Multicomponent reactions, applications in medicinal chemistry & new modalities in drug
discovery
Konstantinidou, Markella
DOI:10.33612/diss.111908148
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MULTICOMPONENT REACTIONS, APPLICATIONS
IN MEDICINAL CHEMISTRY & NEW MODALITIES
IN DRUG DISCOVERY
Markella Konstantinidou
2020
The research was financially supported by the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014 – 2020) under the Marie Skłodowska – Curie Grant “AEGIS” (Accelerated Early staGe Drug Discovery, Agreement No. 675555).
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, Faculty of Mathematics and Natural Sciences, University of Groningen, The Netherlands.
Ebook : PDF zonder DRM (PDF without DRM) ISBN: 978-94-034-2333-3
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Cover design: Danai Konstantinidou Layout: Douwe Oppewal, www.oppewal.nl Printing: Ipskamp printing
© Copyright 2020, Markella Konstantinidou. All rights reserved. No part of this thesis may be reproduced in any form or by any means without prior permission of the author.
Multicomponent reactions,
applications in medicinal
chemistry & new modalities in
drug discovery
PhD Thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. C. Wijmenga
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Friday 14 February 2020 at 14.30 hours
by
Markella Konstantinidou
born on 21 April 1989
Prof. T.A. Holak
Assessment Committee Prof. F.J. Dekker
Prof. P.H. Elsinga Prof. R.V.A. Orru
To
to my family
TABLE OF CONTENTS
Outline of the thesis 9
Chapter 1 Inhibitors of programmed cell death 1 (PD-1): a patent review (2010-2015) 17 Chapter 2 Immune checkpoint PD-1/PD-L1: is there life beyond antibodies? 27 Chapter 3 Glutarimide alkaloids through multicomponent reaction chemistry 43
Chapter 4 β-carbolinone analogues from the Ugi silver mine 91
Chapter 5 Pd-catalyzed de novo assembly of diversely substituted indole-fused polyheterocycles
111
Chapter 6 Sequential multicomponent synthesis of 2-(imidazo[1,5-α]pyridine-1-yl)-1,3,4-oxadiazoles
141
Chapter 7 1,3,4-Oxadiazoles by Ugi-tetrazole and Huisgen reaction 167 Chapter 8 Rapid discovery of novel aspartyl protease inhibitors using an anchoring
approach
193
Chapter 9 PROTACs - A game-changing technology 229
Chapter 10 Discovery of proteolysis targeting chimeras for the cyclin-dependent kinases 4 and 6 (CDK4/6)
255
Chapter 11 Design and synthesis of proteolysis targeting chimeras for the leucine-rich repeat kinase 2 (LRRK2)
277
Summary and future perspectives 305
Samenvatting en toekomstperspectieven 313
Appendix About the author Publications Conferences Acknowledgements 322 323 324 325
OUTLOOK
Medicinal chemistry plays a key role in the drug discovery process, including the early stages of hit identification, the lead optimization (hit-to-lead) and process chemistry. It is considered a multi-disciplinary field and medicinal chemists are key players in interactions with computational chemists, biologists and pharmacologists. In the last few decades, the two main approaches used in drug design (high-throughput screening (HTS)[1] and fragment-based drug discovery (FBDD) [2-3]) had also an effect on medicinal chemistry. The first approach required a large number of compounds for screening, which gave rise to combinatorial chemistry. On the contrary, in the second approach, a smaller number of compounds was needed in the first screening steps, but medicinal chemists had the non-tedious task of designing routes for growing, merging and linking fragment hits together towards drug-like molecules. Nowadays, the growing interest of pharmaceutical industries and academia in difficult or “undruggable” targets, has brought into the research fields a considerable amount of protein – protein interactions (PPIs).[4-5] PPIs tend to lack well-defined binding sites and are largely flat, hydrophobic areas. Therefore, medicinal chemistry also needed to shift from small molecules designed for typical, well-defined binding sites to new modalities. In the last few years, the medicinal chemistry toolbox was enriched with macrocycles, stapled peptides, antisense oligonucleotides and proteolysis targeting chimeras (PROTACs).[6]
In this thesis, new targets in medicinal chemistry, in particular the PPI of PD-1/PD-L1, novel synthetic methodologies towards scaffolds with diverse biological applications and lastly PROTACs, as a highly promising new modality in drug discovery, are discussed.
The accumulation of biological data and better understanding of the immune checkpoints has made the field of immune-oncology a very promising and competitive area in cancer research. [7] In particular, the identification of monoclonal antibodies (mAbs) targeting the PD-1/PD-L1 axis and the first approvals by FDA in 2014 have revived the field. Although monoclonal antibodies for these targets have shown impressive clinical outcomes, there are still certain disadvantages. In general, mAbs are not orally bioavailable and have a high molecular weight, which leads to poor diffusion, especially in large tumors. Production costs are also very high. In chapter 1, promising small molecules targeting the PPI of PD-1/PD-L1 that were disclosed in patents in the last couple of years are discussed. In chapter 2, a structural analysis, is provided, based on co-crystal structures of mAbs, small molecules and macrocycles that aim to block the interaction.
In the drug discovery process, time has always been a key factor. The development of medicinal chemistry and the hit-to-lead optimization are still considered a rate-limiting step. In an interesting analysis regarding the type of reactions most commonly employed in drug discovery, it was shown that there is a tendency to rely on known synthetic routes, with a high prevalence of amide coupling reactions and C-C coupling steps.[8-9]As a result of this trend, certain types of molecular shapes are prevailing and the chemical space explored is limited. There is still a constant need for
11
optimizing reaction schemes, reducing the required time and number of steps and minimizing waste. Most of these requirements are met by multicomponent reaction chemistry (MCR), which in contrast to classical synthetic routes relies at using at least 3 starting materials in a single synthetic step to access complex scaffolds and covers rapidly unexplored chemical space.
routes relies at using at least 3 starting materials in a single synthetic step to access complex scaffolds and covers rapidly unexplored chemical space.
Figure 1. Advantages of multi-component reactions.
Multi-component reaction chemistry can significantly accelerate the synthesis of derivatives and allows the coverage of large chemical space. In most of the cases the reaction conditions are mild and inert atmosphere or dry solvents are not required. Moreover, functional groups are well-tolerated, thus the necessity of protecting and deprotecting steps is kept to a minimum. It is noteworthy that MCR scaffolds can withstand a large number of post-MCR modifications, including cyclizations, macrocyclizations and Pd catalyzed reactions[10-12], just to name a few commonly used strategies. Depending on the
choice of starting materials, properly selected functional groups can be employed at a secondary MCR.
The application of MCR synthetic methodologies is used either to improve an existing synthetic route or to access a scaffold that is not accessible with classical synthetic routes. In chapter 3, a synthetic route for glutarimide alkaloids was designed. The existing procedures don’t provide an easy access neither to the natural products nor to their derivatives. In the described MRC-based methodology, the key step is an Ugi reaction, with two points of variations, thus significantly enabling the synthesis of derivatives.
