E X A M C H E M I C A L G E N E T I C S , January 8, 2014 G U B B E N S

Q u e s t i o n s n a t u r a l p r o d u c t s f r o m Streptomycetes (TWO pages, A through E, 20 points)

NH2

Figure 1: Molecular structure of Gobicliel in A

Sireptomyces sp. NRRL F-4415 was f ound t o p roduce a c o m p o u n d cal led Gobiche l in A. As ind ica ted in

Figure 1, Gobiche l in A can be regarded as a l inear chain consis t ing o f six subun i ts .

a) (3 pt) Explain w h e t h e r Gobichel in A is p roduced by an NRPS comp lex , a PKS complex , o r t h e

r ibosome?

The side chain o f subun i t 4 is d r a w n in the oppos i te d i rec t ion c o m p a r e d t o the side chains o f 3 and 5.

This is t o indicate a d i f fe rence in s te reochemis t ry . However , t he researchers did no t ver i f y th is

exper imen ta l l y . Instead, they looked at t h e gene sequence o f t h e c luster cod ing fo r Gobiche l in A

synthesis.

b) (3 pt) W h i c h fea tu re d id the researchers f ind in the ex tens ion m o d u l e cod ing f o r i nco rpo ra t i on

o f subun i t 4 t h a t explains its s te reochemis t ry?

To con f i rm the compos i t i on o f Gobiche l in A, t h e researchers also t r i ed t o i nco rpo ra te stable i so tope-

labeled c o m p o u n d s . Upon inco rpo ra t i on o f [2,3,3-D3]serine t hey f o u n d the resul t s h o w n in Figure 2.

+ 6 D a

6 7 0 6 8 0

Figure 2: LC-MS analysis of Gobichelin A upon incorporation of [2,3,3-D3]serine

c) (5 pt , answer all subquest ions t o ob ta in fu l l po in ts ! ) Obvious ly , one ser ine is i nco rpo ra ted at

pos i t ion 5. Based on t h e resul t in Figure 2 and the s t ruc tu re in Figure 1 , exp la in h o w many

ser ines are i nco rpo ra ted in t o t a l and at wh ich add i t iona l pos i t ions? Can you pred ic t any

add i t iona l fea tures t h a t w o u l d be present in t h e ex tens ion modu le(s ) cod ing f o r t he add i t i ona l

posi t ion(s)?

1

+ 7 D a

Figure 3: Structure of ornithine Figure 4: LC-IVIS analysis of Gobichel in A

upon incorporation of-[2,3,3,4,4,5,5-D7]ornithine

As observed in Figure 4, o rn i t h ine (see Figure 3 f o r its s t ruc tu re ) can also i nco rpo ra ted in Gobiche l in

A at t he f inal pos i t ion 6. However , t he f inal ex tens ion m o d u l e conta ins an adeny la t i on d o m a i n

specif ic f o r A/ -hydroxy l -orn i th ine (hydroxy l a t t a c h m e n t t o the side cha in -amine) . It t u rns o u t t h a t t he

gene c luster conta ins a separate gene cod ing fo r o rn i t h ine -5 -monooxygenase , wh i ch conver ts

o rn i t h i ne t o A/ -hydroxy l -orn i th ine, p r io r t o i nco rpo ra t i on .

d) (6 pt , answer all subquest ions to ob ta in fu l l po in ts ! ) Draw t h e A/ -hydroxy l -orn i th ine a f te r

i nco rpo ra t i on as a t tached by a t h ioes the r br idge t o t h e PCP d o m a i n o f t h e f ina l ex tens ion

m o d u l e (Don ' t d r a w t h e en t i re pan te the ine mo ie t y , jus t t h e th ioes te r ) . Also, ind icate w h e r e t h e

r ema inde r o f t he Gobiche l in A chain is a t t ached . Next , ind icate by an a r r o w h o w gob iche l in A is

t e r m i n a t e d f r o m t h e synthetase comp lex to ob ta in the f inal s t ruc tu re in Figure 1. Explain

w h e t h e r th is is a cycl izat ion or reduc t ion react ion?

raXD raUE rahO

RakA

RakB

RakC

RakD

RakE

RakF

RakG

RakH

RakI

RakL

RakN

AT ^KR ACP

AT J « r , D H

KS

C

KS

C

C

ACP

TE

nMT

H o N

Figure 5: Ral<icidin synthesis cluster and molecular structure. n M T = A/ -methy l t ransferase

e) (3 pt) Consider t h e c o m b i n e d NRPS/PKS synthesis c luster fo r rakic id in f r o m Sireptomyces

lilacinus NRRL B-1968 (Figure 5) . In terest ing ly , every ex tens ion m o d u l e is coded on a separate

gene. Explain wh i ch gene f r o m Figure 5A/B codes f o r t h e i nco rpo ra t i on o f t he subun i t ind ica ted

by t h e box in Figure 5C?

