enhancing bioactive-peptide by conformationally controlled...

Enhancing bioactive-peptide by conformationally controlled mimeticsLITERATURE SEMINAR #3

Y.KAMIMURA

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1. Introduction2. Major secondary structures and its mimetics• ɲ-helix• ǆĂŵƉůĞŽĨɲ-helix mimetic PPI inhibitor • ɴ-sheet• ǆĂŵƉůĞŽĨɴ-sheet mimetic PPI inhibitor • Turns• ǆĂŵƉůĞŽĨɴ-turn mimetics

3. Summary4. Appendix

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Bioactive PeptidesIntroduction

Enkephalin:Endogenous opioid peptide

Ciclosporin:Immunosuppressants

Actinomycin D:Tumorsuppressants

ʘ-Conotoxin MVIIA:Neurotoxin

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HN

NH

HN

NH

HN

NH

HN

H2NO

O

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

OHO

OSS

NH2H2N

O

NH

NH2

HOHOHOSomatostatin:Growth Hormone-Inhibiting Hormone

IPeptides show strong, wide spectrum of bioactivity.

Advantages of peptide as bioactive substance

9Limitless diversity9The ease of synthesis and analysis9Inherent biological relevance

Introduction

https://www.chem-station.com/odos/2013/11/-merrifield-solid-phase-peptid.htmlMorimoto; J., Fukuda; Y., Sando; S., et al., J.A.C.S, 2019, 141, 146122020/8/5

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Zhou; P., Meng; Y., et al ., Mol. Omics, 2019, 15, 280

Disadvantages of peptide as bioactive substance Introduction

Räder; A.F.B., Kessler; H., et al., Bioorg. Med. Chem., 2018, 26, 2766Fujiwara; Y, Kanai;M., et al, ChemRxiv, 2020

Zhou; P., Meng; Y., et al ., Mol. Omics, 2019, 15, 280Sano; H, Imagawa; A., J. Diabetes Inv., 2019, 11, 2812020/8/5

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X Low bioavailability

X Membrane permeability

X Low stability in cell/vivo

X High flexibility

NH

HN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHO

O

OH

OH

HN NH2

NH

HN

H2NNH

SMe

HN

H2N

NH

OH

OH2N

NHFITC

X Immunogenicity

LANA-FITC

Pathway of the intestinal uptake of peptides

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Räder; A.F.B., Kessler; H., et al., Bioorg. Med. Chem., 2018, 26, 2766

PeptidomimeticsIntroduction

Räder; A.F.B., Kessler; H., et al., Bioorg. Med. Chem., 2018, 26, 2766Verhoork; S.J.M, Coxon; C.R, et al., Chem. Euro. J., 2018, 25, 177

Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896Chang; Y.S., Sawyer; T.K, et al., PNAS, 2013, 110, E34452020/8/5

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Pelay-Gimeno; M, et al., ACIE, 2015, 54, 88962020/8/5

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N-substituted alanine



10

Mimicking Peptide-Protein InteractionsIntroduction


11

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ɲ-helix and its mimeticsDŝŵĞƚŝĐƐŽĨƐĞĐŽŶĚĂƌǇƐƚƌƵĐƚƵƌĞɲ-helix


13

ɲ-helix in p53-MDM2 interaction

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Chène ; P., Nat. Rev. Cancer, 2003, 3, 102Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896

DŝŵĞƚŝĐƐŽĨƐĞĐŽŶĚĂƌǇƐƚƌƵĐƚƵƌĞɲ-helix

Example: Sidechain Crosslinking

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Bernal; F., Walensky; L.D., et al., Cancer Cell, 2010, 18, 411Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896



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Nutlin-3a


ISAH-p53-8 can also bind to HDMX.IHDM2 inhibition by Nutlin-3a can be compromised by overexpression of HDMX which do not bind to Nutlin-3a.

Bernal; F., Walensky; L.D., et al., Cancer Cell, 2010, 18, 411Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896


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Bernal; F., Walensky; L.D., et al., Cancer Cell, 2010, 18, 411Bernal; F., Verdine; G.L., et al., JACS, 2007, 129, 2456

WT SAH-p53-8SAH-p53-5

IMembrane permeableIStabilizedILow flexibility and high affinity

SAH-p53-5

Sidechain crosslinking

Verhoork; S.J.M, Coxon; C.R, et al., Chem. Euro. J., 2018, 25, 177Pelay-Gimeno; M, et al., ACIE, 2015, 54, 88962020/8/5


Thiol based cross-links Ring-closing metathesis based cross-link

Lactam cross-links

ɲ-helix in Invasion of malaria parasite to host cell

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Angrisano; F., Baum; J., et al., PLOS ONE, 2012, 7, e32188Boucher; L.E., Bosch; J., J. Str. Bio., 2015, 190, 93


Actin filament

N-terminal capping

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Aurora; R., Rose; G.D., Protein Sci., 1998, 7, 21 Presta; L.G., Rose; G.D., Science, 1988, 140, 1632


Pham; T.K., Kim; Y., Bioorg. Chem., 2020, 101, 104024

IHydrogen-bond acceptor amino acids such as Asn, Asp are enriched at N-ƚĞƌŵŝŶĂůŽĨɲ-helix.

