Inhibitor of Apoptosis Protein (IAP) Antagonists in Anticancer Agent

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Inhibitor of Apoptosis Protein (IAP) Antagonists in Anticancer Agent Discovery: Current Status and Perspectives Hui Cong, Lijuan Xu, Yougen Wu, Zhuo Qu, Tengfei Bian, Wannian Zhang, Chengguo Xing, and Chunlin Zhuang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01668 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Journal of Medicinal Chemistry

Inhibitor of Apoptosis Protein (IAP) Antagonists in Anticancer Agent Discovery: Current Status and Perspectives Hui Cong†‡¶, Lijuan Xu†‡¶, Yougen Wu#§, Zhuo Qu,† Tengfei Bian§, Wannian Zhang†‡, Chengguo Xing§*, Chunlin Zhuang†‡* †School

of Pharmacy, Ningxia Medical University, 1160 Shengli Street, Yinchuan 750004, China

‡School

of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China

#College

of Tropical Agriculture and Forestry, Hainan University, 58 Renmin Avenue, Haikou 570228, China

§Department

of Medicinal Chemistry, University of Florida, 1345 Center Drive, Gainesville, FL 32610, USA

ACS Paragon Plus Environment

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ABSTRACT Apoptosis, an important form of programmed cell death (PCD), is a tightly regulated cellular process to eliminate unwanted or damaged cells. Resistance of apoptosis is a hallmark of cancer cells. Inhibitor of apoptosis proteins (IAPs) is a class of key apoptosis regulators that promote cancer cell resistant to apoptosis, particularly in cancer treatment. Disrupting the binding of IAPs with their functional partners therefore is a promising strategy to restore the apoptotic response to pro-apoptotic stimuli, particularly those introduced by standard cancer therapies. The most successful example is the use of small molecules to mimic the IAPbinding motif of an endogenous IAP antagonist, second mitochondria-derived activator of caspase (SMAC). Here we will review the functions of IAPs; the structural interactions of IAPs with SMAC; four generations of SMAC-mimetic IAP antagonists; and representative antagonists in clinical evaluations, focusing on research articles over the last 15 years. Outlooks and perspectives on the associated challenges are provided as well.


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Table of Contents Graphic Representative IAP Antagonists in Clinical Trials O N

N O NH

O

N S

N

O

O

NH

NH

O

O

NH

NH F Second generation

First generation

HN O N H

O

HN

O

O N

O N

S O

O S N O O

NH

N

O

N

N

N O

NH

HN

O

NH F Third generation

Fourth generation


N

OH


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1. IAPs are key regulators of apoptosis. Apoptosis, a form of programmed cell death (PCD), is an essential cellular process for normal development and homeostasis, which maintains a balance between cell death and growth.1 Dysregulated apoptosis is involved in the pathological etiology of a variety of diseases, including neurodegenerative diseases, viral infections, and cancers.2, 3 Resistance to apoptosis in cancer cells is a major challenge that limits the success of cancer treatment.4 Therefore, restoring the pro-apoptotic pathway in cancer cells is a promising strategy for anticancer drug discovery.5

Figure 1. Functional domains of mammalian IAPs: typical IAP structure and the structures of XIAP, c-IAP1 and c-IAP2.

Inhibitor of apoptosis proteins (IAPs) are a class of negative apoptotic regulators in mammalian cells.6, 7 IAP family proteins, which were first identified in baculoviruses, are characterized by the presence of one or more of the 70–80-amino-acid zinc-binding baculoviral IAP repeat (BIR) domains.8, 9 To date, eight members of the mammalian IAP family have been identified: neuronal IAP (NIAP), cellular IAP1 (c-IAP1), cellular IAP2 (c-IAP2), X-chromosome-linked IAP (XIAP), survivin, ubiquitin-conjugating BIR domain enzyme (BRUCE), melanoma IAP (ML-IAP), and IAP-like protein 2 (ILP2).6, 10 These proteins are frequently overexpressed in cancer cells and are associated with poor prognoses.11, 12 In this review, we focus on c-IAP1, c-IAP2 and XIAP, which play a predominant role in regulating apoptosis in cells, particularly malignant cells.13 As shown in Figure 1, a typical IAP commonly contains two kinds of BIR domains (type 1 and 2), a ubiquitin-associated ACS Paragon Plus Environment

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(UBA) domain and a RING domain. BIR1, a type 1 BIR domain, interacts with tumor necrosis factor receptorassociated factors 1 and 2 (TRAF1 and TRAF2),14, 15 which may regulate TRAF-mediated NF-κB and MAPK activation.16 BIR2 and BIR3 of IAPs are type 2 BIR domains. The BIR2 domain of XIAP binds to and inhibits the activation of caspase-3 and caspase-7 and the BIR3 domain specifically binds to and inhibits caspase 9.17 Unlike XIAP, c-IAP1 and c-IAP2 can bind to caspase-3 and caspase-9 but do not inhibit these enzymes.18 All three members have one UBA domain, which can bind to ubiquitin chains and thus participate in downstream biochemical processes.19 The RING domain enables E3 ubiquitin ligase activity to the IAPs.20 An additional caspase recruitment domain (CARD) is presented in c-IAPs, but the function of this domain is unclear with one study suggesting that this domain contributes to the auto inhibition of RING domain activity.21



TNF Stress TNFR1

TRAF2

Mitochondria

Cytochrome C

c-IAP1/2

RIP1

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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SMAC mimetics Caspase 8

Caspase 9

Caspase 3/7

XIAP Apoptosis SMAC mimetics Figure 2. Regulation of caspase-dependent apoptosis and NF-κB signaling by IAP and SMAC proteins.

Extensive studies demonstrate that XIAP, c-IAP1 and c-IAP2 regulate apoptosis through a number of pathways (Figure 2).6, 7 XIAP is a key regulator in inhibiting both forms of apoptosis - the death receptormediated pathway and the mitochondria-mediated pathway. It functionally binds to and inhibits caspase 3, caspase 7 and caspase 9. c-IAP1 and c-IAP2 mediate TRAF2 and receptor-interacting protein 1 (RIP1) ubiquitination. Through the interaction of cIAPs and TRAF2, TNF factor receptor 1 (TNFR1)-mediated caspase-8 activation and apoptosis are triggered.22


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In many cancers, IAPs bind to and inhibit activated caspases and high levels of IAP result in resistance to apoptosis. BIR domains of IAPs are required for such protein-protein interactions (PPIs) to inhibit apoptosis. Therefore, the discovery of PPI inhibitors for IAPs in cancer cells is hypothesized to be a potential strategy to overcome the resistance of cancers to chemotherapy.23, 24

