Development of Inhibitors of the Programmed Cell Death-1

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Development of Inhibitors of the Programmed Cell Death-1/ Programmed Cell Death-ligand 1 Signaling Pathway Tianyu Wang, Xiaoxing Wu, Changying Guo, Kuojun Zhang, Jinyi Xu, Zheng Li, and Sheng Jiang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00990 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 25, 2018

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Journal of Medicinal Chemistry

Development of Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-ligand 1 Signaling Pathway Tianyu Wang, † Xiaoxing Wu, † Changying Guo, † Kuojun Zhang, † Jinyi Xu, † Zheng Li, ‡ Sheng Jiang*,† †

State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry,

School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China ‡

Department of Nanomedicine,

⊥ Center

for Bioenergetics, Houston Methodist

Research Institute, 6670 Bertner, Houston, Texas 77030, United States

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract The clinical success of inhibitors targeting the PD-1/PD-L1 pathway has made this an active field in cancer immunotherapy. Currently, most drugs targeting this pathway are monoclonal antibodies. Small-molecule inhibitors, as the alternative to monoclonal antibodies are expected to overcome the disadvantages of mAbs which include production difficulties and their long half-life. Recently, progress has been reported on anti-PD-1/PD-L1 small-molecule inhibitors. In this paper, we review the development of inhibitors targeting the PD-1/PD-L1 pathway, focusing mainly on peptide-based and nonpeptidic small-molecule inhibitors. The structures and the preclinical and clinical studies of several peptide-based small-molecule candidate compounds in clinical trials are discussed. We also illustrate the design strategies underlying reported nonpeptidic small-molecule inhibitors and provide insight into possible

future

exploration.

Development

of

small-molecule

drugs

for

anti-PD-1/PD-L1 activity with specific cancer applications is a promising and challenging prospect.


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Introduction Cancer immunotherapy, re-manipulating the immune system for therapeutic treatment of cancer, has been pursued for over 100 years. In the early 20th century, Ehrlich first developed a chemical theory to explain the formation of antitoxins, or antibodies, which he described as “magic bullets” to fight the toxins released by the pathogens.1 In 1950s, Burnet and Thomas introduced the concept of immunological surveillance with which the immune system can recognize and destroy somatic cells transformed by spontaneous mutations within the body and which provided a theoretical basis for cancer immunotherapy.

2, 3

The T-cell growth factor interleukin 2

(IL-2) was successfully identified in 1976 and patients with metastatic renal cancer and melanoma treated with high-dose IL-2 achieved effective cancer regression. The landmark discovery and trial of IL-2 demonstrated that immune manipulation can lead to cancer remission and contributed to the application of basic science knowledge of T cell regulation into the development of new cancer immunotherapy.4-6 With recent advances in immuno-oncology, from bench to bedside, many different types of cancer immunotherapy have been explored. These include monoclonal antibodies (mAbs), blockade.

7, 8

cancer vaccines,

10-13

9

adoptive T-cell therapy and immune checkpoint

Following the success of proof-of-concept clinical trials, cancer

immunotherapy was highlighted as the Breakthrough of the Year in 2013 by the journal Science.14 The recent clinical success of immune checkpoint blockade in cancer patients has made it an important trend in the development of immunotherapeutic drugs. Under



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normal physiological conditions, immune checkpoints offer inhibitory signals to cause anergy or exhaustion in T cells for the maintenance of self-tolerance and protection of peripheral tissues from damage during immune responses.15, 16 However, tumor cells can hijack certain immune-checkpoint pathways, and evade immune surveillance by suppressing activation of the immune cells. Immune-checkpoint pathways are generally initiated by ligand-receptor interactions and consequently, the blockade of immune checkpoints by specific inhibitors can reactivate the body’s antitumor immunity.10

Cytotoxic

T

lymphocyte

antigen-4

(CTLA-4)

is

the

first

immune-checkpoint receptor to have been clinically targeted, and this resulted in the approval in 2011 by the US Food and Drug Administration (FDA) of ipilimumab, a mAb targeting CTLA-4, for the treatment of metastatic melanoma.17 Encouraged by the approval of ipilimumab, other immune-checkpoint inhibitors are under active study. Among them, the blocker of programmed cell death-1 (PD-1, also known as CD279) and its ligands18 exhibits a promising antitumor efficacy in clinical trials. Several anti-PD-1 mAb drugs, such as nivolumab and pembrolizumab have been approved for the treatment of melanoma,19-23 and atezolizumab and durvalumab for the treatment of urothelial carcinoma.24, 25 Compared to CTLA-4, inhibition of PD-1 pathway has chronically stimulated state, therefore, anti-PD-1 drugs own increased antitumor activity and relatively favorable toxicity profile.22, 26 Additionally, various promising immune checkpoints such as T cell membrane protein 3 (TIM3, also known as HAVcr2) and lymphocyte activation gene 3 (LAG3, also known as CD223) have been reported as potential targets with which to enhance antitumor immunity.27, 28


