Ligand Discovery and Affinity Maturation with Single Stranded DNA Encoded Chemical Libraries
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ETH Library Ligand Discovery and Affinity Maturation with Single Stranded DNA Encoded Chemical Libraries Doctoral Thesis Author(s): Bassi, Gabriele Publication date: 2020 Permanent link: https://doi.org/10.3929/ethz-b-000476062 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use.
DISS. ETH NO. 27142
(The number is mentioned on the invitation for your doctoral examination.)
LIGAND DISCOVERY AND AFFINITY MATURATION WITH SINGLE
STRANDED DNA ENCODED CHEMICAL LIBRARIES
A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
presented by
GABRIELE BASSI
Master of science in chemistry, Università degli studi di Pavia
born on 22.06.1992 citizen of Italy
accepted on the recommendation of
Prof. Dr. Dario Neri,
Prof. Dr. Gisbert Schneider,
P.D. Dr. Jörg Scheuermann,
20201. Summary
Drugs are chemical or biological compounds, able to specific interact with one or more target
proteins, hence driving a pharmacological benefit for the prevention, diagnosis or treatment of
a pathology. In the past, the isolation of novel drugs has often started from the screening of
bioactive compounds from nature, on the basis of trial and error approaches. More recently,
however, also thanks to advances in molecular biology and in the production of recombinant
proteins, it has become important to discover protein ligands by screening organic molecules
in large compound collections, called “chemical libraries”.
At present, it is customary for large pharmaceutical companies to try and identify specific
protein ligands (called “hits”) starting from chemical libraries that may contain up to one
million compounds. Similar activities are prohibitively expensive for academic groups. In
many cases, high-throughput screening assays are used. Once a hit compound is identified,
suitable modifications using medicinal chemistry are performed in order to generate improved
molecules, which may deserve to be tested both in vitro and in vivo. If the so-called “lead
compounds” exhibit the required specificity and potency, alongside other parameters which are
important for clinical development, the product candidates may progress towards late-stage
industrial activities. In many cases, however, screening campaigns based on high-throughput
screening fail to delivery hits. For this reason, there is a considerable interest in the
development of novel methodologies for the construction and screening of large chemical
libraries.
DNA-Encoded chemical libraries (DELs) are collections of small organic molecules,
individually coupled to DNA fragments which act as amplifiable identification barcodes. DELs
can be very large (i.e., containing millions to billions of compounds), as they can be synthesized
using split&pool procedures, starting from a moderate number of starting building blocks.
In this thesis, I describe the synthesis and validation of a novel DEL (termed GB-DEL),
featuring DNA in single-stranded format and containing 366.600 compounds. The library was
based on a stereoisomeric scaffold (i.e., a derivative of glutamic acid), whose compact structure
facilitated chemical transformations and yielded library members of small size (i.e., typically
smaller than 500 Dalton). The GB-DEL library was screened against a number of different
proteins and provided ligands against targets of pharmaceutical interest. I have discovered and
5characterized ligands directed against carbonic anhydrase IX (CAIX), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP-1) and placental alkaline phosphatase, which are tumor- associated antigens suitable for pharmacodelivery applications. Be I have isolated small organic binders against proteins involved in DNA repair and cell metabolism such as FANCD2/FANCI-associated nuclease I (FAN1) and alpha amino adipic semi aldehyde synthase (ASS), as well as enzymes involved in cellular signaling (such as PI3K and CREBBP). GB-DEL hybridization with chemically-modified complementary DNA strands, carrying a protein-binding moiety resulted in self-assembled chemical structures displaying three sets of building block and facilitated the identification of affinity-matured compounds. In a dedicated study, I have investigated whether it would be more convenient to use DELs in single-stranded DNA format, or rather convert the DNA into a double-stranded format using Klenow polymerization. Comparative selection experiments revealed that both approaches could be productive in yielding specific protein ligands, but the use of single-stranded DNA barcodes produced slightly superior selection fingerprints. In one chapter of the thesis, I have reported the discovery and validation of a potent and selective inhibitor of placental alkaline phosphatase (PLAP), a tumor associated antigen frequently expressed in tumors of the female reproductive tract. One of the compounds isolated from a DEL (i.e., from the NF-DEL library, produced by my colleague Nicholas Favalli) inhibited PLAP with an IC50 = 32 nM. Importantly, the product had not detectable inhibition of the closely related tissue non-specific alkaline phosphatase (TNAP), which is expressed on the surface of many normal cells. The PLAP ligand was conjugated to fluorescein and specifically bound to PLAP-positive tumors in vitro. When injected into tumor-bearing mice, the fluoresceinated molecule targeted cervical cancer in vivo, as evidenced by ex vivo fluorescence microscopy investigations. Finally, I have also screened the GB-DEL against tyrosinase and TYRP-1, two proteins selectively expressed in melanocytes and, in pathological context, in melanoma lesions. We discovered novel ligands capable of selective binding to both proteins and used a previously described tyrosinase inhibitor (Thiamidol™) as starting point for affinity maturation methodologies. 6
Collectively, the thesis shows that DEL technology can facilitate the discovery of ligands to a
variety of different proteins of biological or pharmaceutical interest.