INucleation of helix by such interaction induce ƚŚĞĨŽƌŵĂƚŝŽŶŽĨɲ-helix.

Example: N-terminal capping

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Douse; C.H., Tate; E.W., et al., ACS Chem. Biol., 2014, 9, 2204


&ŽůĚĂŵĞƌƐ;ɲɴ-peptides)

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Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896Pu; J., Jiang; S., et al., viruses, 2019, 11, 705


ǆĂŵƉůĞ&ŽůĚĂŵĞƌƐ;ɲɴ-peptides)

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Morimoto; J., Fukuda; Y., Sando; S., et al., JACS, 2019, 141, 14612


(Enfuvirtide, as control)

Foldamers (Peptoids)

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Morimoto; J., Fukuda; Y., Sando; S., et al., JACS, 2019, 141, 14612Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896


Example: Foldamer (Peptoid)

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Morimoto; J., Fukuda; Y., Sando; S., et al., JACS, 2019, 141, 14612Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896

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ɴ-sheet

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Pelay-Gimeno; M, et al., ACIE, 2015, 54, 8896

ɴ-sheet mimetics

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LANA peptide

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PDB: 1ZLA X-ray Structure of a Kaposi's sarcoma herpesvirus LANA peptide bound to the nucleosomal coreWhite; M.K., Khalili; K., Clin. Microbiol. Rev., 2014, 27, 463

DŝŵĞƚŝĐƐŽĨƐĞĐŽŶĚĂƌǇƐƚƌƵĐƚƵƌĞɴ-sheet

ILANA peptide is nucleosome binding site of latency-associated nuclear antigen-1 (LANA-1)ILANA-1 is a multifunctional protein involved in tumorgenesis by Kaposi's sarcoma-associated herpesvirus(KSHV).

H2NHN

NH

HN

NHO

O

O

O OHN

O

NH

HN

ONH

OHN

ONH

O

HN

H2NNH

HOOH

SMe

NHNH2

HN

HO

HO

O

HN

H2N

NH

NH

HN

NH

HN

NHO

O

O

O OHN

O

NH

HN

ONH

OHN

OHN

O

HN

H2NNH

HOOH

SMe

NHNH2

HN

HO

HN

H2N

NH

O

Example: Macrocyclization

Yakushiji; F., Ichikawa; S., et al., Bioorg. Med. Chem., 2020, 30, 1268392020/8/5


9

Competition to LANA(1-23)-FITC, Kd=2.5uM

Kd,(LANA(1-23)-FITC)=200nM

IC50,(9)=614nM

IC50,(12)=204nM

ISimple macrocyclization increased the affinity 3-fold.12

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Turn and its mimetics

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Mimetics of secondary structure: Turn

IdƵƌŶƐĂƌĞŽĨƚĞŶĨŽƵŶĚĂƐƐƵďƐƚƌƵĐƚƵƌĞƐŽĨɴ-sheets.ITurns often play crucial role in presenting hotspot residues.

ůĂƐƐŝĨŝĐĂƚŝŽŶŽĨɴ-turns by Venkatachalam

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I

II

VIII

I’

II’

VIa1

VIa2

VIb

Bravern; A.G., Sci. Rep., 2016, 6, 33191

DŝŵĞƚŝĐƐŽĨƐĞĐŽŶĚĂƌǇƐƚƌƵĐƚƵƌĞɴ-turn

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Schonbrunn; A., Enc. Bio. Chem., 2013, pp.262Weckbecker; G, Bruns; C., et al., Nat. Rev. Drug Discov., 2003, 2, 999


Kumar; U., Singh; S., Int. J. Mol. Sci., 2020, 21, 2568Chanson; P., Salenave; S., Orphanet J. Rare Dis., 2008, 3:17

Somatostatin

Pituitary gigantism

Acromegaly

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Rivier; J., Guillemin; R., et al., J. Med. Chem., 1975, 18, 123