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Currently the development of mAbs for the blockade of PD-1/PD-L1 have achieved great progress and relevant researches have been already reviewed extensively and intensively.29-31 Compared to mAbs, small-molecule drugs exhibit significant advantages such as the possibility of oral administration, stability, membrane permeability and non-immunogenicity. For this reason, the field of small-molecule inhibitors of immune checkpoint has attracted many research and discussions, especially PD-1/PD-L1.32-35 We have been conducting research into the development of inhibitors targeting the PD-1/PD-L1 pathway, especially the peptide-based and nonpeptidic small-molecule inhibitors, as alternatives to mAbs. We are focused on small-molecule inhibitors and their design strategies to provide an insight into further exploration of anti-PD-1/PD-L1 small-molecule drugs for cancer immunotherapy. This paper is a review of the relevant research in this area. An Overview of the Programmed Cell Death 1/Programmed Cell Death-Ligand 1 Signaling Pathway As the member of an immunoglobulin (Ig) superfamily, the protein PD-1 is a homologue of CD28 consisting of 288 amino acids containing a single Ig-like variable domain and a cytoplasmic region. It has 21-33% sequence identity with CTLA-4 and CD28.36, 37 Structural and biophysical data show that, in contrast to CTLA-4 and other Ig family members that are disulfide-linked homodimers,38 PD-1 is monomeric both in solution and on the cell surface. PD-1 possesses an ectodomain with multiple loop structures and strands between the loops as shown in Figure 1. As an immune-checkpoint receptor, PD-1 is expressed on a subset of thymocytes



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and upregulated on the surface of activated T cells, B cells, natural killer (NK) T cells, monocytes, and dendritic cells (DCs).39, 40 The functional role of PD-1 was initially determined to be induction of apoptosis. Because PD-1-deficient mice develop autoimmune diseases, it was thought that PD-1 might be crucial for the maintenance of immunologic homeostasis which maintains peripheral tolerance and prevents autoimmunity.41, 42 PD-1 expression is also considered as a marker for highly reactive tumor-specific T cells, and anti-PD-1 therapy has shown that the therapeutic responses were associated with PD-1 presenting on the surface of cytotoxic T cells in the tumor microenvironment.43

Figure 1.

Crystal structure of the extracellular domain (PDB ID: 2M2D) and structure-based sequence of human PD-1 (hPD-1).

Association of PD-1 with its ligands PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273) triggers an inhibitory pathway. As the member of the B7 family, PD-L1 is expressed in nonlymphoid organs and upregulated in

response

to

activated

T

cells,

antigen-presenting

cells

(APCs)

and

nonhematopoietic cells.44-46 The expression and function of PD-L2 are similar to those of PD-L1, and PD-L2 shares 34% identity with PD-L1. Both ligands have short cytoplasmic tails with an uncharacterized motif for signal transduction, suggesting


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that both ligands do not transduce any signal upon interaction with PD-1.47 Engagement of PD-1 by PD-L1 impedes TCR signaling and CD28 co-stimulation and inhibits the activation of human T cells by anti-CD3.46 T cell receptor (TCR)mediated proliferation and cytokine production by CD4 T cells are inhibited by engagement of PD-1 by PD-L2.48, 49 The interaction of PD-L1/PD-1 is similar with PD-L2/PD-1 with the affinities of KD values as 10.4 and 11.3 nM respectively, but striking differences were observed at the level of the association and dissociation characteristics.50, 51 The aberrant expression of PD-L1 and PD-L2 in tumor cells impairs body’s antitumor immunity, resulting in the immune evasion of the tumor cells.52 Many types of tumor cells and haematopoietic cells within the tumor microenvironment can upregulate the expression of PD-L1 by activating oncogenic pathways such as the phosphoinositide

3-kinase/protein

kinase

B

(PI3K/AKT)

pathway53,

the

nucleophosmin-anaplastic lymphoma kinase (ALK) pathway54 (innate immune resistance) or in response to interferons (IFN), mostly IFN-γ, in the tumor microenvironment (adaptive immune resistance).10,