In chapter 4, a one-pot procedure is discussed regarding the synthesis of beta-carbolinone analogues. The intermediate of the initial Ugi reaction undergoes an intramolecular cyclization towards the desired scaffold. The one-pot protocol reduces the number of purification steps.
In chapter 5, a successful combination of an Ugi reaction with a palladium-catalyzed cyclization to access tetracyclic indoloquinolines, a class of natural alkaloid analogues, is shown. Commercially available starting materials can be used and a library of derivatives was rapidly synthesized.
Figure 1. Advantages of multicomponent reactions.
Multicomponent reaction chemistry can significantly accelerate the synthesis of derivatives and allows the coverage of large chemical space. In most of the cases the reaction conditions are mild and inert atmosphere or dry solvents are not required. Moreover, functional groups are well-tolerated, thus the necessity of protecting and deprotecting steps is kept to a minimum. It is noteworthy that MCR scaffolds can withstand a large number of post-MCR modifications, including cyclizations, macrocyclizations and Pd catalyzed reactions[10-12], just to name a few commonly used strategies. Depending on the choice of starting materials, properly selected functional groups can be employed at a secondary MCR.
The application of MCR synthetic methodologies is used either to improve an existing synthetic route or to access a scaffold that is not accessible with classical synthetic routes. In chapter 3, a synthetic route for glutarimide alkaloids was designed. The existing procedures don’t provide an easy access neither to the natural products nor to their derivatives. In the described MCR-based methodology, the key step is an Ugi reaction, with two points of variations, thus significantly enabling the synthesis of derivatives.
In chapter 4, a one-pot procedure is discussed regarding the synthesis of beta-carbolinone analogues. The intermediate of the initial Ugi reaction undergoes an intramolecular cyclization towards the desired scaffold. The one-pot protocol reduces the number of purification steps.
In chapter 5, a successful combination of an Ugi reaction with a palladium-catalyzed cyclization to access tetracyclic indoloquinolines, a class of natural alkaloid analogues, is shown. Commercially available starting materials can be used and a library of derivatives was rapidly synthesized.
In chapter 6, the focus is the scaffold of 2-(imidazo[1,5-α]pyridine-1-yl)-1,3,4-oxadiazoles, a scaffold of biological importance for topoisomerase II inhibitors and 5HT4 partial agonists. The existing synthetic routes require 6 steps in total and several purifications to access this type of scaffold. The novel designed protocol is based on simple building blocks for an Ugi-tetrazole reaction. With in
situ deprotections and cyclizations, a diverse library of derivatives was synthesized. Remarkably,
only one purification is required in the last step.
In chapter 7, Ugi-tetrazole and Huisgen reactions were combined to access the privileged scaffold of 2,5-disubstited 1,3,4-oxadiazoles. A large number of functional groups was tolerated and great diversity was achieved through the three possible variation points. The synthesis showed good scalability and post-modifications were also well-tolerated.
In chapter 8, an application of MCR scaffolds on a medicinal chemistry target is presented. As target proteins the aspartic proteases were selected and in particular the member called endothiapepsin. The aim was to develop an anchor-centered docking approach in order to rationally design, select and optimize our selected scaffold. A series of Ugi-tetrazole products were designed, synthesized and biologically evaluated. Co-crystal structures of potent inhibitors with the target protein were obtained. MCR in this case gives rapid access to the library of potential inhibitors. Moreover, the developed docking protocol allows the enumeration of tailor-made virtual libraries from commercially available starting materials. This protocol gives access to novel virtual libraries that can be developed for diverse biological targets.
The last part of this thesis is focusing on an exciting new modality in drug discovery that has evolved rapidly after its first description in 2001. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules comprising of a ligand targeting a protein of interest, a ligand targeting an E3 ligase and a connecting linker. The aim is instead of inhibiting the target to induce its proteasomal degradation. The concept relies on the natural protein degradation by ubiquitination, and it is proven so far to work effectively on a number of targets that are traditionally classified as challenging or even “undruggable”. In chapter 9, the advantages of PROTACs over classical inhibitors are discussed and an analysis of the existing co-crystal structures of ternary complexes is presented. Special cases, such as homoPROTACs, PROTACs targeting the Tau protein and the first PROTACs that entered clinical trials are discussed.
In chapter 10, the aim is to design, synthesize and evaluate the biological effects of PROTACs targeting the cyclin-dependent kinases 4 and 6 (CDK4/6). Using the FDA approved dual CDK4/6 kinase inhibitor, abemaciclib, after structural modifications, degraders were designed. A small
library, including different types of linkers was synthesized. Preliminary biological data indicate that the designed PROTACs are highly capable of degrading the protein of interest. In chapter 11, the focus is on the design and synthesis of PROTACs targeting leucine-rich kinase 2 (LRRK2), which has emerged as a potential target for Parkinson’s disease. The rational for the design and synthesis is discussed. A hypothesis is presented regarding the features that make this kinase target challenging.
REFERENCES
1. L.M. Mayr, P. Fuerst, J.Biomol. Screen. 2008, 13(6), 443 – 448. 2. D.A. Erlanson, Top Curr. Chem. 2012, 317, 1 – 32.
3. D.A. Erlanson, S.W. Fesik, R.E. Hubbard, W. Jahnke, H. Jhoti, Nat. Rev. Drug Discov. 2016, 15(9), 605 – 619.
4. Z. Giovanna, D.E. Thurston, Future Med. Chem. 2009, 1(1), 65 – 93.
5. D.E. Scott, A.R. Bayly, C. Abell, J. Skidmore, Nat. Rev. Drug Discov. 2016, 15(8), 533 – 550.
6. E. Valeur, S.M. Guéret, H. Adihou, R. Gopalakrishnan, M. Lemurell, H. Waldmann, T.N.Grossmann, A.T. Plowright, Angew. Chem. Int. Ed. Engl. 2017, 56(35), 10294 –10323.
7. C. Voena, R. Chiarle, Discov. Med. 2016, 21(114), 125 –133. 8. D.G. Brown, J. Boström, J. Med. Chem. 2016, 59(10), 4438 – 4458.
9. N. Schneider, D.M. Lowe, R.A. Sayle, M.A. Tarselli, G.A. Landrum, J. Med. Chem. 2016, 59(9), 4385 – 4402.
10. A. Dömling, Chem. Rev. 2006, 106, 17 – 89
11. E.M.M. Abdelraheem, S. Shaabani, A. Dömling, Drug Discov. Today Technol. 2018, 29, 11 – 17 12. S. Saranya, K.R. Rohit, S. Radhika, G. Anilkuma, Org. Biomol. Chem. 2019, 17, 8048 –8061.