2

E X A M C H E M I C A L G E N E T I C S , January 8, 2014 F L O R E A

Please write down your name on every answer page. There are 20 points.

Activity-based protein profiling and Post translational modifications

You h a v e j u s t g r a d u a t e d as e x p e r t in t h e f i e ld o f a c t i v i t y - b a s e d p r o t e i n p ro f i l i ng a n d M i l l e n n i u m p h a r m a c e u t i c a l s is i n t e r e s t e d t o h i re y o u as sc i en t i s t . You r f i r s t a s s i g n m e n t is t o se t up a s c r e e n i n g p l a t f o r m f o r t h e d i s c o v e r y o f nove l p r o t e a s o m e i n h i b i t o r s . I n y o u r c h e m i c a l t o o l b o x y o u wi l l f i n d f l u o r o g e n i c s u b s t r a t e s a n d a c t i v i t y - b a s e d p r o b e s (ABP) .

F l uo rogen i c s u b s t r a t e s f o r m e a s u r i n g p e p t i d a s e a c t i v i t y cons i s t o f a f l u o r o p h o r e ( a m i n o - c o u m a r i n e g r o u p ) a t t a c h e d to t h e C - t e r m i n u s o f a s h o r t p e p t i d e v ia a p e p t i d e b o n d . T h e s t r u c t u r e b e l o w is an e x a m p l e o f a f l u o r o g e n i c s u b s t r a t e .

Chemical Formula: C36H49N5O7 Exact Mass: 663.3632

Molecular Weight: 663.8160

a) D r a w t h e a m i n o - c o u m a r i n e s t r u c t u r e and t h e f i r s t a m i n o ac id a t t a c h e d t o

i t a n d s h o w t h e p o s i t i o n o f t h i s p e p t i d e b o n d ( 1 p t )

b) G i ve t h e 1 l e t t e r code f o r t h e a m i n o ac ids in t h e p e p t i d e . See t h e p e p t i d e

t a b l e on t h e las t p a g e . ( 1 p t )

A l t h o u g h t h e f l u o r o g e n i c s u b s t r a t e is v e r y use fu l f o r k i ne t i c s s t u d i e s , i t d o e s n o t d i s t i n g u i s h b e t w e e n t h e c o n s t i t u t i v e a n d i m m u n o s u b u n i t s o f t h e p r o t e a s o m e . Y o u d e c i d e t o l ook f o r m e c h a n i s m - b a s e d i n h i b i t o r s t h a t b i nd c o v a l e n t l y t o t h e p r o t e a s o m e a c t i v e s i t es , t h e so ca l led a c t i v i t y - b a s e d p r o t e i n p ro f i l i ng (ABPP) .

c) Make a s c h e m a t i c d r a w i n g f o r a c o m p e t i t i v e g e l - b a s e d ABPP e x p e r i m e n t a n d e x p l a i n h o w y o u can se lec t a p o t e n t i n h i b i t o r (2 p t )

I n s o m e cases , a t t a c h m e n t o f an a f f i n i t y p u r i f i c a t i o n t a g d i m i n i s h e d t h e cel l p e r m e a b i l i t y o f y o u r ABP.

d ) W h i c h t a g is m o s t l y used f o r a f f i n i t y p u r i f i c a t i o n ? ( 1 p t )

A f t e r m a n y s c r e e n s , y o u f i n d o u t t h a t e p o x o m i c i n is a v e r y p o t e n t , c o v a l e n t p r o t e a s o m e i n h i b i t o r w h i c h is p r o p o s e d as d r u g c a n d i d a t e f o r t h e t r e a t m e n t o f m u l t i p l e m y e l o m a . H o w e v e r , t h e t o x i c o l o g y d e p a r t m e n t ra ises s o m e c o n c e r n s b e c a u s e c o v a l e n t l y b i n d i n g d r u g s m a y c a u s e i d i o s y n c r a t i c d r u g r e l a t e d t o x i c i t y .

e ) D e s c r i b e in a f e w s e n t e n c e s w h a t i d i o s y n c r a t i c d r u g r e l a t e d t o x i c i t y m e a n s . W h i c h is t h e m o s t i m p o r t a n t p r o t e a s e in t h i s p rocess? (3 p t )

P r e s e n t a t i o n o f a p e p t i d e t o t h e i m m u n e s y s t e m o b e y s v e r y s t r i c t r u l es . T h e e p i t o p e m u s t be o f a c e r t a i n l e n g h t a n d b i o t i n is n o t t o l e r a t e d . T h e c o n c e n t r a t i o n o f e p i t o p e s is e x t r e m e l y low so t h e r e is n e e d f o r e n r i c h m e n t p r i o r t o a n a l y s i s .