55

In addition to its binding to

PD-1, PD-L1 also inhibits T cell activation and cytokine production through its interaction with additional receptors such as B7-1.56 These findings suggest that the PD-1/PD-L1 pathway could be an ideal target with which to restore antitumor immunity and inhibitory approaches for cancer therapy.57-59 On the other hand, the role of PD-L2 in tumor immunity remains unclear. Reportedly, PD-L2, but not PD-L1 elicits direct activating effects on dendritic cells and this is thought to enhance



immune responses. Because tumor cells barely upregulate the expression of PD-L2, few studies have focused on inhibitors targeting PD-L2 to block its interaction with PD-1. However, a study from the Merck Inc. showed that PD-L2 expression is independently associated with improved clinical response to the anti-PD-1/PD-L1 antibody pembrolizumab through high objective response rates and longer progression-free survival in patients with head and neck squamous cell carcinoma.60 The relevance to anti-PD-1 therapy suggests PD-L2 could provide clinical benefits in these patients.

Figure 2. Mechanism of the inhibition of the interaction between PD-1 and PD-L for cancer therapy. The structure of the full human PD-1/PD-L1 complex has been reported,61-63 and shows that the receptor-ligand interaction is mediated by residues of C’CFG strands within PD-1 and PD-L1. As shown in Figure 3A, the interactions involve nonpolar residues (Val64, Ile126, Leu128, Ala132, Ile134) in the front sheet of PD-1 and those of the front sheet of PD-L1 (LIle54, LTyr56, LMet115, LAla121, LTyr123). This


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hydrophobic region is exposed to solvent on the antigen-binding site. Moreover, three major hot spots on the PD-L1 surface have been identified by Holak et al. (Figure 3B).63 The first is a pocket composed of LGlu58, LArg113, LTyr123, which can accommodate an aromatic ring. The second hot spot is located nearby LMet115 and LAla121

which can be filled by a branched aliphatic moiety. The third is an extended

groove between Asp122 and Asp26 in which multiple hydrogen bonds could be anchored.63



Figure 3. (A) The crystal structure of the complex of hPD-1 (green ribbon)/hPD-L1 (blue ribbon) complex (PDB ID: 4ZQK). (B) Three main hot spots (red circle) on the PD-L1 surface (gray). The first spot comprises Glu58, Arg113 and Tyr123. The second is formed by Met115 and Ala121. The third spot is composed of a shallow groove between Asp122 and Asp26. Residues interacted with PD-1 of PD-L1 are colored blue (PDB ID: 5C3T).

Inhibitors targeting the PD-1/PD-L1 Pathway mAbs Targeting the PD-1/PD-L1 pathway. Since the clinical approval of rituximab and trastuzumab, antibody-based immunotherapy has become a significant field of research.64, 65 Recently, promising clinical results of mAbs targeting the immune-checkpoint pathway including the PD-1/PD-L1 pathway have been obtained.6, 30, 35, 66-68 Table 1 lists the mAbs launched and in promising clinical trials targeting the PD-1/PD-L1 pathway. As described previously, both anti-PD-1 and anti-PD-L1 mAbs have shown promising antitumor


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activity in the treatment of melanoma, non-small cell lung cancer (NSCLC) and urothelial carcinoma (UC). Table 1. mAbs Currently Launched and in Promising Clinical Trials targeting the PD-1/PD-L1 Pathway Generic name or Code name

Target

Organization

Clinical Indicationsa

Highest Phase

Nivolumab

PD-1

Bristol-Myers Squibb/ Ono

Unresectable melanoma (2014) squamous and non-squamous NSCLC, advanced RCC (2015) classical Hodgkin’s lymphoma, renal cell carcinoma recurrent or metastatic head and neck cancer (2016) unresectable or metastatic UC, HCC, metastatic CRC (2017)

Launched-2014

Pembrolizuma b

PD-1

Merck

Unresectable or metastatic melanoma (2014) NSCLC (2015) Squamous head and neck cancer, metastatic NSCLC (2016) CRC, classical Hodgkin’s lymphoma, gastric or GEJ adenocarcinoma (2017)

Launched-2014

Atezolizumab

PD-L1

Roche

Locally advanced or metastatic UC and NSCLC

Launched-2016

Durvalumab

PD-L1

AstraZeneca

Locally advanced or metastatic UC (2017) and NSCLC (2018)