2. SMAC is an endogenous IAP antagonist, and the binding interactions between SMAC and IAPs provide concrete insights for drug design. Second mitochondria-derived activator of caspases (SMAC), also known as direct IAP-binding protein with low pI (DIABLO), is well defined as the endogenous antagonist of IAPs and was first discovered by Dr. Xiaodong Wang and Dr. David Vaux in 2000.25, 26 SMAC is a mitochondrial protein, which is released from the mitochondria into the cytosol during apoptotic induction. Subsequently, SMAC interacts with the BIR2 and BIR3 domains of IAPs at the IAP-binding motif (IBM), thereby antagonizing IAPs and resulting in caspase3/7/9 activation and apoptosis.6, 25-29 Determination of the structures of XIAP BIR3 with the SMAC protein (PDB: 1G73)29 or a 9-residue peptide from SMAC (PDB: 1G3F)28 has provided concrete insights for designing small-molecule SMAC mimetics. As shown in Figure 3, two units of SMAC, each consisting of a three-helix bundle, generate a homodimeric tertiary structure. The four N-terminal residues (Ala1–Val2–Pro3–Ile4, abbreviated as AVPI) of SMAC bind to a surface groove on XIAP BIR3 via a combination of intermolecular hydrogen bonds and van der Waals interactions. The amino group (-NH2) of the Ala1 residue forms four strong hydrogen bonds with the neighboring residues in BIR3 (Glu314, Gln319, Trp310), and the backbone carbonyl group (-C=O) forms an additional hydrogen bond with the indole NH group of Trp323. The methyl group (-CH3) of Ala1 fits into a small hydrophobic pocket that might be important for binding activity. The amino and carbonyl groups of Val2 form hydrogen bonds with Thr308, while its isopropyl side chain is solvent exposed, making this position suitable for further optimization. Van der Waals interactions have been demonstrated between the pyrrole ACS Paragon Plus Environment


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ring of Pro3 and the Trp323-Tyr324 loop. Another hydrogen bond is formed between Ile4 and the carbonyl group of Gly306, and the isobutyl side chain of Ile4 forms hydrophobic interactions with the region containing Leu292, Lys297 and Lys299.

Figure 3. Crystal complex (PDB: 1G73) and detailed interactions of SMAC, XIAP-BIR3 (presented as a surface model and the carbons in gray), and the AVPI peptide (carbons in yellow, nitrogens in blue, and oxygens in red). Blue dashed lines represent hydrogen bonds. The figures were generated by PyMOL.

The N-terminal residues of known binding partners of this groove with high sequence homology, such as Grim (AIAY), Hid (AVPF), and Reaper (AVAF), have been demonstrated essential to maintain strong binding ACS Paragon Plus Environment

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affinities with BIR3-containing proteins.30, 31 Therefore, compounds mimicking this binding motif might serve as prototypical IAP antagonist therapeutic candidates. Four generations of such antagonists will be discussed herein, including peptides and peptidomimetics based on AVPI, nonpeptidic conformation-constrained monovalent IAP antagonists, bivalent IAP antagonists, and nonalanine IAP antagonists.

3. Four generations of SMAC-mimetic IAP antagonists 3.1 First-generation SMAC-based peptides and peptidomimetics designed based on AVPI. The length and variety of amino acids in SMAC were first evaluated for the interaction to the BIR3 domain, and a structure-activity relationship (SAR) was demonstrated (Figure 4).27-29, 32 The mature SMAC (59-239 residues) exhibits a binding affinity with a Kd of approximately 420 nM. The activity almost remains when the C terminus is shortened to the 9-mer AVPIAQKSE. Further shortening the C terminus to the 5-mer AVPIA (Kd = 640 nM) and 4-mer AVPI (Kd = 480 nM) result in no loss of binding affinity to the BIR3 domain of XIAP, indicating that the C terminus of SMAC is not essential for BIR3 binding. The role of each amino acid in the 4-mer peptide is also determined. (1) The binding affinity decreases or is completely lost when the alanine residue is replaced with glycine, serine or methionine while a 2-fold improvement is observed with 2aminobutyric acid (a non-natural amino acid), indicating a hydrophobic interaction between the methyl group of the alanine and the BIR3 domain. However, a larger hydrophobic side chain is not suitable in this pocket. Acetylated peptides or a replacement with propionic or isobutyric acid at the N terminus leads to a complete loss of binding affinity, suggesting the importance of the positive charge and hydrogen bonds for binding. (2) Many amino acid residues could be tolerated at the valine position, in agreement with the crystal structure of the SMAC-BIR3 complex that no close interactions with valine are observed. Replacement with arginine shows an approximately 3-fold increase in binding affinity while replacement with glycine, proline, or aspartate at this position leads to nearly complete loss of activity, indicating that the positive charge may be important to the binding affinity. Methylation of the imino group between alanine and valine leads to ACS Paragon Plus Environment


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complete loss of binding, highlighting the importance of the hydrogen bond with Thr308. (3) The van der Waals interaction between the proline residue of the peptide and the Trp323-Tyr324 loop plays an important role in binding. Proline or substituted pyrrolidine is typically used in peptides and peptidomimetics to constrain the compound in an appropriate configuration, which is discussed in the following sections. (4) At the isoleucine position, a relatively large pocket for hydrophobic interaction is observed in the crystal structure of the complex. Therefore, a hydrophobic residue at this position, for instance, tryptophan, leucine, valine or phenylalanine, is favorable for binding, but a charged or polar residue such as arginine, glutamate, aspartate, histidine, glutamine, and asparagine, is not favorable for binding. N-methylation of isoleucine also decreases the binding affinity, due to the loss of the hydrogen bond, but the effect is much weaker than that observed with the imino group between alanine and valine. Simultaneous replacement of valine and isoleucine to arginine and phenylalanine, ARPF, leads to a 24-fold increase in affinity (Kd = 20 nM) compared with that of the SMAC AVPI peptide. Although they exhibit very high binding affinities, these peptides have a common drawback, i.e., poor cell permeability, similar to other peptidic compounds.33 Therefore, extensive modifications using non-natural amino acids or substituents were carried out to identify potent and cell-permeable peptidomimetics (Figure 4).34 Only a few optimizations were performed at the alanine and valine positions. Monomethylation of the terminal free amino group is well tolerated, but di-methylation significantly decreases the protein binding activity, indicating the importance of the intermolecular hydrogen bonds formed by the amino group. As observed in the crystal structure, the isopropyl group of valine is solvent exposed. A tert-leucine or cyclohexyl group would be suitable at this position without compromising the loss of binding affinity. At the proline position, replacement with a variety of amino acids leads to dramatic loss in binding affinity.34 Azetidine, bearing a four-membered ring, and piperidine, bearing a six-membered ring, exhibit a 2- and 10-fold decreases in the Kd value, respectively. The R-4-hydroxyproline analog exhibits decreased binding affinity, which is consistent with the hydrophobicity of the Trp323-Tyr324 loop. Herein, the introduction of a phenyl ACS Paragon Plus Environment