You a r e a s k e d b y t h e t o x i c o l o g y d e p a r t m e n t t o use y o u r h i t m o l e c u l e f r o m t h e s c r e e n t o d e v e l o p an ABPP s y s t e m t h a t can d e t e c t t h e m o l e c u l e s t h a t m i g h t c a u s e t h e i m m u n o l o g i c a l s ide e f f e c t s .

f ) W h i c h ABPP m e t h o d w o u l d y o u choose? ( 1 p t )

h ) D e s c r i b e in d e t a i l t h e w o r k f l o w f o r t h i s e x p e r i m e n t . W h e r e d o e s t h e c o m p o u n d b i n d , h o w it is p r o c e s s e d , p r e s e n t e d , a n a l y s e d a n d ident i f ied"? (3 p t )

Be low y o u see a t a b l e w i t h t h e f i r s t 4 5 a m i n o ac ids o f t h e m o u s e p r o t e a s o m e a c t i v e p - s u b u n i t s .

p l : TTIMAVQFDGGVVLGADSRITTTGSYIANRVTDKLTPIHDRIFCCR

P l i : TT IMAVEFDGGVVMGSDSRVSAGEAWNRVFDKLSPLHERIYCAL

P2 : T T I A G W Y i ^ D G I V L G A D T R A T E G M V V A D K N C S K I H F I S P N I Y C C G

p 2 i : T T I A G L V F Q D G V I L G A D T R A T N D S V V A D K S C E K I H F I A P K I Y C C G

p 5 : T T T L A F K F R H G V I V A A D S R A T A G A Y I A S Q T V K K V I E I N P Y L L G T M

p 5 i : T T T L A F K F Q H G V I A A V D S R A S A G S Y I S A L R V N K V I E I N P Y L L G T M

P 5 t : TTTLAFRFRHGVIAAADTRSSCGSYVACPASCKVIPVHQHLLGTT

T h e r e s u l t o f y o u r e n r i c h m e n t e x p e r i m e n t s a re p r e s e n t e d b e l o w .

16

biot in l [ e thg l y ] ; pho sph Ile Ile T h r l L e u ' i S . c ' 5 .

b; b; b, b, b, ^ : N

OH O yy y y y y , 1̂0̂ 11 ^2 b̂4 b,5 b̂e

c

9B3.31

(b,+2H)^

S44.98

(b,+2H)

494.60! |[b,+2Hr

438.051

\2t (b3+2HV 624.76 Dj

875.11

706.26 b

be 988.30

by 1089.28 b,

b,4 655

a 1336.71 1248.201

b„ 456

b„ 409 328 D

3991

507,

664

636

566

( y , -COOH) 919

b,6

817

861 (Vo-H^O)

600 700 m/z

800 1000 1200 tn/z

1400 1600 1800

i) Exp la in in w o r d s w h a t t h i s MS f r a g m e n t a t i o n resu l t s s h o w s h e r e (3 p t )

j ) . D e t e r m i n e t h e s e q u e n c e o f t h e p r o t e a s o m e s u b u n i t f r o m w h i c h t h i s a c t i v e

s i te g e n e r a t e s (5 p t )

Table of amino acids

A Ala Alanine 71.03 C Cys Cysteine 103.01 D Asp Aspartic acid 115.03 E Glu Glutamic Acid 129.04 F Phe Phenylalanine 147.07 G Gly Glycine 57.02 H His Histidine 137.06 I lie Isoleucine 113.08 K Lys Lysine 128.09 L Leu Leucine 113.08 M Met Methionine 131.04 N Asn Asparagine 114.04 P Pro Proline 97.05 Q Gin Glutamine 128.06 R Arg Arginine 156.1 S Ser Serine 87.03 T Thr Threonine 101.05 V Val Valine 99.07 w Trp Tryptophan 186.08 Y Tyr Tyrosine 163.06

E X A M C H E M I C A L G E N E T I C S , January 8, 2014 VAN K A S T E R E N

Bio-Orthogonal Chemistry

a) Compare and cont ras t t he s t r a i n -p romo ted 3+2 cyc lo -add i t ion (click react ion) and t h e copper

catalysed 3+2 cyc loadd i t ion react ion as b ioo r thogona l react ions. Focus on react ion rates,

i nco rpo ra t i on in to b iomolecu les , tox ic i ty , s tabi l i ty o f t h e p roduc ts , compa t i b i l i t y w i t h l iv ing

systems (4 po in ts)

b) Chlamydia trachomatis is one o f t h e leading causes o f sexual ly t r a n s m i t t e d bacter ia l disease.