CHAPTER
1
INHIBITORS OF PROGRAMMED CELL DEATH 1 (PD-1):
A PATENT REVIEW (2010-2015)
This chapter is published
Tryfon Zarganes -Tzitzikas, Markella Konstantinidou, Yongzhi Gao, Dobroslawa Krzemien, Krzysztof Zak, Grzegorz Dubin, Tad A. Holak and Alexander Dömling
Expert Opinion on Therapeutic Patents 2016, 6, 973 – 977
ABSTRACT
The PD-1/PD-L1 axis is hijacked by viruses and uncontrolled fast growing cells to suppress the immune surveillance. In cancer for example, the malignant cells express PD-L1, which binds to the PD-1 receptor expressed on immune T-cells. Binding of PD-1 to PD-L1 determines a downregulation of T-cell effector functions in cancer patients inhibiting the antitumor immune response and leading to T-cell exhaustion. In viral diseases, a similar mechanism is used by viruses to undermine the effective immune recognitions and answer. Current medications directed towards the PD-1/ PD-L1 axis include monoclonal antibodies. These have shown impressive clinical results in the treatment of several types of tumors. Here, we review the patent literature of non-biologics targeting the protein-protein interaction PD-1/PD-L1 from 2010 until early 2016.
INTRODUCTION
The search for the magic bullet in cancer immunotherapies is ongoing since more than 100 years, but only recent clinical success of immune checkpoint directed antibodies significantly revived the field of immune-oncology.[1] A key protein target in this area is the protein-protein interaction between PD-1 and its ligand PD-L1. Functionally, PD-1, also called programmed death-1 protein, comprises an immune checkpoint located on T-cells. The PD-1/ PD-L1 axis is hijacked by viruses and tumor/cancer cells to suppress the immune surveillance. For example, PD-L1 is expressed on tumor cells and also on immune cells (e.g. myeloid tumor-infiltrating cells). Binding of PD-1 to PD-L1 determines a downregulation of T-cell effector functions in cancer patients, inhibiting the antitumor immune response and leading to T-cell exhaustion.[2] In viral diseases, a similar mechanism is used by viruses to undermine the effective immune recognitions and answer.[3] Current medication directed towards the PD-1/PD-L1 axis includes monoclonal antibodies. These have shown impressive clinical results in the treatment of several types of tumors, including melanoma and lung cancer.[4] Currently, two humanized monoclonal antibodies targeting PD-1 are approved by the regulatory bodies, pembrolizumab and nivolumab.[5] Multiple additional clinical trials either as single agents or in combination with other agents are ongoing to extend their indication areas. Therapeutic antibodies, however, exhibit several disadvantages such as limited tissue and tumor penetration, very long half-life time, lacking of oral bioavailability, immunogenicity, and difficult and expensive production. Moreover, current PD-1/PD-L1 axis directed monoclonal antibodies lead to a tumor response only in a fraction of cases and tumor types. Therefore, a search for non-biologics, including small molecules, peptides, cyclo-peptides and macrocycles is ongoing and will be reviewed here.
DISCUSSION
Recently the co-crystal structure between the human PD-1 and PD-L1 has been determined for the first time.[6] The Å-resolution crystal structure provides a possible starting point for the design of molecules against the protein-protein interaction (PPI). The interface between the two proteins is extended (~1.700 Å2), hydrophobic and flat, without deep binding pockets, which makes the interface likely a difficult target for small molecules. The hydrophobic interface also increases the chances to discover false positive hits considerably. Moreover, direct competitor PD-1/PD-L1 antagonists are potentially very hydrophobic molecules, which can lead to downstream development issues, including toxicity, selectivity and poor water solubility, just to name a few. Nonetheless, currently several series of small molecules, peptides and cyclo-peptides have been disclosed targeting the PD-1/PD-L1 PPI. In the following, we focus on discussing first small molecules, followed by peptide and cyclic peptide derivatives.
A group from Harvard University has discovered sulphonamide derivatives (1) and (2) to work in
1
a similar fashion as reference mAbs[7] (Figure 1). The two compounds are active antagonists in an IFNγ-release assay in transgenic mouse T cells that express PD-1.
Figure 1. Small-molecule antagonists of the PD-1 pathway.
Workers from the company Bristol-Myers Squibb (BMS) have disclosed scaffold (3) (Figure 2) binding to PD-L1.[8] It consists of a tri-aromatic structure, including a mono-ortho substituted biphenyl substructure. Moreover, another phenyl ring is connected to the biphenyl and contains also a methylene amine moiety. The biological activity of the claimed compounds was established by a homogenous time-resolved fluorescence (HTRF) binding assay in which Europium cryptate-labeled anti-Ig was used. To assess selectivity, the PPIs PD-1/PD-L2 and PD-L1/CD80 were tested as well. Typical examples are BMS-8, BMS-37, BMS-202, BMS-230 and BMS-242. No further in vitro or in vivo assays have been described supporting the biological activity of compounds based on scaffold (3) (Figure 2). The structural basis of BMS-202 and BMS-8 as PD-1/PD-L1 antagonists was recently rigorously proven by a cocrystal structure and other biophysical methods.[9]
Certain compound classes have been recently described to interfere with the time-resolved fluorescence resonance energy transfer (TR-FRET) PD-1/PD-L1 assay during a high throughput screening campaign, producing false positive hits.[10] Examples include the salicylates NCI 211717 and NCI 211845. Mechanistically, the interaction of the chelator moiety salicylate with the cryptand-ligated europium FRET donor leading to a change in the assay signal is suggested (Figure 3).
Figure 3. False positive PD-1/PD-L1 inhibitors.
Several PD-1/PD-L1 antagonists based on peptide structures including bioisosteres such as oxadiazole and urea have also been disclosed. Workers from Aurigene Ltd. described cyclic peptidomimetic compounds as immunomodulators able to interfere with the programmed death (PD-1) signalling pathway. The general formula (4) is exemplified in 20 explicit examples. It consists of a central 1-oxa- or 1-thia-3,4-diazole fragment with a serine or threonine side chain in position 2 and another amino acid or dipeptide linked in position 5. Dipeptides are bound by a urea unit. In the majority of examples the first aminoacid in position 5 is asparagine, glutamine, aspartic acid or glutamic acid. The biological activity of the compounds was tested by using a rescue assay of mouse splenocytes in the presence of recombinant mouse PD-1/PD-L1. Compound (5) for example was able to rescue the mouse immune cells to 92% at 100 nM (Figure 4).
Figure 4. Peptidomimetic inhibitors of the PD-1/PD-L1 interaction with oxa- and thiadiazole core moieties.
1
Figure 5. a) Hydrolysis-resistant D-peptide antagonist to target the PD-1/PD-L1, b) macrocyclic peptidic
inhibitor, c) peptide antagonist of PD-L1 (10) along with his sequence similarities to PD-1.