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group improves the binding affinity by 2-fold compared with the nonsubstituted analog. The Rtetrahydronaphthyl amide peptidomimetic (1) and R-indane amide peptidomimetic (2) at the isoleucine position have Kd values of 12 and 29 nM, respectively, with XIAP-BIR3 (Figure 4). The configuration is important for the activity, as the S-diastereomeric analogs markedly reduce the binding affinities. In addition to exhibiting biochemical potency, these compounds also show decent cell permeability. Compounds 1, 3 and 4 could rescue BIR3-mediated caspase inhibition with EC50 values of 290, 310, and 240 nM, respectively. Compounds 1 and 3 can also inhibit MDA-MB-231 cancer cell growth with IC50 values of 68 and 13 nM, respectively, and they exhibit modest inhibition of tumor growth in an MDA-MB-231 xenograft mouse model, suggesting the therapeutic potential of these compounds in cancer treatment.34 A c-IAP-selective peptidomimetic antagonist (5) was developed by Genentech.35 This compound exhibits high affinity for the BIR3 domains of c-IAP1 and c-IAP2 and more than 2000-fold selectivity over XIAP. This antagonist is mostly used as a tool compound because c-IAP-selective antagonists are less potent than other antagonists in inducing apoptosis in cancer cells.35 Compound 6 (also known as GDC-0152), a pan-IAP antagonist that has been advanced into a phase I clinic trial (NCT00977067), binds to c-IAP1, c-IAP2, XIAPBIR3, and ML-IAP with Kd values of 17, 43, 28, and 14 nM, respectively.36 This compound promotes cIAP1 degradation, induces caspase-3/7 activation, inhibits MDA-MB-231 breast cancer cell growth without affecting normal mammary epithelial cells, regresses tumor growth in a mouse model, and has excellent preclinical predictions of pharmacokinetics, mean plasma clearance and distribution. Compound 7 (also known as LCL161) is another peptidomimetic that is in several clinical trials but sufficient data are unavailable currently.13 A recent study, using a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), determined the binding affinity of this compound with the BIR3 domains of XIAP, cIAP1, and c-IAP2 with IC50 values of 52.7, 10.4 and 12.9 nM, respectively.37 In this study, a thermodynamicsdriven approach was used to first identify compound 8 as a Lys-covalent XIAP-BIR3-selective inhibitor with



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an IC50 value of 16.6 nM and more than a 12-fold selectivity toward c-IAPs. This compound also exhibits anticancer activity in multiple cancer cell lines, especially those that are resistant to LCL161. Takeda Pharmaceutical developed a series of bicyclic octahydropyrrolo[1,2-a]pyrazine peptidomimetics using a bioisostere-based strategy to replace the proline.38, 39 The proline ring is expanded to a six-membered piperazine ring, and a fused five-membered ring is introduced onto the piperazine to hydrophobically interact with the Trp23-Tyr324 region. Further optimization leads to the development of compound 9 (also named T-3256336), which exhibits potent c-IAP1 (IC50 = 1.3 nM) and XIAP-BIR3 (IC50 = 200 nM) inhibitory activity and cancer cell growth inhibition (GI50 = 1.8 nM) against MDA-MB-231 breast cancer cells. This compound has good metabolic stability and an oral bioavailability of 50% and causes complete tumor regression in an MDA-MB-231 tumor xenograft model.39 However, this compound has low membrane permeability (1.0 nm/s, the membrane width permeabilized per second) into cells expressing multidrug resistance 1 (MDR1), which mediates drug efflux (efflux ratio: 24).38 Therefore, a tricyclic hexahydropyrazino[1,2-a]indole scaffold was developed to enhance the membrane permeability into MDR1-expressing cells. Compound 10, with an electron-withdrawing chloride group that decreases the electron density of the bridgehead nitrogen, exhibits significantly improved membrane permeability (64 nm/s) and decreases the MDR1 substrate activity (efflux ratio: 1.4). Moreover, this compound exhibits strong biochemical activity and tumor growth inhibition (TGI) in MDA-MB-231 cells, representing a suitable lead for further evaluation.


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Journal of Medicinal Chemistry Decrease O

O

Decrease

O

HO

NH2

*

O

*

N

OH

O

O

NH2

*

*

*

N

N

NH2

O

R

S

*

Increase

O

HN

N

O N H

NH

H 2N

Decrease NH

N

H 2N

Increase

*

*

HN

NH

NH2

N

O

N NH

NH

O

N

NH

NH

O

O

N

H 2N

O NH

O

N

O

O OH

OH

O NH

O

O OH

*

HO

O

O

H 2N OH

*

O

Decrease

NH

HN

HN

*

OH

O

NH

*

O

NH

OH

OH

O HO O

Increase

NH

Sightly decrease

NH

O

NH2

O

O

O

NH

*

NH

*

O

NH

O

O

NH

HO

HO

O

OH

OH O NH

NH

O

O

O

Representative SMAC peptidomimetics O H N

N O NH

O

O

O

NH

O

O NH

O

1 XIAP-BIR3 Kd = 12 nM Caspase 9 inhition: EC50 = 290 nM MDA-MB-231: IC50 = 68 nM MDA-MB-231 model: 40 mg/kg/day, 16 d, ~50%

3 XIAP-BIR3 Kd = 16 nM Caspase 9 inhition:EC50 = 310 nM MDA-MB-231: IC50 = 13 nM MDA-MB-231 model: 20 mg/kg/day, 16 d, ~50%

2 XIAP-BIR3 Kd =29 nM

NH

O

N

H N

N O

S

O NH N

O

N S

O

O S HN

6 (GDC-0152) c-IAP1-BIR3 Kd = 17 nM c-IAP2-BIR3 Kd = 43 nM XIAP-BIR3 Kd = 28 nM XIAP-BIR2 Kd = 112 nM ML-IAP-BIR Kd = 14 nM

7 (LCL161) c-IAP1-BIR3 Kd = 10.4 nM c-IAP2-BIR3 Kd = 12.9 nM XIAP-BIR3 Kd = 52.7 nM

O

NH

5 c-IAP1-BIR3 Kd = 16 nM c-IAP2-BIR3 Kd = 85 nM XIAP-BIK3 Kd > 3400 nM

Cl

N N

NH

NH

N

N

N F

O

N

F F

O

NH #

NH

O

O

O

N

O #

O

O

4 XIAP-BIR3 Kd = 5 nM Caspase 9 inhition: EC50 = 240 nM

O

O S O F

N

NH

NH

H N

N O

NH

Covalent

O

O

O

O

H N

N

NH

NH

NH

H N

N

H N

N

NH NH

N

O O

O

NH NH

N

NH

8 XIAP-BIR3 IC50 = 16.6 nM c-IAP1-BIR3 IC50 > 200 nM c-IAP1-BIR3 IC50 = 353 nM

NH

O NH

F


O O

9 (T-3256336) XIAP-BIR3 Kd = 200 nM c-IAP1-BIR3 Kd = 1.3 nM MDA-MB-231: GI50 = 1.8 nM MDA-MB-231 model :100 mg/kg/bid 14 d, regression, F = 50% Apparent permeability: 1.0 nm/s Efflux ratio: 24

O

10 XIAP-BIR3 Kd = 220 nM c-IAP1-BIR3 Kd = 2.8 nM MDA-MB-231:GI50 = 15 nM Apparent permeability: 64 nm/s Efflux ratio: 1.4

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Figure 4. Systematic summary for the different regions of the first-generation SMAC-based AVPI peptides and peptidomimetics. *: Structures with stars contain non-natural substituents. Chemical structures of representative potent and cell-permeable SMAC peptidomimetics as IAP antagonists. Substituents in different regions are colored as follows: alanine (blue), valine (pink), proline (black), and isoleucine (red). #: Compound has been advanced into clinical evaluations.