HN

NH

HN

NH

HN

NH

HN

H2NO

O

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

OHO

OSS

NH2H2N

O

NH

NH2

HOHOHO

Me Me

HN

NH

HN

NH

HN

NH

HN

H2NO

O

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

OHO

O

NH2H2N

O

NH

NH2

HOHOHO


Intrinsic macrocyclization

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Veber; D.F, Hirschmann; R., et al., JACS, 1976, 98, 2367

RHN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

O

R1

XY

NH2H2N

O

NH

NH2

HOHOHO


Stabilization of macrocycle

8

91011

1213

76

543

lD-ĂŵŝŶŽĂĐŝĚĂƚŝнϭƉŽƐŝƚŝŽŶŝƐŬŶŽǁŶƚŽďĞƐƚƌŽŶŐŝŶĚƵĐĞƌŽĨɴ-turn structure.

lIntroduction of D-Trp at position 8 improved the activity by 8-fold.

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Rivier; J., Vale; W., et al., Biochem. Biophys. Res. Commun., 1975, 65, 746


D-Trp at i+1 position improved the activity

HN

NH

HN

NH

HN

NH

HN

H2NO

O

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

OHO

OSS

NH2H2N

O

NH

NH2

HOHOHO

8

91011

1213

7654

3

14

21

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Veber; D.F, Hirschmann; R., et al., PNAS, 1978, 75, 2636


Search for active conformation

HN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

O

NH2

NH

NH2

HOHO

SS

III, IIa (CysїCys(Acm))VI, V (Cys=Cys(Acm))

HN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

O

NH2H2N

O

NH

NH2

HOHOHO

SS

HN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

O

NH2

NH

NH2

HOHO

S S

IV, IIb (Cys=Cys(Acm))

8

910

11

12

13

76

5

4

HN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

O

NH2H2N

O

NH

NH2

HOHOHO

8

910

11

12

13

76

5

4

Cyclic analog

8

9101112

13

765

4

8

9

10

11

12

13

76

5

4

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Veber; D.F, Hirschmann; R., et al., Nature, 1979, 280, 512


Extraction of the essential structure

HN

NH

HN

NH

HN

O

O

O

O

O OHN

O

HN

NH

OHN

ONH

OHN

O

NH2

NH

NH2

HOHO

SS

I

8

91011

12

13

76

5

4

HN

NH

HN

O

O

O OHN

O

HN

NH

OHN

O

NH

NH2

HO

SS

III

8

91011

12

76

5

HN

NH

HN

O

O

O OHN

O

HN

NH

OHN

O

NH

NH2

HOIIa

8

91011

76

12

5

lShorter bi-cyclic peptide resulted in ca. 2.5-fold increase in the activity.

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Veber; D.F, Hirschmann; R., et al., Nature, 1981, 292, 55Lewis; I., Bruns; C., et al., J. Med. Chem., 2003, 46, 2334

NH

HN

O

O OHN

O

HN

NH

O

NH

NH2

HO

NO


DĂĐƌŽĐǇĐůŝǌĂƚŝŽŶďǇɴ-turn inducing amino acids

8

NH

HN

O

O OHN

O

HN

NH

O

NH

NH2

NO

O NH

H2N

OPasireotide

lIncorporation of turn inducing sequence resulted in improved effect p.o.

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Biron; E., Kessler; H. et al., ACIE, 2008, 47, 2595


NH

NO

O ON

O

HN

N

O

NH

NH2

HO

NO

11

67 8

9

10

8

Further improvement by N-Methylation

lThe conformation shown above seems to have effect on bioavailability.lThe turn structure is the same as orally available peptide cyclosporin A.lAlteration of turn type resulted not only in the loss of binding affinity but also in the loss of bioavailability.

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Räder; A.F.B., Kessler; H., et al., Bioorg. Med. Chem., 2018, 26, 2766


Relation between secondary structure and bioavailability

Biron; E., Kessler; H. et al., ACIE, 2008, 47, 2595

NH

NO

O ON

O

HN

N

O

NH

NH2

HO

NO

Me

Me

Me

11

67 8

9

10

Phe11їD-Phe

Loss of binding affinityLoss of bioavailability

type II’ɴ-turn

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Beck; J.G, Kessler; H., et al., JACS, 2012, 134, 12125Räder; A.F.B., Kessler; H., et al., Bioorg. Med. Chem., 2018, 26, 2766



lNone of the parameters could not solely explain the tendency.lMethylation of N atom adjacent to D-Ala was common among the permeable peptides.

lϮŽĨϯďŝŽĂǀĂŝůĂďůĞĂŶĚĐŽŶĨŽƌŵĂƚŝŽŶĂůůǇƵŶŝĨŽƌŵƉĞƉƚŝĚĞƐŚŽǁĞĚɴ//-ɴs/turn structure.

lThe structure is the same as cyclosporin A and the somatostatin analog.