Positional isomeric 1-oxa-2,4-diazoles (6) with otherwise similar sidechains were also disclosed by the same company. Compound (7) exhibited 91% rescue in the above mouse splenocyte assay (Figure 4). Aurigene Ltd. described also small peptidomimetics comprising of 3-4 amino acid moieties with a hydrazine and an urea linker with general structure (8). A total of 14 compounds were explicitly exemplified. [11] Compound (9) for example was able to rescue the mouse immune cells to 84% at 100 nM (Figure 4).
A team from Zhengzhou and Tsinghua Universities described the discovery of the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 interaction.[12] The optimized compound
DPPA-1 could bind to PD-L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that DPPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo (Figure 5).
Researchers at Aurigene Ltd. developed compound (10) for the treatment of cancer. Sequences of the extracellular domain of PD-1 critical for the PD-L1/PD-L2 binding interaction, overlapping or in close proximity to the known ligand-binding regions, were identified and used as starting points for the design and evaluation of 7- to 30-mer peptides derived from human and murine PD-1 sequences. Compound (10) is highly effective in antagonizing PD-1 signalling, with in vivo exposure upon subcutaneous dosing. It is claimed to inhibit tumor growth and metastasis in preclinical models of cancer and to be well tolerated with no obvious toxicity at any of the tested doses. The structure of compound (10) is shown in Figure 5. [13]
BMS chemists disclosed macrocyclic peptides with general structure (11) that inhibit the PD-1/ PD-L1 protein-protein interaction with nanomolar potency in HTRF assays, as well as cellular binding assays. (Figure 5).[8] In addition, the peptides also demonstrate biological activity in cytomegalovirus (CMV) recall and HIV Elispot assays demonstrating their utility in ameliorating hyperproliferative disorders such as cancer. The peptides are competing with the binding of PD-L1 with anti-PD-1 monoclonal antibody nivolumab (BMS-936558, MDX-1106) that are known to block the interaction with PD-1, enhancing CMV-specific T-cell IFNγ secretion and enhancement of HIV-specific T cell IFNγ secretion.
EXPERT OPINION
Small molecules and peptide antagonists of the PD-1/PD-L1 interaction are highly sought after, since they could have considerable therapeutic advantages over the current clinically used mAbs. The PD-1/PD-L1 target consists of a very hydrophobic, large and flat protein-protein interaction and would be classically described as ‘undruggable’ by small molecules. Nonetheless, several compound classes have been described as PD-1/PD-L1 antagonists. Currently the majority of compound classes seem to competitively antagonize the PD-1/PD-L1 interaction and no allosteric mechanism was established. It should be also mentioned that care has to be taken during the screening process to avoid false positives by carefully triaging compound hits. The sequence and structural differences between mouse and human PD-1/PD-L1 should be taken into account when recombinant proteins or mouse models are used to screen compounds. The hydrophobicity and large molecular weight of direct PD-1/PD-L1 antagonists might also result in development issues such as low target selectivity, poor oral bioavailability, low solubility, fast metabolism and toxicity. For example, many compounds based on scaffold (3) have rather high cLogP. The nature of the PD-1/PD-L1 interaction has also resulted into several distinct peptides, including linear and cyclic peptides derived from interfacial epitopes or discovered by other techniques. Small molecule
1
PD-1/PD-L1 antagonists might be advantageous over current mAbs due to their easy diffusion across physiologic barriers such as the blood–brain barrier and plasma membranes resulting in an overall better tumor tissue uptake. In contrast, large biomolecules such as mAbs largely rely on tumor endothelium permeability to penetrate solid tumors, and endocytic consumption can strongly affect their biodistribution. A recently described PD-1-targeting high affinity small protein, 100x smaller than a mAb and lacking its Fc moiety, penetrates deeper into tumors as seen by fluorescence microscopy and, unlike antibodies, does not cause unwanted depletion of PD-L1-positive T-cells that mediate antitumor immunity.[14] PD-1/PD-L1 antagonists are proven useful in the indication area of cancer and potentially useful in viral or bacterial infections and more recently therapeutic application in Alzheimer’s disease (AD) are also claimed. In the first area mAbs are well established as therapeutic agents, not so in the field of viral and bacterial infections. Small molecules and peptides antagonizing PD-1/PD-L1 might therefore easily penetrate the later fields. Moreover, in AD small molecules might be advantageous due to their better blood-brain-penetration. The area of non-mAb based PD-1/PD-L1 targeting is at the very beginning and currently none of the discussed compound classes is reported to have progressed further to clinical trials yet. Thus it remains to be seen if the protein- (and cell-) based activities translate
in vivo and lead to an advancement in cancer treatment and/or in the anti-infective or AD areas
in clinical trials. Advanced techniques such as radiotracers for immunoPET imaging of PD-1 checkpoint expression on tumor-infiltrating lymphocytes and PD-L1 in general might therefore become very valuable to assess the prognostic value of PD-1/PD-L1 axis targeting molecules in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.[15]
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3. C.L. Day, D.E. Kaufmann, P. Kiepiela et al. Nature 2006, 443, 350 – 354.
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7. A. H. Sharpe, (President and Fellows of Harvard College), WO2011/082400 A3, 2011.
8. a) L. S. Chupak, Myers Squibb Company), WO2015/034820 A1, 2015; b) L.S. Chupak, (Bristol-Myers Squibb Company), WO2015/160641 A2, 2015; c) A.F. Abdel-Magid ACS Med. Chem. Lett. 2015,
6, 489 – 490; d) M. M. Miller (Bristol-Myers Squibb Company), WO2014/151634 A1, 2014.
9. K.M. Zak, P. Grudnik, K. Guzik et al. Oncotarget 2016, 7, 30323 – 30335. 10. R.P. Hanley, S. Horvath, J. An et al. Bioorg. Med. Chem. Lett. 2016, 26, 973 – 977.
11. a) P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), WO2015/033301 A1, 2015; b) P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), WO2015/033299 A1, 2015; c) P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), WO2015/036927 A1, 2015; d) P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), WO2013/144704 A1, 2013; e) P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), WO2015/044900 A1, 2015; f) P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), WO2015/033303 A1, 2015.
12. H.N. Chang, B.Y. Liu, Y.K. Qi et al. Angew. Chem. Int. Ed. 2015, 54, 11760 – 11764.
13. a)P.G.N. Sasikumar, (Aurigene Discovery Technologies Limited), US2011/0318373 A3, 2011; b) P.G. Sasikumar, L.K. Satyam, R.K. Shrimali et al. Cancer Res. 2012, 72, 2850.
14. R.L. Maute, S.R. Gordon, A.T. Mayer et al. PNAS 2015, 112, 6506-6514.
15. a) K. Baruch, A. Deczkowska, N. Rosenzweig et al. Nature Medicine 2016, 22, 135 – 137; b) A. Natarajan, A.T. Mayer, L. Xu et al. Bioconjugate Chem. 2015, 26, 2062 – 2069.
1
CHAPTER
2
IMMUNE CHECKPOINT PD-1/PD-L1:
IS THERE LIFE BEYOND ANTIBODIES?