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3.2 Second-generation conformation-constrained monovalent IAP antagonists. Dr. Shaomeng Wang and colleagues first published bicyclic lactam structures as IAP antagonists in 2004 to mimic the geometry of the AVPI peptide.40,

41

A representative structure optimization workflow is

summarized in Figure 5. First, optimization of the isoleucine position of compound 11 results in compound 12, which has a Kd value of 290 nM, showing a 2-fold improvement over the Kd value of compound 11. Then, cyclization of valine and proline forms [6,5] bicyclic compound 13 and its diastereoisomer. The binding affinities of these compounds to the XIAP BIR3 protein are determined, with Kd values of 4470 nM and higher than 100 μM respectively, indicating the importance of the conformational constraint to binding.40, 41 An analog (14) optimized at the alanine and isoleucine positions using ethyl and diphenylmethyl groups exhibits the best Kd values (350 nM) with XIAP BIR3 in [6,5] bicyclic series and can enhance apoptosis induced by cisplatin in PC-3 human prostate cancer cells.41 Next, one or two additional carbon atom(s) are inserted into the six-membered ring to afford [7.5] or [8.5] bicyclic analogs, to better mimic the binding conformation of the AVPI peptide.42, 43 A [7.5] bicyclic compound (15) exhibits a Kd value of 150 nM with XIAP BIR3. Further optimization by introduction of a diphenylmethyl group and methylation of its primary amino group obtains compound 16 with an excellent binding affinity, a Kd value of 61 nM.42 This compound biochemically suppresses the inhibition of caspase-9 activity by XIAP-BIR3 and exhibits an IC50 of 100 nM in the inhibition of MDA-MB-231 cancer cell growth. The cellular activity of these SMAC mimetics is affected by methylation of the amino group at the alanine position.33, 42 The [8.5] bicyclic series are another class of highly potent and cell-permeable SMAC mimetics.43 Among these compounds, those with diphenylmethyl (17 and 18), Rindane amide (19) and R-tetrahydronaphthyl amide (20) groups exhibit high biochemical potency toward the XIAP BIR3 domain, with Kd values of 67, 26, 15 and 14 nM, respectively. Co-crystallization of compound 17 with the XIAP BIR3 protein (PDB: 2JK7) provides the structural basis for further design of bicyclic monovalent SMAC mimetics. As shown in Figure 5, six hydrogen bonds are observed with Glu314, Asp309, Trp323, Thr308, and Gly306 residues. The ethyl group at the N terminus fits into the small hydrophobic pocket, mimicking ACS Paragon Plus Environment


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the methyl group in the SMAC AVPI peptide. The diphenylmethyl group inserts into the hydrophobic pocket formed by the Leu292-Lys297-Lys299 region. The eight-membered lactam constrains the molecule in the correct configuration and forms weak contacts with Trp323, leaving space for further optimization at the ring region. Considering the modest area under the curve (AUC) and oral bioavailability of compound 18 (also known as SM-122, Kd = 26 nM),44 multiple optimizations were carried out on the bicyclic rings to improve drugability.44-48 Replacement of the original [8.5] bicyclic core with a diazabicyclic core (structure not shown in Figure 5) results in a compound with similar binding affinity to that of compound 17.44 Introduction of substituents on the nitrogen leads to various feasible derivatives. Among these derivatives, compound 21 (also known as SM-337) shows high binding potencies with XIAP-BIR3, c-IAP1, and c-IAP2, with Kd values of 8.4, 1.5, and 4.2 nM, respectively. This compound inhibits MDA-MB-231 cancer cell growth with an IC50 value of 31 nM, which is 17-fold more potent than that of compound 18. The pharmacokinetic (PK) profile of this compound is also much better than that of compound 18, with an oral bioavailability of 24% at a dose of 30 mg/kg in rats, compared with 14% observed for compound 18. Furthermore, an isovaleryl analog (22, also named SM-406 or AT-406 or Debio-1143) exhibits potent binding to XIAP-BIR3, c-IAP1, and c-IAP2 with Kd values of 66.4, 1.9, and 5.1 nM, respectively. Preclinical evaluations have shown that this compound has an excellent drug profile, with good PK properties observed in four different species (mice, rats, nonhuman primates, and dogs), high effectiveness in the induction of apoptosis in MDA-MB-231 xenograft tumors, and complete TGI using the regimen of oral gavage daily, 5 days a week for 2 weeks at 100 mg/kg. This compound has been evaluated as an oral agent alone or in combination with other agents in five phase I trials (NCT03270176, NCT01930292, NCT01265199, NCT02022098, and NCT01078649). Similarly, a phenylacetylamino group (23) is introduced at the carbon of the eight-membered lactam to generate a new chiral center.47 A fluorescence polarization (FP)-based binding assay shows that the R configuration was 2-3 times more potent than the corresponding S isomer. Compound 23 binds to XIAPBIR3, c-IAP1, and c-IAP2 proteins with Kd values of 36, 7,000 times more potent than the AVPI peptide (IC50 = 10396 nM).55 SM-164 is 1,000 times more potent than SM-122 as an apoptosis inducer in HL60 tumor cells. In the MDA-MB-231 xenograft model, this compound induces rapid c-IAP1 degradation and strong apoptosis in tumor tissues, and the tumors completely regress with no obvious toxicity observed in normal mouse tissues.57, 58 In addition, a follow-up study described in detail that the linker has a major effect on intracellular concentrations, cell permeability, and overall cellular activity.59 Compound 33 (also known as AZD5582) is designed from peptidomimetic 2 using the P4-P4 strategy with 1,3-bisacetylene linkage.60 This compound exhibits potent bindings to the BIR2 domain of XIAP (IC50 = 21 nM) and the BIR3 domains of c-IAP1, c-IAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively); causes c-IAP1 degradation (IC50 = 0.1 nM); and significantly inhibits MDA-MB-231 cell growth (IC50 < 0.06 nM). Compound 34 (also named as TL32711 and Birinapant), manufactured by TetraLogic Pharmaceuticals, interacts with the BIR3 domains of XIAP, c-IAP1, c-IAP2 and the single BIR domain of ML-IAP with Kd values of 50, ~1, 36 and ~1 ACS Paragon Plus Environment


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nM, respectively.61 This compound potently induces c-IAP1 and c-IAP2 degradation (5 and 151 nM, respectively) and inhibits TNF-mediated NF-κB activation. Compound 34 also promotes the degradation of cIAP1 and inhibits the TNF-mediated NF-κB signal transduction pathway more effectively than its monovalent counterpart (highlighted in blue box). These results indicate that specific bivalent IAP antagonists but not monovalent compounds are capable to effectively degrade cIAP1 and are preferred for the targeting of TNF-dependent signaling for diseases.62 This compound has been advanced into several clinical trials.13, 63

N O

NH

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Tethering site detection O

N O

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NH

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Monvalent counterpart OH

NH

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31 (SM-157) XIAP-BIR2-BIR3 IC50 = 376 nM HL60 IC50 = 2000 nM

18 (SM-122) XIAP-BIR2-BIR3 IC50 = 438 nM XIAP-BIR2 IC50 = 5600 nM HL60 IC50 = 1400 nM

O F

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O O

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NH N N O O H N

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34 (Birinapant) # XIAP-BIR3 Kd = 50 nM c-IAP1-BIR3 Kd = ~1 nM c-IAP2-BIR3 Kd = 36 nM ML-IAP-BIR Kd = ~1 nM

O

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2 33 (AZD5582) XIAP-BIR2 IC50 = 21 nM XIAP-BIR3 IC50 = 15 nM c-IAP1-BIR3 IC50 = 15 nM c-IAP2-BIR3 IC50 = 21 nM MDA-MB-231: GI50 < 0.06 nM c-IAP1 degradation: EC50 = 0.1 nM

Figure 8. Bivalent antagonists linked using the P4-P4 strategy, highlighted in red. #: Compound has been advanced into clinical evaluations.