Launched-2017

Avelumab

PD-L1

Merck/ Pfizer

Metastatic Merkel cell carcinoma and locally advanced or metastatic UC

Launched-2017

Cemiplimab

PD-1

Sanofi/ Regeneron

Metastatic CSCC, NSCLC and second-line cervical cancer

Pre-Registered

Camrelizumab

PD-1

Jiangsu Hengrui

Non-squamous NSCLC and squamous esophageal carcinoma

Phase Ⅲ

IBI-308

PD-1

Innovent Biologics

NSCLC

Phase Ⅲ

Tislelizumab

PD-1

BeiGene

Unresectable HCC

Phase Ⅲ



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Spartalizumab

PD-1

Novartis

Advanced melanoma

Phase Ⅲ

JNJ-63723283

PD-1

Janssen Research & Development

UC

Phase Ⅲ

MGA-012

PD-1

Incyte/ MacroGenics

Metastatic CRC, metastatic Merkel cell carcinoma

Phase Ⅲ

AGEN-2034

PD-1

Agenus

Cervical Cancer

Phase Ⅲ

M-7824

PD-L1/ TGF-β

Merck

Advanced solid tumors

Phase Ⅲ

CX-072

PD-L1

CytomX Therapeutics

Solid tumors, lymphomas

Phase Ⅲ

Sym-021

PD-1

Symphogen


Phase Ⅲ

LZM-009

PD-1

Livzon

Solid tumors

Phase Ⅲ

BI-754091

PD-1

Boehringer Ingelheim

Solid tumors

Phase Ⅲ

XmAb-20717

PD-1

Xencor

Solid tumors

Phase Ⅲ

PF-06801591

PD-1

Pfizer


Phase Ⅲ

MGD-013

PD-1

MacroGenics

Solid tumors, neoplasms

Phase Ⅲ

TSR-042

PD-1

Tesaro/ AnaptysBio

Solid tumors

Phase Ⅲ

AMP-224

PD-1

GlaxoSmithKlin e/ MedImmune

CRC

Phase Ⅲ

MSB-2311

PD-L1

MabSpace Biosciences

Solid tumors

Phase Ⅲ

FS-118

PD-L1/ LAG-3

F-star/ Merck

Advanced cancer

Phase Ⅲ

FAZ-053

PD-L1

Novartis

Advanced cancer

Phase Ⅲ

KN-035

PD-L1

Suzhou Alphamab

Solid tumors

Phase Ⅲ

LY-3300054

PD-L1

Lilly

Solid tumors

Phase Ⅲ

a

hematologic

RCC, renal cell carcinoma; HCC, hepatocellular carcinoma; CRC, colorectal cancer; GEJ,

gastroesophageal junction; CSCC, cutaneous squamous cell carcinoma.

So far, the available inhibitors of PD-1/PD-L1 pathway in clinical development are all mAbs. However, due to disadvantages such as their long half-life and unchecked immune response, mAbs targeting PD-1/PD-L1 pathway can lead to immune-related adverse events. These events can cause damage to normal organ systems and tissues,


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and typically involve the skin, gastrointestinal, hepatic, pulmonary, and endocrine systems.69 Moreover, lack of oral bioavailability, emergence of tumor resistance, and difficult and expensive production of the mAbs also limit their current clinical application.66 The search for non-mAbs and low-molecular-weight inhibitors of PD-1/PD-L1 has thus become an active field. However, developing small-molecule protein-protein interaction (PPI) inhibitors is difficult due to the lack of small-molecule starting point and well-defined binding pockets.70 Nevertheless, various small-molecule PPI inhibitors which have reached clinical development show that certain classes of PPI are amenable to small-molecule inhibition.71 And small-molecule inhibitors of PD-1/PD-L1 pathway have achieved substantial progress during the last few years. Peptide-based inhibitors targeting the PD-1/PD-L1 pathway. Liu et al. from Tsinghua University developed the first proteolysis-resistant D-peptide anti-PD-L1 antagonists.72 In their initial study, this group obtained a PD-L1 affinity peptide based on the technology of phase display, which used the ectodomain of PD-L1 expressed by E. coli for screening by a 12-mer peptide library displayed on M13 phage. The obtained peptide, FPNWSLRPMNQM, administered by hypodermic injection, has good affinity for PD-L1 and antitumor activity.73 However, due to its rapid degradation by proteolytic enzymes in the blood plasma, it failed to show any activity when administered by intraperitoneal injection. In order to overcome the stability problem and using the D-version of the IgV domain of PD-L1 as bait, the optimized compound DPPA-1 which binds to PD-L1 with an affinity of 0.51 µM was



investigated (Table 2). Flow cytometry experiments demonstrated that DPPA-1 can block PD-1/PD-L1 interaction at the cellular level. Tumor growth and survival experiments showed that DPPA-1 significantly inhibits the growth of implanted CT26 in mice and no loss of body weight was detected. Table 2. Amino Acid Sequences of D-peptide from PD-L1-binding Phase and the Corresponding KD Values Name Sequence KD (µM)a D PPA-1 NYSKPTDRQYHF 0.51 D PPA-2 KHAHHTHNLRLP 1.13 D PPA-3 AAKMDGHLHGGQ NBb D PPA-4 MRNRERYPKPYY 22.0 D PPA-5 TLYQRPSTNLER NBb a

KD values measured by surface plasmon resonance (SPR) spectroscopy. bNB=No binding.