This chapter is published
Markella Konstantinidou, Tryfon Zarganes –Tzitzikas, Katarzyna Magiera-Mularz, Tad A. Holak and Alexander Dömling
Angewandte Chemie International Edition 2018, 57, 4840 - 4848
ABSTRACT
The PD-1/PD-L1 interaction has emerged as a significant target in cancer immunotherapy. Current medications include monoclonal antibodies, which have shown impressive clinical results in the treatment of several types of tumors. The co-crystal structure of human PD-1 and PD-L1 is expected to be a valuable starting point for the design of novel inhibitors, along with the recent crystal structures with monoclonal antibodies, small molecules and macrocycles.
1. INTRODUCTION: PD-1/PD-L1 PATHWAY
The development of cancer is monitored by the immune system. Most tumors are eliminated by the process of immune surveillance. In this process, T-cells play a major role; their activation stimulates an immune response against cancer cells. The T-cell activation requires two signals: a specific peptide epitope of the antigen must be presented on the major histocompatibility complex (MHC) of an antigen-presenting cell (APC) and it must form a complex with the T-cell receptor. A second signal occurring from the interaction of co-stimulatory molecules of activation is necessary. In the absence of co-stimulatory molecules, T-cells enter the unresponsive state of clonal anergy.[1] Tumors tend to evade immuno-surveillance by down-regulating both MHC and co-stimulatory molecules and also up-regulating co-inhibitory molecules.[2] Mechanistic hallmarks by which tumors avoid immune surveillance are called immune checkpoints or co-inhibitory pathways and recently, they have emerged as a promising approach in cancer immunotherapy.
Programmed death-1 / PD-1 (or CD279) is an immune checkpoint receptor and belongs to the B7-CD28 family of receptors.[3] Upon binding to either of its two ligands, PD-L1 (known also as CD274 or B7-H1) and PD-L2 (known also as CD273, B7-DC or PDCD1LG2), a co-inhibitory signal is delivered.[4] PD-1 is a 55-kDa monomeric type I surface transmembrane glycoprotein. The protein is composed of an extracellular IgV domain, a transmembrane domain, and an intracellular cytoplasmic domain, which contains two tyrosine-based immunoreceptor signaling motifs; the inhibitory motif (ITIM) and the switch motif (ITSM).[5-7] Both motifs, can be phosphorylated upon PD-1 engagement and in turn recruit Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2.[8] The 40-kDa PD-L1 and the 25-kDa PD-L2 are both type I transmembrane proteins, containing extracellular IgV and IgC domains and a transmembrane domain. They lack an identifiable intracellular signaling domain.[9] The two ligands share 37% identity with each other, but differ significantly in their affinity for PD-1 and their tissue specific expression.
2. ANTIBODIES APPROVED AND IN DEVELOPMENT
Currently, there are antibodies targeting both PD-1 and antibodies targeting PD-L1 under clinical investigation either as monotherapy or in combinations with other immune checkpoint inhibitors, monoclonal antibodies (mAbs), chemotherapy, vaccines and radiation. The first monoclonal antibodies targeting PD-1 approved by FDA in 2014 were pembrolizumab and nivolumab; both for the treatment of advanced melanoma. An overview of FDA-approved mAbs in the field is provided in Table 1. The current focus in clinical trials is to improve efficacy and patient response by searching for drug combinations, and thus close to 1,000 clinical trials are ongoing just for checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1.[10]
IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
Table 1. PD-1/PD-L1 directed FDA approved monoclonal antibodies (www.fda.gov, last update 23/9/2017)
mAb Indication
Pembrolizumab (Keytruda®) melanoma, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), Hodgkin lymphoma, urothelial carcinoma, solid tumors, gastric or gastroesophageal junction adenocarcinoma
Pembrolizumab (Keytruda®)
with pemetrexed and carboplatin non squamous non-small cell lung cancer (NSCLC)
Nivolumab (Opdivo®) melanoma, non-small cell lung cancer, renal cell carcinoma (RCC), squamous cell carcinoma of the head and neck (SCCHN), Hodgkin lymploma, urothelial carcinoma, colorectal cancer
Nivolumab (Opdivo®) with
ipilimumab(Yervoy®)* melanoma
Atezolizumab (Tecentriq™) urothelial carcinoma, non-small cell lung cancer (NSCLC), bladder cancer
Avelumab (Bavencio ®) metastatic Merkel cell carcinoma, urothelial carcinoma
Durvalumab (Imfinzi ®) urothelial carcinoma
*Ipilimumab is an anti-CTL4 mAb, approved in 2011 by FDA for melanoma
3. BIOMARKERS FOR PD-1/PD-L1
Following the clinical success of immune checkpoint inhibitors, the establishment of biomarkers in immunotherapy has emerged as an imperative need. Although dramatic survival benefits, mostly for patients with melanoma and less in other types of cancers, have been observed, a rather small percentage of patients currently respond to PD-1/PD-L1 directed treatments. Therefore, biomarkers play a crucial role in predicting a patient’s response, understanding the mechanisms of action, and avoiding immune-related adverse effects (irAEs). Cancer biomarkers have been successfully established in cases of KRAS mutation, HER2 expression and estrogen receptor expression just to name a few. Currently, PD-L1 is under investigation as a predictive biomarker of response for PD-1/PD-PD-L1 immunotherapy.
In a recent study, the tumor expression of PD-L1 was shown to be significant different in different types of cancer. Over-expression of PD-L1 is correlated with better response to PD-1/PD-L1 inhibition in melanoma, non-small cell lung cancer (NSCLC) and, renal cell carcinoma (RCC).[11] A meta-analysis, including data from 20 clinical trials for melanoma, lung cancer and genitourinary cancers, showed that in the overall sample, a significant correlation was observed between PD-L1 expression and overall response rate (ORR), which was significantly higher in PD-PD-L1 positive patients treated with nivolumab or pembrolizumab.[12] Notably, however, clinical response has also been demonstrated in patients with PD-L1 negative tumors.[13]
Moreover, although the up-regulation of PD-L1 in selected solid tumors can be detected by immunohistochemistry (IHC) on both tumor and immune cells, confusion arises regarding the
significance of this detection. PD-L1 is not present simultaneously on tumor and immune cells in all types of cancer.[14] The fact that the expression of PD-L1 is inducible complicates the situation even further. Therefore, it is possible for PD-L1 to be expressed heterogeneously even within a tumor.[11] So far, the methods used to evaluate PD-L1 status, differ significantly. Interestingly however, in October 2015, following the accelerated approval of pembrolizumab for metastatic NSCLC, the FDA approved PD-L1 IHC 22C3 pharmDx (Dako North America) as the only predictive companion diagnostic for selecting NSCLC patients for pembrolizumab. The approval was based on an analysis showing that patients with at least 50% of their tumor cells expressing PD-L1 were most likely to respond to treatment. To observe the PD-L1 expression in a spatially and temporally resolved manner, techniques other than immunohistochemistry, for example, the modern imaging technique positron emission tomography (PET), could be beneficial.