O NH

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32 (SM-164) XIAP-BIR2-BIR3 IC50 = 1.4 nM HL60 IC50 = 1.0 nM

N

F

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The Wang group also reported the synthesis of bivalent antagonists starting from compound 22 using the P2-P2 strategy (Figure 9).64, 65 This compound exhibits high affinity to the BIR2 and BIR3 domains in XIAP, and computational model predicts that the solvent-exposed amide group in the eight-membered ring might be suitable for tethering. Therefore, compound 35 was designed using a linker similar to SM-164, exhibiting a Kd value of 2 nM with XIAP containing the BIR2 and BIR3 domains. This compound is also cell permeable, with an IC50 value of 3.4 nM against MDA-MB-231 cells, and efficiently induces the degradation of c-IAP1 and c-IAP2 in cancer cells at concentrations as low as 1 nM; it also activates caspase-3 and caspase-8, and cleaves PARP at single-digit nM concentrations.64 Further SAR studies of this tethering strategy leads to the potent compound 36 (SM-1200). Compared with SM-164, the major advantage of this compound is its PK profile, exhibiting a 17-fold increase in the AUC in rat plasma and an 8-fold decrease in clearance. In addition, compound 36 exhibits 99% tumor regression at 10 mg/kg (i.v.) in the MDA-MB-231 xenograft model when administered weekly for 4 weeks with 6 out of 7 mice remaining tumor free 27 days after the final dose.65 Compound 38 (AEG40730) is a peptidomimetic generated by tethering two molecules of compound 37 (AEG40599).66 It potently binds to the BIR3 domains of the XIAP, c-IAP1 and c-IAP2 proteins, but the potency of BIR2 binding has not been reported.



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NH

O N O

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22 (SM-406/AT-406) XIAP-BIR3 Kd = 55 nM XIAP BIR2 Kd = 5500 nM

O HN

35 XIAP-BIR2-BIR3 Kd = 2 nM MDA-MB-231: IC50 = 3.4 nM

H N

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O N 37 (AEG40599) XIAP-BIR3 Kd = 1059 nM c-IAP1-BIR3 Kd = 49 nM c-IAP2-BIR3 Kd = 86 nM

O NH

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F

F F

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HN

38 (AEG40730) XIAP-BIR3 Kd = 100 nM c-IAP1-BIR3 Kd = 17 nM c-IAP2-BIR3 Kd = 34 nM

Figure 9. Bivalent antagonists linked using the P2-P2 strategy, highlighted in pink.

Bristol-Myers Squibb published tetrahydroisoquinoline-based heterodimeric bivalent compounds tethered at the proline site (Figure 10).67, 68 Tethering of compounds 1 and 39 using a carboxybenzamide linker results in compound 40, which exhibits a high binding affinity with the XIAP and c-IAP1 proteins containing BIR2 and BIR3 domains. This compound also exhibits a TGI value of 100% in the A875 tumor xenograft model, while ACS Paragon Plus Environment

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monovalent inhibitors (e.g. GDC-0152, compound 6) are inactive.67 Subsequent studies examined a series of highly potent analogs with truncated linkers.68 Compound 41, obtained by directly tethering 1 and 39, exhibits improved biochemical and cell-based activity. In addition, complete tumor regression is demonstrated without substantial weight loss in both the A875 melanoma and H1703 lung xenograft models. HN O N H

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NH 40 XIAP-BIR2-BIR3 IC50 = 10 nM c-IAP1-BIR2-BIR3 IC50 = 4 nM MDA-MB-231: IC50 = 1.5 nM A875: IC50 = 6.8 nM Caspase 3: EC50 = 28 nM A875 model: TGI = 100%

41 XIAP-BIR2-BIR3 IC50 = 4.9 nM c-IAP1-BIR2-BIR3 IC50 = 1.5 nM MDA-MB-231: IC50 = 0.3 nM A875: IC50 = 0.8 nM Caspase 3: EC50 = 10 nM A875 model: TGI = 112% H1703 model: TGI = 124%

Figure 10. Bivalent antagonists linked using the P3-P3 strategy, highlighted in orange.

3.4 Fourth-generation nonalanine IAP antagonists identified by fragment-based drug discovery (FBDD). Instead of using the AVPI peptide as a starting point, four nonalanine fragments of millimolar binding affinities with XIAP BIR3 were identified by Astex Pharmaceuticals using an FBDD-based strategy.69 Among these fragments, fragment 42 (Figure 11A) exhibits a promising binding mode with XIAP BIR3 despite of weak binding and low ligand efficiency (LE), leading to further fragment optimizations. Fragment 43 was then designed based on the AVPI peptide with greatly improved IC50 values toward the XIAP BIR3 and c-IAP1 BIR3 proteins. Based on the co-crystal structure of the complex (Figure 11B), this fragment locates at the AVP ACS Paragon Plus Environment


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positions of the BIR3 protein but the binding pocket is not fully occupied. Structure-based drug design (SBDD) was then utilized to obtain an early lead compound 44, exhibiting dual inhibition of the XIAP and c-IAP1 BIR3 proteins (IC50 = 160 and 10 nM). The LE is higher than the benchmark level of 0.3. At the cell level, this compound is also active in MDA-MB-231 and EVSA-T cancer cells, with EC50 values of 590 and 100 nM, respectively. In the MDA-MB-231 xenograft model, this compound significantly reduces the tumor size when administered intraperitoneally. Further optimization results in the potent, orally bioavailable, compound 45 (also named AT-IAP), which is a dual antagonist of XIAP and c-IAP1 (IC50 = 5.1 and 0.32 nM).70 As shown in Figure 11C, the morpholine mimics the iso-propyl group of the valine amino acid of SMAC and occupies the valine-binding pocket. In addition to common hydrogen bonds labeled in the figure, a water molecule is observed in the co-crystal structure, mediating an H-bond interaction between the tertiary amine of the piperazine ring and the NH of Trp323. The piperazine-indoline forms a suitable conformation, with the fluorinated phenyl group located on the hydrophobic side of the binding pocket. Although AT-IAP has acceptable oral bioavailability in mice (F = 22%) and antitumor growth activity in mouse MDA-MB-231 xenograft models, the oral bioavailability at 5 mg/kg is only 2% in cynomolgus monkeys, and the hERG inhibition rate at 30 μM is 70%. Therefore, in order to improve the metabolic stability and cardiac safety profile, further optimization was performed and results in the IAP antagonist clinical candidate 46 (also named ASTX660). The X-ray co-crystal structure (Figure 11D) reveals that 46 binds to XIAP in a manner similar to AT-IAP. This compound exhibits a favorable preclinical profile and is currently being evaluated in a phase I/II clinical trial (NCT02503423).71, 72