Recently, Zhu et al. reported a peptide targeting PD-L1. TPP-1 whose amino acid sequence is SGQYASYHCWCWRDPGRSGGSK, which was screened and identified through a bacterial surface display method, binds to PD-L1 with an affinity of KD=94 nM.74 Experiments with a xenograft mouse model using H460 cells and increased expression of IFN-γ and granzyme B indicated that TPP-1 could attenuate the inhibitory effect of PD-L1 on the T cells and reactivate T cells in vivo, inhibiting tumor growth. Li and co-workers at East China University of Science and Technology developed a computational method for de novo design of anti-PD-1 peptides with residues Y56, R113, A121, D122 and Y123 of PD-L1 as key anchors.75 The sequence and binding affinities to the PD-1 of four selected peptides are shown in Table 3. The most potent of these, Ar5Y_4 (GNW WDYNSQRAQLYNQ) has a KD value of 1.38±0.39 µM. The SPR competitive binding assay and the production by T cells of IL-2 analyzed by an


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enzyme-linked immune sorbent assay (ELISA) indicated that Ar5Y_4 can inhibit the interaction of PD-1/PD-L1 and restore the suppressed function of Jurkat T cells. Table 3. Amino Acid Sequence, Purity and Corresponding KD Values of the Computational de novo Peptides. Name

Sequencea

Purity (%)b

KD (μM)c

Ar5Y_1

FNW WDYSWKSERLKEAYDL

96.66

3.39±0.85

Ar5Y_2

FNW WDYSLEELREKAKYK

95.80

3.14±0.92

Ar5Y_3

TEK KDYRHGNIRMKLAYDL

96.71

3.13±0.45

Ar5Y_4

GNW WDYNSQRAQLYNQ

98.24

1.38±0.39

a

Anchor residues are underlined, residues corresponding to anchor residue A121 are in bold.

b

Determined by HPLC. cKD value is shown as the mean ± SD from three independent experiments.

Scientists from Aurigene, an Indian biotechnology company, developed several peptides or peptidomimetics as PD-1/PD-L1 pathway inhibitors. In 2014, Aurigene, together with Pierre Fabre Pharmaceuticals disclosed AUNP-12, a peptide based on the PD-1 ectodomain, which is highly effective in antagonizing PD-1 signaling with desirable in vivo exposure following subcutaneous administration. By analysis of the related patent,76 the most possible structure of AUNP-12 appears to be compound 1 (Figure 4), which shows the highest inhibition of tumor growth and metastasis in preclinical models of cancer, and has a safer toxicological profile.

Figure 4. Possible structure of AUNP-12.



Small peptidomimetics have also been developed by Aurigene (Figure 5). Compounds 2 and 3 are tripeptide or tetrapeptide peptidomimetics containing a hydrazine or urea linker77,

78

Compound 2 shows high inhibition of PD-L1 and PD-L2 (rescue of

splenocyte effector function, with EC50=30 and 40 nM, respectively) and significantly suppresses tumor growth. Compound 3, rescued anti-CD3/CD28-stimulated C57BL/6 mice with splenocyte proliferation in the presence of recombinant mouse ligand PD-L1 by 87% at 100 nM in carboxyfluorescein succinimidyl amino ester (CFSE) proliferation assays. CFSE is a dye which diffuses passively into cells and binds to intracellular proteins. Compound 4, comprised of a modified BC loop (amino acids 24-30 of the extracellular domain of hPD-1, sequence: SNTSESF), reduces lung metastasis by 64% in mice bearing melanoma B16F10 cancer cells.79

Figure 5. Small peptidomimetics developed by Aurigene.

Cyclic peptidomimetic compounds were also disclosed by the same company (Figure 6).80, 81 Compounds such as formula 5 have a -CO-(CH2)n-NH- or an amide bond as a linker and a further modified BC loop. Compound 6 induces mouse splenocyte proliferation in presence of human breast MDA-MB-231 cancer cell lines overexpressing PD-L1 in CFSE assays and lung metastasis by 54% in mice bearing


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melanoma

B16F10

cancer

cells.

Compounds

7

and

8

rescue

anti-CD3/CD28-stimulated mice splenocyte proliferation in the presence of recombinant mouse ligand PD-L1 by 81% and 82% respectively at a concentration of 100 nM in CFSE proliferation assays.