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Beck; J.G, Kessler; H., et al., JACS, 2012, 134, 12125Räder; A.F.B., Kessler; H., et al., Bioorg. Med. Chem., 2018, 26, 2766



Shielding of solvateable NH of lipophilic peptide improve permeability

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Rezail; T., Lokey; R.S., et al., JACS, 2006, 128, 2510Marelli; U.K., Kessler; H., et al., Chem. Euro. J., 2015, 21, 8023


lBlocking of NH does not generally improve bioavailability.

Turn inducing amino acids

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Non-ProteinogenicProteinogenic

NH

HN

O OHN

O

HN

O

R1R2

R3R4

i+1

i+2i+3

i


NH

HN

O ON

O

HN

O

R1R2

R4

i+1

i+2i+3

i

NH

N

O ON

O

HN

O

R4

i+1

i+2i+3

i

R

NH2

O

H


NH

N

O ON

O

HN

O

R1

R4

i+1

i+2i+3

i

NH

N

O ONH

O

HN

O

R1

R4

i+1

i+2i+3

i

R3

Tendency of turn induced by Pro-Xaa sequence

lInduction of turn can be controlled, and indeed there are some major and strong turn inducer, the structure of turn induced will usually be highly dependent on the context.

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Metrano; A.J., Miller; S.J., et al., JACS, 2017, 139, 492

N-Methylation

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48 DŝŵĞƚŝĐƐŽĨƐĞĐŽŶĚĂƌǇƐƚƌƵĐƚƵƌĞɴ-turn

NH

HN

O ON

O

HN

O

R1R2

R4

i+1

i+2i+3

i

NH

HN

O ON

O

HN

O

R1R2

R4

i+1

i+2i+3

i

R

NH

N

O ON

O

HN

O

R1

R4

i+1

i+2i+3

i

NH

N

O ON

O

HN

O

R1R2

R3R4

i+1

i+2i+3

i NH

N

O OHN

O

HN

O

R1R2

R3R4

i+1

i+2i+3

iNH

N

O ON

O

HN

O

R1R2

R4

i+1

i+2i+3

i

lN-Methylation can be seen as a surrogate of proline.

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SummarylStereopopulation controlled mimetics of peptide by the various artificial motifs improve the properties of peptides.l Affinity, Bioavailability and membrane permeability, Metabolic stability, Immunogenicity

lFor biological activity, rigidifying the structure at its active form is crucial.lFor bioavailability and membrane permeability,

l Conformational rigidity and scarcity of solvateable NH (N-methylation, intramolecular hydrogen bonding, steric shielding)

l Lipophilicity (lipophilic sidechains)l ^ƉĞĐŝĨŝĐĐŽŶĨŽƌŵĂƚŝŽŶ;ĞŐɴ//-ɴs/ turn structure with cis peptide bond)

have a positive effect in general.lThe effect of each factor depends on the dominant transport pathway.lFor metabolic stability, unnatural structure is effective on the whole.

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Synthesis of SAH-p53-x

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Structure of Enfuvirtide

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56 Appendix

Synthesis Oligo N-substituted Glycine

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Simon; R.J., Bartlett; P.A., et al., P.N.A.S, 1992, 89, 9367

PyBOP or PyBrop

SPPS

Only example of highly potent inhibitor made of oligo-NSG

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58 Appendix

Zuckermann; R.N., et al., J. Med. Chem., 1994, 37, 2678

ɲ1-adrenergic receptor

ʅ-opiate receptor

Synthesis of Oligo N-substituted Alanine

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59 Appendix

Morimoto; J., Fukuda; Y., Sando; S., et al., J. A. C. S, 2019, 141, 14612

Other foldamers

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60 Appendix

Gangloff; N, Luxenhofer; R., et al., Chem. Rev., 2016, 116, 1753

C,D-ƚǇƉĞŵŝŵĞƚŝĐƐŽĨɲ-helixAppendix

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C,D-ƚǇƉĞŵŝŵĞƚŝĐƐŽĨɲ-helixAppendix

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ƉƉĞŶĚŝǆWŽƉƵůĂƚŝŽŶŽĨɴ-turns

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Bravern; A.G., Sci. Rep., 2016, 6, 33191

enhancing bioactive-peptide by conformationally controlled...

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