 value of brazilin and
brazilein on WiDr cancer
cells were 41 µM and 52
µM respectively.

The combination of ½
IC50 of Bi-Cisp reduced
cells viability up to 64%
and showed synergistic
effect with CI value less
than 1 (CI = 0.8).

2
Both combination of Bi-Cisp
and Be-Cisp showed
3 synergistic effect on WiDr
cells through induction of
➢ ½ IC50 of Be induced S-phase arrest
apoptosis
➢ ½ IC50 of Bi induced G2/M-phase arrest
➢ Combination of Be-Cisp and Bi-Cisp enhanced S-phase arrest

25
(Handayani et al., 2019)
SAPPAN WOODS

In the Boyden chamber

2
assay, gelatin zymography,
and subsequent
immunoblotting assay, the
combination Bi and Dox
inhibited migration, possibly
through downregulation of
MMP9, MMP2, HER2, Rac1,
and p120 protein
1 expression.
Bi inhibited MCF-7/HER2
cell growth in a dose-
dependent manner with an
IC50 of 54 ± 3.7 µM.
Furthermore, the
combination of Bi and Dox
showed a synergistic effect
(CI

(Jenie et al., 2018)

26
BLACK PEPPER
The Anti-aging of Black Pepper
Activity Reference
Reduces the level of radical (DPPH assay) Zarai et al., 2013
Antioxidant Lowers peroxide and thiobarbiturate acid better than BHA and Singh et al., 2010
BHT
Reduce the level of radical and increase superoxide dismutase Jeena et al., 2014
and GST in liver on female balb/c mice
Lowers the level of radical Saha and Verna, 2015
Increases SOD, CAT, POX, GST, APOX, and GPDH in Ephestia Syahriari et al., 2018
kuehniella
Lowers the level of ROS on CHO-K1 cell lines Sari et al., 2018
Modulation Decreases GSH on limfosit cells Vijaykumar et al, 2015
of ROS Level
ROS

Increases ROS on HeLa, MCF-7 and ZR-75-1 cell lines Jafri et al, 2019
Deng et al., 2016
Lowers senescence cell on CHO-K1 and NIH-3T3 cell lines Nugraheni, 2019
Anti- Reduces senescence cell and increase in hippocampal volume Banji et al, 2013
senescence
and protection of hippocampal neurons on Wistar rats

27
(CCRC, Pharmacy UGM)
BLACK PEPPER
▪ Piperine
▪ Isopiperine
▪ Chavicine
▪ Isochavicine
▪ α-Caryophyllen
▪ β-Caryophyllen Major
▪ Limonene Constituents
▪ α-Pinene
▪ β-Pinene
▪ Sabinene
▪ 3-Carene
▪ Champene
Effect of BPEO on intracellular ROS level of ▪ P-cymene BLACK PEPPER
Cytotoxic effect of BPEO on CHO-K1 cells CHO-K1 cells

Anti-aging
Natural
Activities
products
Antioxidant

Modulation of Cosmeceutical
ROS level products

Anti
senescence

Senescence inhibition effect of

28
BPEO on CHO-K1 cells
(CCRC, Pharmacy UGM)
Research of Black Pepper and its's application

Minyak merica hitam merupakan esensial oil yang berbau khas serta
memiliki banyak manfaat diantaranya meningkatkan sirkulasi udara,
memberikan sensasi hangat, dan lain-lain.
Minyak atsiri kaya akan monoterpen dan seskuiterpen, yang memiliki
manfaat antioksidan, melindungi dari kerusakan yang disebabkan oleh
radikal bebas, memperbaiki kerusakan ini.

Massage oil Body wash Body lotion

Black pepper applications/end users

• Aromatherapy
• Skin care
• Essential oil
• Dietary Supplements Soap

• Others Essential oil

Supplement

29
RICE BRAN

RICE BRAN
(Tocotrienol)

4T1 CYTOTOXICITY NIH3T3

ROS SENESCENCE
A B

GST ASSAY
CELL CYCLE APOPTOSIS AND DOCKING

CO
CHEMOTHERAPEUTIC ANTI AGING
AGENT

(A) Dox significantly increased intracellular ROS, whereas RBE did

not
(B) Dox showed extremely enhanced senescence evidence in 4T1
30
cells, RBE treatment with Dox resulted in a deficient number of SA- (Zulfin et al., 2021)
β-Gal-positive cells (CCRC, Pharmacy UGM)
 APPLICATION OF
BIOINFORMATICS
in Herbal Chemoprevention Research and Development
 PROBLEM FOCUS Laboratory based methods

In vitro experiment
Bioflavonoids

Data laboratory
In vivo experiment Based-paper

Pharmacology Chemical experiment

Citrus reticulata Pharmacologycal effects

• Journals Databases Original article/

• Genomic database Software tools Critical
• Protein database
• Chemical database AI Review paper
Target mechanisms
• Herbal database
Data mining

Bioinformatics based methods 32

AI: Artificial Intelligence
Alur penelitian
PGV-1 Piperine

Combination of Piperine
and PGV-1

In vitro assay Bioinformatic study

TNBC cells (4T1)