Figure 6. Cyclic peptidomimetic compounds developed by Aurigene. Formula 5: L1 is selected from -CO-(CH2)n-NH-, -NH-(CH2)n-CO- or an amide bond; Am1, Am2 are amino acid residues; X is selected from Lys, Glu or Ser; Y is selected from Glu, Gln or Lys; Z is amino acid residues-L2; L2: -NH-(CH2)n-CO- or absent.

Aurigene has also developed several macrocyclic peptidomimetic compounds.82, 83

Five such compounds were reported to rescue the anti-CD3/CD28-stimulated

mouse splenocyte proliferation by up to 90% at a 100 nM concentration in the presence of recombinant mice ligand PD-L1 in CFSE assays (Figure 7). The peptidomimetics showing the highest inhibition were compounds 9 and 10 which rescued 95% and 94% splenocyte proliferation at 100 nM in presence of PD-L1.



Figure 7. Macrocyclic peptidomimetic compounds developed by Aurigene.

Since nivolumab was approved by the FDA for the treatment of unresectable melanoma, Bristol-Myers-Squibb has developed non-mAbs inhibitors targeting the PD-1/PD-L1 pathway. A series of macrocyclic inhibitors containing 13-15 residues involved in the PD-1/PD-L1 interaction have been reported.84-90 BMS-986189, a macrocyclic peptide with a 45-residue N-methylated backbone with 14 amide bonds and 1 thioether bond, is a PD-L1 antagonist which entered into a Phase I clinical trial in 2016 for the treatment of serve sepsis. Although the full structure of BMS-986189 has not been disclosed, the patents indicate that formula 11 is a possible skeleton (Figure 8). Compound 12 shows the highest binding affinity to PD-L1 using a PD-1/PD-L1 homogenous time-resolved fluorescence (HTRF) binding assay.


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Figure 8. Macrocyclic peptidomimetic compounds developed by BMS company.

Holak and co-workers from Jagiellonian University have characterized three macrocyclic-peptide PD-1/PD-L1 antagonists that were later developed by Bristol-Myers-Squibb (Figure 9).91 Compounds 13 and 14 dose-dependently restore the activity of the TCR-responsive promoter under the control of overexpressed PD-1 with EC50 values of 556 nM and 293 nM, respectively. In comparison, the immunomodulatory effects of the anti-PD-1 mAbs durvalumab and nivolumab were determined to have EC50 values of 0.20 nM and 1.3 nM, respectively. Compound 15, with EC50 = 6.30 µM was the least active. The co-crystal structures of 13 or 14 with PD-L1 demonstrate that no significant structural changes are induced within the PD-L1 receptor upon ligand binding. Analysis of the interactions of the residues of the therapeutic mAbs avelumab and peptides 13 and 14 shows that these peptides mimic about 37% of the PD-L1/mAbs interactions.



Figure 9. Three macrocyclic-peptides PD-1/PD-L1 antagonists reported by Holak.

Nonpeptidic small-molecule inhibitors targeting the PD-1/PD-L1 pathway. Sharpe and co-workers at Harvard University reported sulfonamides as inhibitors of PD-1/PD-L1.92 The activities of sulfamonomethoxine (16) and sulfamethizole (17) are >400 nM in rescuing PD-1 mediated inhibition of IFN-γ production (Figure 10).

Figure 10. Sulfonamides small-molecules inhibitors developed by Sharpe.

Using computer-aided drug design (CADD),93 Sun’s group from China Pharmaceutical University has identified isonicotinic acid derivatives as inhibitors of the interaction of PD-1 with PD-L1. Compound 18 provides 36% inhibition of the


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PD-1/PD-L1 binding at 10 µM in an HTRF binding assay.

Figure 11. Isonicotinic acid derivatives reported by Sun et al. and resorcinol derivatives developed by Li et al.