Currently, the data concerning the potential establishment of PD-L1 as a single biomarker remain controversial. Alternative biomarker approaches, such as the quantification of tumor infiltrating lymphocytes (TILs), the identification of tumor neoantigens, and the mutational load of the tumor biomarkers seem to offer a better correlation with the clinical outcomes. [15]
4. IS THERE A NEED FOR SMALL MOLECULES AND OTHER APPROACHES
BEYOND mAbs?
There are several arguments why it is desirable to search for alternatives to mAbs in immunoncology. Generally, the production cost of mAbs remains extremely high. Moreover, they are not orally bioavailable and their high molecular weight, leads to poor diffusion, especially in large tumors. High-affinity antibodies bind tightly to the antigen on first encounter, meaning that they remain on the periphery of the tumor, which is far from ideal for targeting solid tumors. Furthermore, the Fc portion of IgG antibodies can interact with various receptors on the surface of different cell types, which affects their retention in the circulation.[16] mAbs are immunogenic and can lead to irAEs sometimes with deadly outcome, albeit in rare cases. In general, adverse effects with anti-PD-1 / anti-PD-L1 mAbs are less severe than anti-CTLA-4 mAbs. The common side effects of mAbs of PD-1 / PD-L1 as monotherapy are fatigue, dermatological toxicities, diarrhea, colitis, endocrine and hepatic toxicities, pneumonitis, neurological syndromes and ocular toxicity. The reported grade 3-4 adverse effects range from 7-12% in cases of monotherapy.[17] Rare cases of deaths have been reported with pembrolizumab[18] and nivolumab.[19,20] It must be noted, that the combination approaches seem to lead to elevated toxicity. For instance, the combination of ipilimumab and nivolumab showed notably increased toxicity compared to monotherapy with these mAbs.[17,21] The very long half-lives of PD-1 and PD-L1-directed mAbs can make irAEs difficult to treat. Small and medium sized molecules (such as macrocycles) can potentially overcome these issues. The significance of protein-protein interactions (PPIs) is well-established and although targeting PPIs with small molecules can be challenging there are successful examples of small-molecule modulators of PPIs. [22]
IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
5. CRYSTAL STRUCTURES OF PD-1/PD-L1 AND PD-1/PD-L2
In 2008, the first high-resolution crystal structure complexes regarding this PPI were published. The complex of murine PD-1 and human PD-L1 (PDB ID: 3BIK)[23] and that of murine PD-1 and murine PD-L2 (PDB ID: 3BP5)[24], established the structural foundations of the L1 and PD-1/PD-L2 interactions. However, these structures do not allow assessment of the extent of plasticity in these interactions when starting from the apo-protein components of the complexes. The crystal structure of the extracellular domain of human PD-1 alone was determined in 2011 (PDB ID: 3RRQ).
Despite the fact that murine PD-1 binds in vitro both to murine and human PD-L1, and human PD-1 binds to the PD-L1 of each species, it should be taken into account that the protein sequence identity between murine and human 1 is only 64% and that between murine and human PD-L1 is 77%. This indicates likely differences in the details of the binding modes. This hypothesis was recently confirmed,[25] when the crystal structure of the human PD-1/human PD-L1 complex was reported (PDB IDs: 4ZQK, 5C3T), which indeed documents significant differences in the binding between murine and human PD-1 and the ligand (hPD-L1). This information also allowed the identification of features of three hotspot pockets in human PD-1/PD-L1 that are required for inhibition of this interaction.
PD-1 assumes a ß-sandwich immunoglobulin-variable (IgV)-type topology, with Cys54 and Cys123 forming a characteristic disulfide bridge; however PD-1 lacks the second disulfide common to other family members (CD28, CTLA-4, and ICOS).
Similarly to PD-1, the interacting N-terminal domain of PD-L1 is also characterized by the IgV-type topology. PD-1 and PD-L1 form a 1:1 complex within the crystal, in contrast to CTLA-4 complexes with its ligands, where both interacting partners form homodimers. The interaction of PD-1 and PD-L1 resembles that of IgV domains within antibodies and T-cell receptors, being mediated by the strands from the front faces of interacting domains (GFCC0 b sheets).
In principle, the 2.45 Å-resolution of the reported crystal structure[25] provides a perfect starting point for the rational structure-based drug design (SBDD) of molecules against this PPI. However, the interface between the two proteins is rather large (~1.700 Å2), hydrophobic and flat, without deep binding pockets, which makes it a difficult target for small molecules. Moreover, the hydrophobic interface also increases the chances of discovering false positive hits considerably. Nonetheless, small molecule interrupters of the PD-1/PD-L1 protein – protein interaction have been described recently (see below).
6. C0-CRYSTAL STRUCTURES WITH MONOCLONAL ANTIBODIES
Recently, co-crystal structures of monoclonal antibodies targeting PD-1 or PD-L1 were reported, which shed light on their molecular interactions.
For pembrolizumab, an IgG4 antibody, a crystal structure with the full-length antibody was described (PDB ID: 5DK3).[26] The complex of the pembrolizumab antigen-binding fragment (Fab) with hPD-1 (PDB ID: 5JXE)[27] revealed that the stoichiometry is 1:1. Furthermore, the structural superposition of this complex with hPD-1 / hPD-L1 shows overlapping surface regions, thus indicating that the antibody can antagonize hPD-L1 by competing for binding to hPD-1. One more crystal structure of pembrolizumab with hPD-1 (PDB ID: 5B8C)[28] was obtained with higher resolution. It is in good agreement with the previous one and provides additional data regarding the interfacial water molecules at the binding interface, which have an impact on both the affinity and specificity of the interaction.
Moreover, a comparison of the crystal structure of PD-1/nivolumab Fab complex (PDB ID: 5GGR) with that of PD-1/pembrolizumab (PDB ID: 5GGS)[29] indicated that the epitopes of both antibodies directly occupy part of PD-L1 binding site and can thus outcompete PD-L1 for binding to PD-1.
Avelumab, an IgG1 antibody, utilizes both heavy (VH) and light chain (VL) to bind to the IgV domain of the PD-L1 in its complex with hPD-L1 (PDB ID: 5GRJ).[30] The contribution of the light chain is greater than that of the heavy chain. Moreover, the binding epitope region of avelumab on hPD-L1 overlaps with the hPD-1 binding region, thus indicating that the partially overlapping pattern results to the blocking mechanism.