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O

A N HN

Fragment optimization

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42 (Fragment hit) XIAP-BIR3 IC50 > 5 mM, LE < 0.20 c-IAP1-BIR3 IC50 > 5 mM, LE < 0.20

N

N O

43 XIAP-BIR3 IC50 = 52% at 1 mM, LE = 0.27 c-IAP1-BIR3 IC50 = 410000 nM

N

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Structure-based optimization

HN

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44 XIAP-BIR3 IC50 = 160 nM, LE = 0.30 c-IAP1-BIR3 IC50 = 10 nM, LE = 0.35 MDA-MB-231: EC50 = 590 nM MDA-MB-231 model: 100 mg/kg/bid, i.p., 14d, ~100% O

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Structure-based optimization

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OH

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F F 45 (AT-IAP) XIAP-BIR3 IC50 = 5.1 nM c-IAP1-BIR3 IC50 = 0.32 nM MDA-MB-231: EC50 = 4.4 nM hERG inhibition: 70% at 30000 nM MDA-MB-231 model: 30 mg/kg/bid, p.o., 22d mouse F = 22% monkey F = 2%

46 (ASTX660) # XIAP-BIR3 IC50 = 2.8 nM c-IAP1-BIR3 IC50 = 0.22 nM MDA-MB-231: EC50 = 1.8 nM CYP3A4 t1/2 = 32 min hERG inhibition: 30% at 30000 nM MDA-MB-231 model: 20 mg/kg/qd, p.o., 22d mouse F = 34% rat F = 29% monkey F = 12%

Figure 11. Structure optimization workflow of nonalanine IAP antagonists (A) and their co-crystal complexes with the XIAP BIR3 protein: (B) Fragment 43 (PDB: 5C7B); (C) AT-IAP (PDB: 5M6L); (D) ASTX660 (PDB: 5OQW). The red dots represent water molecules in the crystal complex.

4. Other representative IAP antagonists. 4.1 Benzoxazepinones and benzodiazepinones are identified as XIAP BIR2 selective antagonists. Hoffmann-La Roche published a series of benzoxazepinones and benzodiazepinones that selectively target BIR2 over BIR3 (Figure 12A).73, 74 The benzazepinone 47 is an initial hit from a high-throughput screening and ACS Paragon Plus Environment


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has selective binding affinity for XIAP BIR2.73 A peptidomimetics SAR study shows that N-methylation of the L-alanine moiety and substituents containing aromatic groups on the azepinone N-substituent are preferred. Compound 48 exhibits high BIR2-selective binding affinity, unacceptably high in vivo clearance (521 mL/min/kg) and low blood stability (7%). The introduction of substituents onto the azepinone ring was thought to be a suitable approach to improve the PK profile. Compound 49, with a spiro-tetrahydropyran moiety, significantly improves the blood stability (88%) and clearance (60 mL/min/kg) but remains unsatisfactory. In addition, replacement of the bromine with a carboxylic acid (50) improves BIR2 potency although unfavorable clearance is observed. To guide further design of new analogs, a co-crystal structure of compound 50 with the XIAP BIR2 domain is obtained revealing features that might contribute to the selectivity for BIR2 over BIR3 (Figure 12B).75 The left side of the figure shows four hydrogen bonds, similar to the structure of the BIR3 domain with other antagonists. In contrast, the fused benzene ring of benzoxazepinone locates in the His223 region, where a tryptophan residue is located in the BIR3 protein. The naphthyl ring system is inserted into the Lys206-Lys208 hydrophobic pocket and forms a hydrogen bond with Lys206 backbone NH. The methoxy group of this residue is inserted into the narrow loop between His223 and Phe224. The other part of the naphtyl moiety locates at the solvent-exposed edge, indicating that relatively bulky groups can be tolerated at this site. Compound 51, with an indole moiety, exhibits excellent selective BIR2 potency, an acceptable clearance rate (41 mL/min/kg) and good oral bioavailability (F = 53%). However, this compound has some safety issues that make it unfavorable as a clinical lead.73, 74 This compound is a potent inhibitor (IC50 = 1.8 µM) of CYP3A4 and demonstrates a time-dependent inhibition (TDI). The potential of CYP3A4 induction (CYP3A4 mRNA > 40% of control at 10 μM) is also unacceptable. Subsequent efforts focused on safety optimization using a related benzodiazepinone scaffold to overcome the above drawbacks.74 Compound 52 is designed to have a substituent on the new nitrogen atom oriented toward a solvent-exposed area in the co-crystal complex (Figure 12C). The substituted phenyl compound retains high selectivity over BIR3 and low CYP3A4 inhibition. Similar to the spiro-tetrahydropyran compound, ACS Paragon Plus Environment

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introduction of a trans methyl group at the 4 position of the benzodiazepinone core might lead to a compound with improved metabolic stability as well. Compound 53 was developed using this strategy to retain potent BIR2 activity and excellent clearance. The CYP3A4 inhibition also decreases compared to compound 51 to an acceptable value of 16 µM. Moreover, no induction is observed at concentrations up to 10 µM.

A Structural optimization

O H 2N

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Br 48 XIAP-BIR2 IC50 = 101 nM XIAP-BIR3 IC50 > 54 M rat blood stability (% remianing at 4h): 7 rat iv PK, CL = 521 mL/min/kg

47 (HTS hit) XIAP-BIR2 IC50 = 14.4 M XIAP-BIR3 IC50 > 54.2 M

49, R = Br XIAP-BIR2 IC50 = 65 nM XIAP-BIR3 IC50 = 23.5 M rat blood stability (% remianing at 4h): 88 rat iv PK, CL = 60 mL/min/kg 50, R = COOH XIAP-BIR2 IC50 = 18 nM XIAP-BIR3 IC50 = 22.6 M rat iv PK, CL = 206 mL/min/kg

H 2N O

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52 XIAP-BIR2 IC50 = 6 nM XIAP-BIR3 IC50 = 24.5 M CYP3A4 inhibition: IC50 = 10.2 M

51 XIAP-BIR2 IC50 = 39 nM XIAP-BIR3 IC50 = 3.06 M rat iv PK, CL = 41 mL/min/kg F = 53% CYP3A4 inhibition: IC50 = 1.8 M > 40% CYP3A4 induction at 10 M

B

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R222 H223

E219

53 XIAP-BIR2 IC50 = 45 nM XIAP-BIR3 IC50 = 30.8 M rat iv PK, CL = 4.3 mL/min/kg CYP3A4 inhibition: IC50 = 16 M No CYP3A4 induction at 10 M

R222 H223

F224 D214

D214

E219

F224

E211

N209 K208

K206

K206

N209 K208

Figure 12. Structure optimization workflow for benzoxazepinone and benzodiazepinone synthesis (A) and ACS Paragon Plus Environment


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the co-crystal complexes of these compounds with the XIAP BIR2 protein: (B) compound 50 (PDB: 4KJV); and (C) compound 52 (PDB: 4KJU).