Li et al. from Beijing Institute of Pharmacology and Toxicology have developed resorcinol derivatives as immune-checkpoint inhibitors of the PD-1/PD-L1 interaction.94 Compound 19 inhibits the PD-1/PD-L1 interaction by 43% at 500 µM in the HTRF binding assay and NMR spectroscopy suggests that the N atom (red) was supposed to interact with PD-1. Other than the aforementioned peptide-based anti-PD-1/PD-L1 inhibitors, the most promising candidates developed by Aurigene are CA-170 and CA-327. CA-170, co-developed by Curis, is a first-in-class, orally bioavailable, dual small-molecule inhibitor of PD-1 and a v-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), an immune checkpoint sharing structural similarity with PD-L1 and co-expressed with PD-L1 in specific tumor cells95. Preclinical ex vivo data show that CA-170 can induce effective proliferation and IFN-γ production by means of T cells that are specifically suppressed by PD-L1 or VISTA. In various in vivo tumor models, CA-170 shows antitumor effects similar to those of antibodies of PD-1 or VISTA. In 2016, Curis initiated the Phase Ⅲ trials of CA-170 for the treatment of advanced solid tumors and lymphomas and established that with a once daily oral



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dosing regimen, CA-170 has a safe toxicological profile. In the same year, Aurigene, in collaboration with Curis, reported CA-327 as an oral small-molecule antagonist targeting PD-L1 and the T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3: an immune checkpoint co-expressed with PD-1 in certain T cells). CA-327 inhibits PD-L1 and TIM-3 with EC50 values of 34 nM and 35 nM respectively. Moreover, CA-327 shows no inhibition of other immune checkpoints such as CTLA-4 or VISTA, good PD modulation comparable to an anti-TIM3 antibody and a combination of anti-PD-1 antibody and anti-TIM3 antibody properties. Currently CA-327 is undergoing IND-enabling studies. Aurigene and Curis have not revealed the structures of CA-170 and CA-327. On the basis of small peptidomimetics comprising of a hydrazine and urea linker, Aurigene further developed a series of tripeptides or their derivatives comprised of a core oxadiazole or thiadiazole with an amino acid side chain along with a hydrazine or

urea

linker

at

different

sites.

Specifically

1,2,4-oxadiazole/thiadiazole

derivatives96-99 and 1,3,4-oxadiazole/thiadiazole derivatives have been reported100-102 as immunomodulators targeting PD-1 (Figure 12). Compounds 20 and 21 rescue splenocyte proliferation 92% and 99% at 100 nM in the presence of PD-L1. In 2018, patents103,

104

from Aurigene shows that these oxadiazole/thiadiazole derivatives

compounds can also modulate the VISTA signaling pathway. Compound 22 rescues 99% IFN-γ release in a human peripheral blood mononuclear cell assay in the presence of VISTA. Compound 23 rescues splenocyte proliferation 97% at 100 nM in the presence of PD-L1 and rescues more than 70% IFN-γ release at 100 nM in the


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presence of VISTA. Besides the urea linker, compounds with amide or thioamide bonds as the linker were also disclosed by Aurigene.105, 106 Compounds 24 and 25 rescue 97% and 91% splenocyte proliferation at 100 nM in the presence of PD-L1.

Figure 12. 1,2,4-oxadiazole/thiadiazole and 1,3,4-oxadiazole/thiadiazole derivatives developed by Aurigene.

In 2018, Guangzhou Maxinovel Pharmaceuticals Company disclosed MAX-10129, as a novel, orally administrated, small-molecule inhibitor of PD-1/PD-L1 interaction. According to the patent,107 a series of aromatic acetylene or ethylene compounds as PD-1/PD-L1

inhibitors

have

been

developed

by

Guangzhou

Maxinovel

Pharmaceuticals. Compounds 26 and 27 (Figure 13), which have IC50 values of 0.018 and 0.036 µM in the inhibition of interaction of PD-1 with PD-L1, are supposed to be the possible structure of MAX-10129. MAX-10129 displayed significant antitumor efficacy in MC-38 murine colorectal carcinoma model with dose dependency. Moreover, MAX-10129 demonstrated synergistic antitumor efficacy in different combinations with an anti-CTLA-4 antibody, Epacadostat (IDO inhibitor), Celebrex (COX-2 inhibitor) and Cisplatin, with the p value of the combo treatment group relative to the vehicle group is less than 0.05.



Figure 13. Aromatic ethylene compounds developed by the Guangzhou Maxinovel Pharmaceuticals company.

Fen and co-workers from Institute of Materia Medica, Chinese Academy of Medical Sciences reported a series of benzyl phenyl ether derivatives (28) as PD-1/PD-L1 inhibitors,108-110 which can inhibit the interaction of PD-1 with PD-L1 with IC50 values in the nanomolar range. Inhibition in the picomolar range was achieved with some substituted pyridine methylene derivatives. For example, compound 29 (Figure 14) inhibits the PD-1/PD-L1 interaction with an IC50 value of 0.08 pM.

Figure 14. Benzyl phenyl ether derivatives as PD-1/PD-L1 inhibitors.