MTT May Grunwald – SA--

assay Giemsa Staining Galactosidase
Overexpressed Predictive target
genes in TNBC proteins

Potential target
proteins
% mitotic
% cell viability % Senescent
catastrophe cells
Cells

Anova one-way Profile of expresssion

Survival rate

34
CITRUS
MMP Expression and its Correlation to Survival Rate of Colon Cancer Patients

UALCAN & GEPIA2 IKK Expression and Its Correlation to

NF-кB . activation canonical Survival Rate of Colon Cancer Patients

Ekspresi MMP3
signaling pathway Ekspresi IKK2
✱✱✱
Ekspresi MMP1 Ekspresi MMP7

400 400 ✱✱✱ 40

400 Normal
✱✱✱ Cancer

Transkrip mRNA per juta

300 300

Transkrip mRNA per juta

Transkrip mRNA per juta

300 30

Transkrip mRNA per juta

200 200 ✱✱✱
200 20

100 100
100
10
0 0
Normal Kanker 0
Normal kanker
Normal Kanker Degradasi
0
Sampel TCGA Sampel TCGA
Sampel TCGA proteaseom Normal Cancer
Ekspresi MMP9 Ekspresi MMP11 Ekspresi MMP12 TCGA Sampel
150 ✱✱✱ ✱✱✱ 400
400
Normal
Kanker
Transkrip mRNA per juta

300
Transkrip mRNA per juta

300
Transkrip mRNA per juta

100
✱✱✱
200 200 Nukleus

50
100 100
(A)

0 0 0
Normal Kanker
Normal Kanker
Sampel TCGA
Normal Kanker
Sampel TCGA Sampel TCGA IKK overexpression in colon cancer correlates with
worsening survival rate in colon cancer patients.“
MMP overexpression in colon cancer UALCAN & IKK is a potential target in the development of
GEPIA2 results : MMP1, 3, 7, 9, 11, and 12 colon cancer chemoprevention"
35
36
CITRUS
Prediction Results of Machine Learning Inhibition of Compounds in Orange Peel on IKK
Hesperidin, asam fleurat, Overexpres
No Compound Prediction value limonin, nobiletin, sed in colon
tangeretin cancer

1 Asam Fleurat 0.62

2 Hesperidin 0.63
295 21 881
3 Limonin 0.67
4 Nobiletin 0.63
5 Tangeretin 0.63
SwissTargetPrediction UALCAN
& GEPIA

Hesperidin
Function Protein Compound
Asam fleurat Proliferation TYMS Hesperidin
MET Tangeretin, Nobiletin,
Cell cycle CDK1 Tangeretin, Nobiletin
NEK2 Tangeretin, Nobiletin
PLK1 Tangeretin, Nobiletin
Tangeretin, Nobiletin,
Nobiletin AURKB "IKK is a potential target in the development
Limonin
Metastasis MMP1 Asam Fleurat, Limonin of colon cancer chemoprevention"
Limonin
MMP3 Tangeteretin, Nobiletin,
MMP7 Hesperidin

Tangeretin
MMP9
Tangeteretin, Nobiletin,
Asam Fleurat
37
MMP12 Hesperidin, Nobiletin
38
CITRUS
A. B. C.

VEGFA, AGTR1, MAPK14, SRC, EGFR,

PTPN1, FN1, TNF, HIF1A, PTGS2,
CASP3, GAPDH, MPO, ALB, AKT1, XDH

Potential target genes of citrus flavonoids and proteins

interaction of NOX4. (A) Protein - protein interaction network
(PPIN) of NOX4 analyzed using STRING-DB (B) a Venn diagram
of citrus flavonoid target genes from the SwissTargetPrediction
and PPIN of NOX4 (C) Schematic diagram of citrus flavonoids
nephroprotective mechanism in kidney injury (CCRC’s
unpublished document).

39
CITRUS
Effects of PGV-1 and diosmin on 4T1 Senescent Cells
ns

80 ✱✱
✱✱✱

% Senescence Cells
60

40

20

0
Untreated PGV-1 4 µM Diosmin 193 µM PGV-1 2 µM +
Diosmin 193 µM

Concentration

PGV-1 and Diosmin compounds with

protein overexpression in TNBC
breast cancer and expression of
predicted target proteins in a
normal cell, cancer cell, and triple-
negative breast cell.
40
GALANGAL
Revealing dual effects of galangal

Galangin target prediction reveal protein targets

underlying anti-cancer effect in breast cancer

GLO1 is potential target that responsible for ROS level and senescence
formation on breast cancer

2-Endo-hydroxy- Ocatanol acetate Epipachysamine B Isocoronarin D

1,8-cineole

Kaempferide Galanal A Galangin

Galangin is galangal potential secondary metabolite that act as

dual effect previously studied on breast cancer and Normal
fibroblast Proposed mechanism, galangal dual effect toward
breast cancer and normal fibroblast 41
GALANGAL 15 ✱
✱