4.2 Macrocyclic antagonists. In addition to the above antagonists, macrocyclic molecules are another series of compounds that act as IAP antagonists (Figure 13). Compound 54, also developed by the Wang group, is a cyclic, bivalent SMAC mimetic that simultaneously modifies the valine and isoleucine positions.76 This compound is much more potent than the AVPI peptide, with Kd values of 4 or 4400 nM toward XIAPs containing the BIR3 or BIR2 domain alone. For the XIAP containing both BIR2 and BIR3 domains, this compound exhibits a biphasic dose-response curve, with IC50 values of 0.5 and 406 nM for the two binding sites. This compound is also cell permeable and effectively inhibits MDA-MB-231 cell growth (IC50 = 2200 nM). The amide linker is later replaced with two triazoles to reduce the peptidic characteristics of the compound.77 Compound 55 (also known as SM-162) is more potent than compound 54 at binding to XIAP, c-IAP1 and c-IAP2. This compound is 5–8 times more potent than compound 54 in inhibiting the growth of MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cells.


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H N

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55 (SM-162) c-IAP1-BIR3 Kd = 11 nM c-IAP2-BIR3 Kd = 24 nM XIAP-BIR2 Kd = 3200 nM XIAP-BIR2-BIR3 Site 1: IC50 = 0.43 nM Site 2: IC50 = 28 nM

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56 XIAP-BIR2-BIR3 IC50 = 22 nM c-IAP1-BIR2-BIR3 IC50 = 5 nM MDA-MB-231: IC50 = 1.5 nM A875: IC50 = 73 nM A875 model: TGI = 148 % at 50 mg/kg

O O

H N

N H 57 XIAP-BIR2-BIR3 IC50 = 0.8 nM c-IAP1-BIR2-BIR3 IC50 = 3 nM A875: IC50 = 19 nM A875 model: TGI > 100% at 5 mg/kg

Figure 13. Macrocyclic molecules as IAP antagonists.

Bristol-Myers Squibb and Ensemble Therapeutics together screened macrocycle libraries obtained by DNAprogrammed chemistry and identified antagonists that exhibited binding to XIAP BIR2 and BIR3 domains.78 Compound 56 is obtained after structural optimization, with similar affinities for XIAP and c-IAP1 proteins containing both BIR2 and BIR3 domains. This compound also exhibits potent proapoptotic activity in several cancer cell lines and causes complete tumor regression at a dose of 50 mg/kg in an A875 xenograft model. Further optimization of the linkers and the side chains of the macrocyclic compounds results in compound 57, with a cyclopropylacylsulfonamide group and a propyl linker.79 This compound exhibits high binding affinities to XIAP and c-IAPs. In addition, both in vitro and in vivo antitumor activities are significantly improved compared with those of the initial lead compound 56. Complete tumor regression (TGI > 100%) is ACS Paragon Plus Environment


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observed upon intraperitoneal administration of the compound at 5 mg/kg in the A875 xenograft model. The PK profile shows that this compound has a short to moderate half-life and very low clearance in mice, rats and dogs.

5. IAP antagonists in clinical development. Currently, there are at least 8 small-molecule IAP antagonists in clinical trials (Figure 14).63, 80 Among these trials, the phase I trial using compound 6 (GDC-0512) for the treatment of solid cancers has been terminated early by the sponsor for reasons unrelated to patient safety or antitumor activity (NCT00977067). Compound 58 (GDC-0917/CUDC427) completed phase I trials on refractory solid tumors or lymphomas, but the results have not been reported (NCT01226277). Compound 7 (LCL161), developed by Novartis, completed phase II trials on triple-negative breast cancer in combination with paclitaxel (NCT01617668) and on relapsed or refractory multiple myeloma (NCT01955434). The pathologic complete response (pCR) rate after 12 weeks of therapy was measured. The results showed that LCL161 with paclitaxel had a pCR rate of 24.9%, compared with the value of 23.4% observed for paclitaxel alone, in patients with positive gene expression signatures, while a value of less than 10% was observed in participants with negative expression signatures. The overall response rate was 0% when LCL161 was used alone to treat myeloma and 17.4% when LCL161 was used in combination with cyclophosphamide. There are at least four phase I or II trials recruiting patients with other indications to test LCL161 in combination with other drugs (NCT02098161, NCT02649673, NCT03111992, and NCT02890069). Compound 22 (SM-406/AT-406) from Ascenta, now partnered with Debiopharm (Debio-1143), is in phase I/II trials for dose finding and efficacy evaluation (NCT02022098). Another phase I trial of this compound in combination with avelumab is recruiting participants with advanced solid malignancies and advanced or metastatic NSCLC after platinum-based therapy (NCT03270176). Compound 34 (TL32711/Birinapant), a bivalent SMAC mimetic developed by TetraLogic, has been advanced ACS Paragon Plus Environment

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into several phase I or II trials as a single agent (NCT00993239, NCT01681368, NCT01486784, and NCT01681368). The first human phase I trial with birinapant as a single agent in 50 patients with advanced solid tumors or lymphoma (NCT00993239) determined the maximum tolerated dose (MTD) of 47 mg/m2 with favorable pharmacodynamics (PD) and PK properties. However, toxicities including headache, nausea, and vomiting, and two cases of Bell's palsy (grade 2) were observed at 63 mg/m2.81 In a phase II trial, the effectiveness of birinapant for ovarian, primary peritoneal, or fallopian tube cancer was evaluated (NCT01681368). Eleven patients received birinapant at 47mg/m2 on days 1, 8, and 15 of 28-day cycles. However, accrual was terminated for lack of a clinical benefit as a single agent in this small population.82 Combinations with gemcitabine (NCT01573780), 5-azacitidine (NCT02147873, NCT01828346), or other standard chemotherapeutics such as irinotecan or docetaxel (NCT01188499), carboplatin (NCT02756130), conatumumab (NCT01940172) and pembrolizumab (NCT02587962) are also in progress to identify optimal synergistic combinations. Compound 59 (also named APG-1387), a bivalent compound developed by Ascentage, has been advanced into two phase I trials to test the safety, tolerability, PK and PD profile.83 The first indication was advanced solid tumors or hematologic malignancies, and the compound was used as a single agent or in combination with systemic anticancer agents (NCT03386526). Another study indicates a dismal prognosis in hepatocellular carcinoma (HCC) patients with copy number alterations of cIAP1, cIAP2, and XIAP.84 Therefore, the use of APG-1387 has been attempted for the treatment of HBV-positive HCC. The result shows that this compound directly induced cancer cell apoptosis and promoted innate antitumor immunity, suggesting that APG1387 could be a good candidate for combination with immune checkpoint inhibitor anti-PD1 antibody treatment to overcome low responses of checkpoint inhibitors in HBV-positive HCC.84 Several phase Ib/II studies will be implemented to further assess the antitumor effects of APG-1387 in combination with pembrolizumab.80 Another trial will explore the tolerability, safety and PK/PD of APG-1387 in chronic hepatitis B patients (NCT03585322, see discussion in Section 6.4.4). ACS Paragon Plus Environment


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Figure 14. IAP antagonists with disclosed structures that have been advanced into clinical trials.