In recent years, the Incyte Corporation has disclosed many new small-molecule compounds that block the PD-1/PD-L1 interaction.111-117 Compounds such as 30 can inhibit the PD-1/PD-L1 interaction with IC50 values ≤100 nM in HTRF binding assays. Compounds 31-37 (Figure 15) are described in different patents and their IC50 values are 100 nM or less.


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Figure 15. Small-molecule compounds developed by Incyte Corporation.

Besides mAbs and peptides, the Bristol-Myers-Squibb company developed small-molecules as inhibitors of the PD-1/PD-L1 pathway. Two patents disclosed a series of triaromatic compounds (Figure 16A, 38) that inhibit the PD-1/PD-L1 interaction.118, 119 Compounds 39 and 40 show IC50 values of 0.146 and 0.018 µM respectively in HTRF binding assays. Studies by Holak et al. showed that based on the crystal structures and other biochemical data,120 compounds 39 and 40 act by directly binding to PD-L1 rather than to PD-1 and PD-L2. The relative affinity of compounds 39 and 40 towards PD-L1 and PD-L2 was assessed using differential scanning fluorimetry. Compounds 39 and 40 stabilize the thermally induced unfolding of PD-L1 by 9.4 ºC and 13 ºC, respectively. However, neither 39 nor 40 significantly affects the melting temperature of PD-L2. The crystal structure of a complex of PD-L1 with 40, shown in Figure 16B suggested that 40 inserts into the cylindrical, hydrophobic pocket at the PD-L1 dimer interface and induces dimerization of PD-L1.



Page 26 of 55

The distal phenyl ring within the biphenyl moiety creates a T-stacking interaction with ATyr56.

The phenylmethyl ring provides hydrophobic interactions with AAla121 and

BMet115

and the alkoxy- pyridine moiety creates π-π stacking with BTyr56 and a

number of polar interactions of chain A. The acetamide moiety of the N-(2-aminoethyl)acetamide group contributes a hydrogen bond to ALys124. NMR data confirmed that 39 and 40 both can interrupt the PD-1/PD-L1 interaction at stoichiometric concentrations.

(A)

HO Br R1

N R2

Z

Ar1

Ar

X Y

2

Ar3

O

N

O 38

Ar1: aryl, heteroaryl, aralkyl or heteroralkyl group; Ar2, Ar3: phenylene group or heteroarylene group having 5 or 6 ring atoms and 1,2,3 ro 4 heteroatoms selected from O, S and N; X, Y: O, S, CHOMe, NOMe, CFH, CF 2, NH or CH2.

39 N H O

N 40


O

H N O

Page 27 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 16. (A) Triaromatic compounds developed by the BMS Company. (B) The crystal structure of PD-L1 (chain A, green; chain B, blue)/40 (yellow) complex (PDB ID: 5J89). Hydrogen bond is shown in yellow dashed line.

Holak et al. recently presented NMR and X-ray characterizations of 41 and 42 (Figure 17A), representatives of the two classes of anti-PD/PD-L1 inhibitors.121 Ligand-induced chemical shift perturbations in the 2D HMQC spectra confirmed the interaction of 41 and 42 with PD-1. The X-ray crystal structures of compounds 41 and 42 complexed with PD-L1 show that 41 and 42 both bind to the dimeric PD-L1. The binding mode of the 41/PD-L1 complex is similar to that of the 40/PD-L1 complex. Compound 41 also locates itself in the cylindrical, hydrophobic pocket created at the interface of two monomers within the dimer. However, the ligand-binding mode of 42 (Figure 17B), which contains a 2,3-dihydrobenzo-[b][1,4]dioxine group, is different. The difluorophenyl ring of 42 provides interactions with AAla121 through a long hydrogen bond and with interacts with

AMet115

ATyr56

while the 2,3-dihydrobenzo-[b][1,4]dioxine ring

and BAla121. The methyl group on the phenyl ring has

hydrophobic interactions with Met115 of chain A and B and with

AAla121.

Additionally, the (S)-4-amino-3-hydroxybutyric acid moiety provides two hydrogen bonds, with AThr20 and BGln66. This structural difference also suggests that the ligand binding site of PD-L1 is more flexible.



Figure 17. (A) Anti-PD/PD-L1 inhibitors characterized by Holak et al. (B) The crystal structure of PD-L1 (chain A, blue; chain B, green)/42 (yellow) complex (PDB ID: 5N2F). Hydrogen bonds is shown in yellow dashed lines.

Scaffold 43 (Figure 18) is another small-molecule inhibitor of PD-1/PD-L1 targeting PD-1/PD-L1 disclosed by BMS company. Compounds 44-46 taken from three patents,122-124 are typical examples of compounds which can inhibit the PD-1/PD-L1 interaction with IC50 values

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