✱✱

Senescence Cell (%)

*p<0,05
10
**p<0,01

0
Doxo 100 nM - + - - -
Gal 60μM - - - + +
PGV-1 4μM - - + - +

Combination of PGV-1 and Galangal

enhances senescence induction
Kontrol Sel
CDK1
TOP2A

AURKB
KIF11
PLK1
CHEK1
CCNB1
CCNE1
CCNB2
QPCT
NEK2

PGV-1 and Galangal target cell cycle machinery proteins

42
PGV-1 4μM+Gal 60μM (Ualcan & Swiss Target Prediction)
BLACK PEPPER KNIME analysis target on MMP9

Compounds Prediction value

(R)-Pinocembrin 1
Piplartine 1
Piperin 0,96
Beta-Caryophyllene 0,95
Anti-senescence effect of Pepper extract on NIH3T3 cells
Gene aging related targets of
Pepper active compounds
(Genecards and swiss-target prediction)
TP53, IL6, TGFB1, TNF, VEGFA, IL1B, LMNA, COL1A1,
MMP2, MMP1, AKT1, ESR1, PTEN, MMP9, TLR4,
MAPK1, ELN, CDKN2A, FN1, IL1A, FBN1, CASP3,
SMAD4, CDKN1A, PIK3R1, TGFBR2, MTOR, COL3A1
FA = Fibroblast aging
FP = Fibroblast photoaging
SW = Skin wrinkle

Proposed mechanism of BP as anti-aging 43

(CCRC, Pharmacy UGM)
44
SUMMARY

❑Drug discovery dan development merupakan proses

yang Panjang dan dapat dibantu dengan perangkat
bioinformatika
❑Herbal Indonesia potensial untuk dikembangkan
sebagai kemoprevensi kanker dan antiaging
❑Bioinformatika sangat dibutuhkan untuk pengebangan
herbal kemoprevensi dan antiaging

45
REFERENCES
Ahlina, F.N., Nugraheni, N., Salsabila, I.A., Haryanti, S., Da’i, M., Meiyanto, E., 2020. Revealing the Reversal Effect of Galangal (Alpinia galanga L.) Extract Against
Oxidative Stress in Metastatic Breast Cancer Cells and Normal Fibroblast Cells Intended as a Co- Chemotherapeutic and Anti-Ageing Agent. Asian Pac. J. Cancer
Prev. APJCP 21, 107–117.

Deore, A.B., Dhumane, J.R., Wagh, R., Sonawane, R., 2019. The Stages of Drug Discovery and Development Process. Asian J Pharm Res Dev 7, 62–67.

Gupta, R., Srivastava, D., Sahu, M. et al. Artificial intelligence to deep learning: machine intelligence approach for drug discovery. Mol Divers (2021).

Handayani, S., Susidarti, R.A., Jenie, R.I., Meiyanto, E., 2017. Two Active Compounds from Caesalpinia sappan L. in Combination with Cisplatin Synergistically
Induce Apoptosis and Cell Cycle Arrest on WiDr Cells. Adv. Pharm. Bull. 7, 375–380.

Jenie, R.I., Handayani, S., Susidarti, R.A., Udin, L.Z., Meiyanto, E., 2018. The Cytotoxic and Antimigratory Activity of Brazilin-Doxorubicin on MCF-7/HER2 Cells.
Adv. Pharm. Bull. 8, 507–516.

Larasati, YA., Yoneda-Kato, N., Nakamae, I., Yokoyama, T., Meiyanto, E, and Kato, J., 2018, Curcumin targets multiple enzymes involved in the ROS metabolic
pathway to suppress tumor cell growth, Sci Rep, 8, 2039.

Lestari, B., Nakamae, I., Yoneda-Kato, N., Morimoto, T., Kanaya, S., Yokoyama, T., Shionyu, M., Shirai, T., Meiyanto, E., Kato, J., 2019a. Pentagamavunon-1 (PGV-1)
inhibits ROS metabolic enzymes and suppresses tumor cell growth by inducing M phase (prometaphase) arrest and cell senescence. Sci. Rep. 9, 1–12.

Utomo, R.Y., Ikawati, M., Putri, D.D.P., Salsabila, I.A., Meiyanto, E., 2020. The Chemopreventive Potential of Diosmin and Hesperidin for COVID-19 and Its
Comorbid Diseases. Indones. J. Cancer Chemoprevention 11, 154–167.

Zaragozá, C., Villaescusa, L., Monserrat, J., Zaragozá, F., Álvarez-Mon, M., 2020. Potential Therapeutic Anti-Inflammatory and Immunomodulatory Effects of
Dihydroflavones, Flavones, and Flavonols. Molecules 25.

Zulfin, U.M., Rahman, A., Hanifa, M., Utomo, R.Y., Haryanti, S., Meiyanto, E., 2021. Reactive oxygen species and senescence modulatory effects of rice bran
extract on 4T1 and NIH-3T3 cells co-treatment with doxorubicin. Asian Pac. J. Trop. Biomed. 11, 174.
THANK YOU