72. Riassunto Un farmaco, per definizione, è un qualsivoglia composto chimico o biologico atto ad interagire specificamente con una o diverse proteine bersaglio in modo da recare un effetto benefico per la prevenzione, diagnosi o trattamento di una patologia. In passato, L’identificazione di nuovi composti biologicamente attivi si basava su un approcio empirico, in cui sostanze naturali venivano isolate e testate al fine di rivelarne una possibile applicazione in campo medico o biologico. Al giorno d’oggi, la scoperta di nuove potenziali molecole candidate allo sviluppo farmaceutico avviene attraverso, grazie anche agli avanzamenti scientifici che hanno portato alla produzione di proteine ricombinanti, a saggi enzimatici ad alta automatizzazione in un processo denominato “High throughput screening”. Durante questa procedura vengono analizzate in singoli esperimenti librerie chimiche, sinthetiche o naturali, contenenti fino ad un milione di composti al fine di identificare liganti specifici (“hits”) a una proteina (target). Una volta identificato e selezionato un hit, il composto viene sottoposto a una prima fase di traformazioni chimiche per renderlo adatto a sperimentazioni in vitro ed in vivo. Una volta identificato il “lead compound con adeguata specificità, bassi profili di tossicità e molti altri parametri importanti per lo sviluppo farmaceutico, può iniziare la cosidetta fase clinica di validazione. Tuttuvia, i costi e la logistica necessaria a una campagna di screening rendono l’high throughput screening impraticabile a livello accademico. In aggiunta, una campagna potrebbe fallire nell’identificare composti attivi causando perdite monetarie sostanziali ad aziende che investono in questa tecnologia. Per queste ragioni, è necessario sviluppare nuove metodologie per lo screening e la sintesi di librerie che abbattano i costi richesti dall’high throughput screening. Le DNA encoded chemical libraries (DELs) consistono in grandi collezioni di composti organici individualmente codificati da DNA che viene utilizzato come “codice a barre” per il riconoscimento dell’identità di ogni singolo composto in soluzione. Questa tecnologia rappresenta un’alternativa innovativa per la sintesi e l’analisi di grandi librerie di composti a costi relativamente contenuti e in un solo eppendorf tube. Le DEL possono contenere fino a 8
miliardi di composti partendo da centinaia di frammenti (“building blocks”) sfruttando la
chimica combinatoriale (split&pool).
In questa tesi è riportata la costruzione e la validazione di una libreria contenente 366'600
composti basata su uno scaffold stereoisomerico (derivato dall’acido glutammico) denominata
GB-DEL. La libreria ha permesso l’identificazione di nuovi liganti per proteine di
considerevole interesse farmaceutico. Tra queste anidrasi carbonica IX (CAIX), tirosinasi
(TYR), proteina tirosinasi correlata 1 (TYRP-1), Placental alkaline phosphatase (PLAP)
cosiddette tumour associated antigens e adatte ad applicazioni di pharmacodelivery, nucleasi
FANCD2/FANCI-associata I (FAN1), ASS correlate a metabolismo cellulare e DNA repair
eprotein coinvolte in signalling cellulare, PI3K and CREB binding protein.
In aggiunta, l’ibridizzazione del filamento di DNA codificante per i composti contenuti nella
libreria con un filamento di DNA a esso complementare, coniugato a liganti noti per diverse
protein target, ha acconsentito la formazione di strutture a doppia elica utili per l’isolamento di
composti con costanti di affinità superiori.
In un progetto correlato abbiamo valutato qualora sia più conveniente costruire librerie in
formato single stranded o double stranded testando le performace con le due differenti varianti.
Partendo dalla GB-DEL (single strand) abbiamo eseguito l’annealing con un oligonucleotide
complementare a essa formando una struttura a doppia elica equivalente. Sorprendentemente,
le librerie a single strand hanno dimostrato performance debolmente ma consistentemente
superiori.
In un capito della tesi, si tratterà degli esperimenti di scoperta e validazione di un inibitore 32
nM contro PLAP, tumour associated antigen espresso in differenti tumori dell’apparato
riproduttivo sia femminile che maschile. Il ligante ha dimostrato di essere specifico anche nei
confronti di un isozima di PLAP, tissue non specific alkaline phosphatase (TNAP) che è
espresso sulla superfice di molte cellule non malate. L’hit compound coniugato alla
fluoresceina, ha dimostrato di legare cellule cancerose positive a PLAP in vitro. Quando
iniettata in topi aventi tumori subcutanei esprimenti PLAP, la molecola fluoresceinata ha
inoltre dimostrato di targettare un modello umano di cancro alla cervice in vivo come
evidenziato da esperimenti di microscopia ex-vivo.
9Per concludere, questa tesi dimostra l’applicabilità delle DNA encoded chemical libraries per la scoperta di nuovi ligandi per proteine di interesse biologico o farmaceutico. 10
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