Its s y m p t o m s inc lude ster i l i ty and bl indness, bu t in fec t ion can o f t e n go unde tec ted unt i l t h e

damage is d o n e . It is also a cont rovers ia l bac te r i um: It is no t k n o w n w h e t h e r it has a

pept idog lycan (PG) and t h e ev idence is con t rad ic to ry :

• It is k i l led by pept idog lycan d is rup t ing ant ib io t ics

• A fu l ly opera t iona l cell wa l l b iosynthet ic mach inery has been d iscovered

• No PG has been f o u n d using s ta in ing m e t h o d s

UDP MurNAc

TI-Ala j D-GIU

Q> CX 1 m-DAP (3 ^^ C \ D-Ala GJ

0 . 1 D-Ala

L-Ala D-Ala o-Ala D-Ala-D-Ala

(DA-DA)

Bio-o r thogona l chemis t ry has recent ly (Nature d o i : 1 0 . 1 0 3 8 / n a t u r e l 2 8 9 2 ; pub l ished 5*^ o f

december ) been used t o shed l ight on th is cont roversy . B io -o r thogona l l y tagged D-Ala- D-Ala

d imers we re fed t o C. trachomatis and labeled w i t h a f l u o r o p h o r e in f ixed cells.

- W h i c h b io -o r thogona l react ion w o u l d you use t o ob ta in t h e best signal to noise

proper t ies? (10 points)

W h i c h chemica l handle w o u l d you in t roduce in to you r D-Ala-D-Ala d imers t o

select ive ly label C. trachomatis PG? (1 po in t )

- W h a t o t h e r m e t h o d s could you select ively t rack Chlamydia in v ivo? (1 po in t )

- The au thors o f th is paper also t r ied label ing w i t h D-Ala m o n o m e r s . This d id no t

w o r k in C. trachomatis. W h i c h enzyme in t h e pep t idog lycan biosynthesis is

t h e r e f o r e no t compa t i b l e w i t h th is b io -o r thogona l tagging s t ra tegy? (1 po in t )

c) A m b e r codon suppress ion is a p o w e r f u l m e t h o d fo r t h e i n t r o d u c t i o n o f unna tu ra l a m i n o

acids f o r b io -o r thogona l tagging at specif ic sites in p ro te ins .

• W h a t are the steps one needs to take t o p roduce a mammal ian -exp ressed p ro te in

w i t h an unna tu ra l a m i n o acid? (1 po in t )

• Wh ich tRNA/ tRNA synhetase pair is t he best fo r achiev ing th is? ( 1 po in t )

• W h a t are t h e ma jo r by-produc ts w h e n expressing a p ro te in w i t h an a m b e r codon? (1

po in t )

E X A M C H E M I C A L G E N E T I C S , January 8, 2014 V A N DER S T E L T

Kinases

Compound 1 (PLX4720)

Question 1 PLX4720 (compound 1) is an Iniiibltor of B-RAF^^°°^ l<inase (ICjo = 13 nl^l). PLX4720 has been tested in two different xenograft models using two different melanoma cell lines. PLX4720 Inhibited tumour growth in the 1205LU model, which expresses B-RAF^^"""^ (See Figure 1, left panel), whereas It was ineffective in blocking tumour growth In C8161 model (See Figure 1, right panel).

l a . PLX4720 Is a type 1 kinase inhibitor. Draw the most important Interactions of PLX4720 with the amino acids in the ATP-blndIng pocket of B-RAF^^°°^; Explain how this compound stabilizes the active conformation of this mutant kinase. (5 pt)

l b . PLX4720 does Inhibit wild type BRAF (IC50 = 160 nM). Explain why PLX4720 Is Ineffective In blocking tumour growth of C8161 melanoma cells, which express wild type B-RAF. (5 pt)

Question 2 2a. PLX4720 cannot be developed into a drug, because it does not reach sufficient plasma concentrations In the blood. I t Is thought that this is due to low solubility. Three new compounds (2, 3 and 4) have been proposed to replace PLX4720. Which one would you select to be synthesized and tested In xenograft models? The compound should be potent and selective. Explain your answer. (5 pt)

2b Vemurafenib Is a B-RAF^^°°'̂ kinase inhibitor, which is on the market to treat metastatic melanoma's. However, resistances develops after 8-12 months of treatment. Explain why the tumours reappear and how can this be combatted. (5 pt)