A crystal structure was also recently disclosed for the anti-PD-L1 mAb durvalumab (PDB ID: 5XJ4). In this case, both heavy and light chains contribute to the binding, resulting in steric clash which deters PD-L1 from binding to PD-1. [31]
A crystal structure of PD-L1 with BMS-936559 Fab, a fully human IgG4 antibody currently in clinical trials, showed that its epitope occupies a large part of PD-1 binding site (PDB ID: 5GGT).[29]
Very recently, a co-crystal structure of atezolizumab, the first anti-PD-L1 mAb approved by FDA, was solved (PDB ID: 5XXY). In this case, the binding involves extensive hydrogen bonding, hydrophobic interactions and π-π stacking or cation-π interactions. Moreover, mutagenenesis studies revealed two hotspot residues of PD-L1 (E58, R113). Overall, atezolizumab competes with PD-1 for binding to the same surface site of PD-L1 (Figure 1). [32]
IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
Figure 1. Complex of PD-L1 (purple) with atezolizumab Fab (cyan heavy chain, pink light chain; PDB ID:
5XXY).
In addition, in 2016 the co-crystal structure of an ultra-high-affinity engineered PD-1 mutant (HAC) with hPD-L1 was described (PDB ID: 5IUS). This complex has a high degree of similarity with the hPD-1/hPD-L1. The main differences are observed in the β4-β5 loop. The high-affinity binding is driven by enthalpic gains, owning to the extensive polar contact network between the mutant and PD-L1.[33] In 2017 a second high-affinitiy mutant PD-1 was described, bearing a single aminoacid substitution (A132L). This leads to an increase of van der Waals interactions.[34]
Furthermore, the crystal structure of a PD-L1 nanobody (single domain antibody) was published (PDB ID: 5JDS). The nanobody KN035 competes with PD-1 for binding to PD-L1 mainly through a single surface loop of 21 amino acids. [35] In general, the binding mode seems to differ between PD-1 and PD-L1 mAbs (Figure 2). A more thorough analysis of the structural biology for PD-1/PD-L1 was recently performed.[36]
Figure 2. Different interaction modes of PD(L)-1. A) Complex of hPD-1 (red) with hPD-L1 (blue; PDB ID:
4ZQK). The amino acids in the hotspots are shown as stick models. The two residues in the red circle are Tyr68 of PD-1 (red) and Tyr123 of PD-L1 (blue). B) Complex of PD-1 (red) with nivolumab Fab (yellow light chain, purple heavy chain; PDB ID: 5GGR). C) Complex of PD-L1 (blue) with avelumab Fab (purple
7. CO-CRYSTAL STRUCTURES WITH SMALL MOLECULES
Bristol-Myers Squibb (BMS) researchers have recently disclosed small molecules that bind to PD-L1 (Scheme 1).[37] The scaffold consists of a tri-aromatic structure, including a mono-ortho-substituted biphenyl substructure. Another phenyl ring is connected to the biphenyl and also contains a methylene amine moiety. The claimed biological activity of the reported compounds was established by a homogenous time-resolved fluorescence (HTRF) binding assay in which Europium cryptate-labeled anti-Ig was used. Typical examples are BMS-8, BMS-37, BMS-200 and BMS-202. No further in vitro or in vivo assays have been described to support the biological activity of compounds based on this scaffold.
The true nature of compounds BMS-202 and BMS-8 as PD-1/PD-L1 antagonists was recently rigorously demonstrated by co-crystal structures with PD-L1 (PDB IDs: 5J89, 5J8O respectively). [38] The obtained crystals diffracted at 2.2 Å resolution. Four protein molecules found in the asymmetric unit were organized into two dimers with one inhibitor molecule located at the interface of each dimer. The inhibitor inserts deep into a cylindrical, hydrophobic pocket created at the interface of two monomers within the dimer. The pocket is open to the solvent on one side of the dimer and restricted by the sidechain of ATyr56 on the opposite side. Overall, the inhibitor-protein interaction is best described as bimodal, being spatially divided into hydrophobic and electrostatic parts according to the bimodal inhibitor design.
Scheme 1. PD-1/PD-L1 inhibitors synthesized by BMS. IC50 values were established by the HTRF binding assay.
IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
Furthermore, two novel crystal structures of BMS-37 and BMS-200 were disclosed.[39] The crystals diffracted at 2.35 and 1.7 Å respectively (PDB IDs: 5N2D, 5N2F). NMR experiments indicated that both compounds bind to PD-L1 and induce its oligomerization in solution. Interestingly, the crystal structures revealed notable differences (Figure 3).
Figure 3. Binding mode of BMS-37 (left) and BMS-200 (right) on PD-L1. Yellow sticks represent ATyr56.
The binding mode of compound 37 follows the one already observed for 8 and BMS-202. All of these are examples of the (2-methyl-3-biphenylyl)methanol scaffold. However, BMS-200, an example of [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol scaffold induced a conformational change in ATyr56. The 2,3-dihydro-1,4-benzodioxinyl group forces the ATyr56 to take a different position, thus turning the previously observed deep, hydrophobic cleft to a deep, hydrophobic tunnel and making part of the compound accessible to solvent. Two novel crystal structures were reported for the optimized derivatives BMS-1001 (PDB ID: 5NIU) and BMS-1166 (PDB ID: NIX).[40] These derivatives in particular showed reduced unspecific cytotoxicity against tested cell lines. Furthermore, it was shown that both BMS-1001 and BMS-1166 have the potential to restore the activation of effector Jurkat T-cells, although less effectively than the monoclonal antibodies. More specifically, the immunomodulatory effects of BMS-1001 and BMS-1166 as EC50 values were 253 nM and 273 nM respectively, whereas for mAbs the values were in the range 0.333 - 115 nM. Nevertheless, these data highlight the great potential of small molecules in this field.
Other small molecules have been claimed to antagonize the PD-1/PD-L1 protein – protein interaction, however their mode-of-action has not been rigorously proven so far. An overview of claimed PD-1/PD-L1 inhibitors from patents is provided here.[41]
8. MACROCYCLES
Several patents belonging to Bristol-Myers Squibb Company claim macrocycles that showed high affinity to PD-L1 at low concentrations.[42]
The majority of the described macrocycles contain either 14 or 13 aminoacid residues (Scheme 2). In most of them a sulfur atom is present and this is used as the starting point for the numbering of the aminoacids. In another patent this sulfur is replaced, either by oxygen or carbon.
Scheme 2. Example of macrocycle with 14 amino acids (Bristol-Myers Squibb Company WO2014151634 A1
compound 16). The IC50 value was determined by HTRF assay. The numbering of amino acids starts from the
position adjacent to the sulfur and continues clockwise.
A comparison of the different structures with the 14-motif reveals that in most cases, the first amino acid is an unaltered neutral amide or bis-amide. Possible alterations include the addition of extra aromatic or aliphatic rings on the amide moiety to make this residue more hydrophobic. The second amino acid is frequently changed and varies from a hydrophobic isoleucine to polar amino acids, including aspartic acid, arginine, lysine, serine or threonine. Amino acids 3 and 4 are mostly constant as hydrophobic moieties with butane chains. Moreover, the backbone nitrogen atoms in positions 3 and 4 are in almost all cases methylated. In position 5, a tryptophan is usually present or if altered it is towards a benzothiophene, a dihydropyrrole ring or an indole ring bearing a carboxylic acid substitution. Morpholine or thiomorpholine also appear, but less frequently. A highly variable position among the patents is amino acid 6, which varies from polar (serine, lysine, tyrosine, aspartic acid, glutamic acid, glutamine) to hydrophobic (alanine, glycine). Position 7 is also highly constant as a tryptophan residue, whereas position 8 is almost always a proline or a hydroxylated proline. Isoleucine is usually found in position 9, but it could also vary towards polar residues (aspartic acid, glutamic acid, lysine, serine, asparagine, glutamine). Amino acid 10 also varies and usually it is a polar or basic residue (histidine, lysine, morpholine, hydroxy-pyrrole, serine, asparagine, glutamine). The next two amino acids are highly constant with a proline in position 11 and an asparagine in position 12 in almost all cases. This is followed by a hydrophobic IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
residue in position 13, usually an alanine or a proline is present. The final position 14 is always aromatic and the most common feature is tyrosine. In some cases, there are also halogens or methoxy substituents on the phenyl ring, but it seems to be less common than the tyrosine.
Scheme 3. 2D-Structures of peptide-57 (15-mer, left) and peptide-71 (14-mer, right). IC50 values were determined by the HTRF assay.
Regarding macrocycles with 13 amino acids, a sulfur bond is always included, as well as the two proline residues in positions 5 and 10. Most likely the latter are responsible for making beta turns in the macrocycles. The main difference from the 14-motif is that there are 5 phenyl rings present (positions 3, 4, 6, 7, 12 and 13) and not three (positions 5, 7 and 14). This feature makes these macrocycles more hydrophobic. Moreover, the tyrosine, which is the most common amino acid in the last position of the 14th motif, is always replaced with a phenyl ring with fluoro substituents in the 13th motif.
The BMS macrocyclic peptides that disrupt the PD-1/PD-L1 interaction were originally studied in HTRF assay.[43] Further studies were performed recently for these macrocycles, including NMR, DSF, crystallography and also a cell assay in order to determine their ability to restore T-cell function. [44] The analysis included peptide-57 (15-mer), peptide-71 (14-mer) and peptide-99 (13-mer). Peptide-57, peptide-71 and peptide-99 showed immumodulatory effects with EC50 of 566 nM, 293 nM and 6.30 μM, respectively in the cell assay, whereas for durvalumab and nivolumab the values were 0.199 nM and 1.27 nM, respectively. Crystal structures were obtained for peptide-57 (PDB ID: 5O4Y) and peptide-71 (PDB ID: 5O45) in a peptide/PD-L1 ratio 1:1 (Scheme 3, Figure 4).
residue in position 13, usually an alanine or a proline is present. The final position 14 is always aromatic and the most common feature is tyrosine. In some cases, there are also halogens or methoxy substituents on the phenyl ring, but it seems to be less common than the tyrosine.
Scheme 3. 2D-Structures of peptide-57 (15-mer, left) and peptide-71 (14-mer, right). IC50 values were determined by the HTRF assay.
Regarding macrocycles with 13 amino acids, a sulfur bond is always included, as well as the two proline residues in positions 5 and 10. Most likely the latter are responsible for making beta turns in the macrocycles. The main difference from the 14-motif is that there are 5 phenyl rings present (positions 3, 4, 6, 7, 12 and 13) and not three (positions 5, 7 and 14). This feature makes these macrocycles more hydrophobic. Moreover, the tyrosine, which is the most common amino acid in the last position of the 14th motif, is always replaced with a phenyl ring with fluoro substituents in the 13th motif.
The BMS macrocyclic peptides that disrupt the PD-1/PD-L1 interaction were originally studied in HTRF assay.[43] Further studies were performed recently for these macrocycles, including NMR, DSF, crystallography and also a cell assay in order to determine their ability to restore T-cell function. [44] The analysis included peptide-57 (15-mer), peptide-71 (14-mer) and peptide-99 (13-mer). Peptide-57, peptide-71 and peptide-99 showed immumodulatory effects with EC50 of 566 nM, 293 nM and 6.30 μM, respectively in the cell assay, whereas for durvalumab and nivolumab the values were 0.199 nM and 1.27 nM, respectively. Crystal structures were obtained for peptide-57 (PDB ID: 5O4Y) and peptide-71 (PDB ID: 5O45) in a peptide/PD-L1 ratio 1:1 (Scheme 3, Figure 4).
Figure 4. Binding of peptide-57 (15-mer, left) peptide-71 (14-mer, right) on PD-L1.
The interaction is described as “face-on binding”. In both cases there is a partial overlap with the PD-1 binding epitope and the binding is dominated by hydrophobic interactions and to a smaller extent polar interactions. Closer inspection of the interactions reveals significant differences between the peptides. For peptide-57, two significant pockets are occupied by bulky indole side-chains, whereas for peptide-71 only one hydrophobic pocket is occupied by the side chain of phenylalanine. The polar interactions vary significantly between the two peptides, but in any case, the binding seems to be driven mainly by the hydrophobic interactions. These novel crystal structures allow comparison of the binding mode with that of monoclonal antibodies and provide valuable structural information for drug design.
9. SUMMARY AND OUTLOOK
Immune checkpoint inhibitors represent an exciting new field in cancer treatment. Following the FDA approval of monoclonal antibodies targeting the PD-1/PD-L1 axis, a plethora of crystal structures was published, revealing the binding modes of antibodies, small molecules and very recently, macrocycles. The crystal structures revealed significant differences, especially for small molecules that induced the dimerization of PD-L1. All these data taken together show significant hotspots and provide the missing pieces of structural information necessary for the rational design of small molecule inhibitors, macrocycles or middle-sized cyclic peptides that may have specific advantages compared to the already approved monoclonal antibodies. Importantly, some small molecules and macrocycles show activity comparable to approved mAbs in more complex cell based assays. Thus, future developments in the area could result in drugs different from mAbs for specific cancer applications or different indications.
IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
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IMMUNE CHECKPOINT PD-1/PD-L1: IS THERE LIFE BEYOND ANTIBODIES?
CHAPTER
3
GLUTARIMIDE ALKALOIDS THROUGH
MULTICOMPONENT REACTION CHEMISTRY
This chapter is published
Markella Konstantinidou, Katarzyna Kurpiewska, Justyna Kalinowska-Tłuscik and Alexander Dömling
European Journal of Organic Chemistry 2018, 47, 6714 – 6719
ABSTRACT
A concise four step synthetic route for glutarimide alkaloids of high biological interest is presented. The scaffold is accessed via an Ugi four component reaction, hereby introducing two points of variation. This is followed by a hydrolysis, a cyclization under mild conditions and an amine deprotection. The diastereomers of the cyclized intermediate can be easily separated, thus leading to optically pure alkaloids. By this route, four natural products and ten derivatives were synthesized. The scope and limitations of the synthetic methodology are investigated.