An open-label, dose-escalation phase I/II study (NCT02503423) of compound 46 (ASTX660) from Astex Pharmaceuticals is recruiting participants with advanced solid tumors and lymphomas to determine MTD, recommended phase II dose (RP2D), recommended dosing regimen and to obtain preliminary efficacy, PK, and target engagement data. AEG40826 (HGS1029), the hydrochloride salt of a small-molecule inhibitor of IAP with an undisclosed structure (proposed to be a bivalent SMAC mimetic), was found to be tolerated for the treatment of advanced solid tumors in a phase I trial (NCT00708006), but no further trials have been ACS Paragon Plus Environment

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reported.85

6. Outlook and Future Strategies. 6.1 Advantages and disadvantages of monovalent and bivalent IAP antagonists. As drug candidates, both monovalent and bivalent SMAC mimetics have their advantages and disadvantages.33 Monovalent antagonists are potent toward XIAP-BIR3 (i.e. 18, Kd = 26 nM) but much weaker binders of XIAP containing the BIR2 and BIR3 domains (i.e. 18, IC50 = 438 nM) and thus generally less effective at inhibiting XIAP compared to the corresponding bivalent antagonists (i.e. 32, IC50 = 1.4 nM). Although monovalent mimetics are approximately 100-1000 times less potent than the corresponding bivalent compounds at the cellular level, these compounds exhibit superior pharmacological properties when administered orally due to their significantly lower molecular weight. Monovalent compounds 21 (SM-337) and 22 (SM-406) have oral bioavailability of 24% and 46%, respectively, in rats.44,

48

Bivalent mimetics,

including macrocyclic antagonists, are mainly administered intravenously, such as compounds 32 (SM-164), 36 (SM-1200), 40-41 and 59 (APG-1387), or intraperitoneally, such as compounds 55 and 56, which is a disadvantage for further clinical development. In terms of mechanism, monovalent and bivalent SMAC mimetics have their own potential advantages. As discussed, the intrinsic apoptotic pathway is activated in response to intracellular damage, leading to activation of caspase-9.86 The extrinsic apoptotic pathway is activated by the binding of death ligands, such as TNFα, resulting in the activation of caspase-8. Both caspase-8 and caspase-9 can activate the terminal caspase-3 and caspase-7.87 Both pathways are inhibited by XIAP via binding of the BIR3 domain to caspase9 and the BIR2 domain to the terminal caspase-3 and caspase-7.17 Most monovalent mimetics are pan-IAP inhibitors that bind to the BIR3 domains of XIAP, c-IAP1, and/or c-IAP2, such as the SM series, and some of these compounds exhibit selectivity for BIR2 over BIR3, such as compounds 47-53. Bivalent compounds mirror the mode of action of SMAC by targeting both the BIR2 and BIR3 domains of XIAP and blocking the ACS Paragon Plus Environment


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inhibition of the activity of caspase-9, caspase-3 and caspase-7 using a single molecule. Thus, selectively targeting the BIR2 domain of XIAP using monovalent mimetics via a distinct mechanism from that of pan-IAP inhibitors and bivalent compounds could result in different PD properties.74 Collectively, the advantages and disadvantages of monovalent and bivalent inhibitors cannot be ignored in clinical studies, and further research is required to obtain detailed insight.

6.2 Challenges in the development of SMAC mimetics in the future. Currently, most SMAC mimetics are pan-IAP antagonists that non-selectively inhibit XIAP, c-IAP1 and c-IAP2, while only a few compounds are selective for XIAP, c-IAP1 or c-IAP1/2.35, 37 Almost all IAP antagonists contain the alanine motif derived from the natural AVPI peptide. Recently, a non-alanine monovalent mimetic is considered for the first time as a clinical candidate (46, ASTX660), providing a novel idea for future design of selective IAP antagonists with promising chemical space in this area.69,

71, 72

A covalent antagonist (8)

selectively targeting the BIR3 domain of XIAP, not c-IAP1/2, indicates that covalent binding with specific residues on the IAP could provide insight into the relative selectivity potentials.37 To date, eight IAP inhibitors have been advanced into clinical trials. These compounds, however, demonstrate minimal to moderate efficacy alone, and most of them are now being re-evaluated in combination with additional standard therapies, such as paclitaxel and gemcitabine (see Section 5 and Section 6.3). In addition, SMAC mimetics also stimulate TNFα-dependent apoptosis.88 Mechanistically, the SMAC mimetic stimulates NF-κB activation, inducing elevated levels of cytokines and chemokines in normal tissues, which might have potential adverse effects in clinical studies. For instance, the phase I trial of compound 6 (GDC-0152) was terminated while it was reported to cause acute induction of TNFα in the plasma of dogs and rats that may result in TNFα-mediated toxicity.89 On the other hand, in several phase I studies of SMAC mimetics, including GDC-0152, no severe TNFα-driven systemic inflammatory response or severe cytokine release syndrome was observed in humans.13 It remains to be determined what may have ACS Paragon Plus Environment

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contributed to such differences. It has also been reported that a bivalent SMAC mimetic, BV6,22 can affect the host microenvironment to increase bone metastasis via unexpected osteoclast activation.90 Based on the apoptotic pathway, any cancer cell expressing TNFR1 should respond to SMAC mimetic treatment because TNFR1 expression is ubiquitous among various cancer cell types. However, screening of a panel of 50 human NSCLC cell lines shows that only 22% are sensitive to SMAC mimetics treatment.91 Therefore, insensitivity to SMAC mimetics is expected to be common, and utilization of combination therapies is likely required to minimize this limit.

6.3 Combinatorial therapies with other agents for the treatment of cancers. Based on the above challenges, IAP antagonists have been extensively evaluated in combination with other cytotoxic agents, kinase-targeted agents, death receptor ligands and radiation therapy.92 Several formulations using combinatorial therapies have been evaluated in clinical trials because of the moderate efficacy of IAP antagonists as single therapies (see Section 4). Many cytotoxic drugs, such as gemcitabine, doxorubicin, etoposide, cisplatin, 5-fluorouracil, vinorelbine, irinotecan and cytarabine, have been tested in combination with IAP antagonists, and synergistic effects on various cancers, including pancreatic, lung, colon, prostate, breast and skin cancer as well as acute leukemia, have been detected, mostly in vitro that requires further validation.91, 93-95 IAP antagonists have also been reported to potentiate the effects of kinase-targeting agents. For instance, phosphatidyl inositol-3 kinase (PI3K) has been reported to potentially upregulate c-IAP2 by inhibition of caspase-3.96, 97 Inhibition of the PI3K pathway by a PI3K inhibitor, LY294002, could regulate c-IAP2 and sensitize resistant cancer cells to a SMAC mimetic and TNFα.98 Fms-like tyrosine kinase-3 (FLT3), which is mutated in approximately 30% of acute myelogenous leukemia (AML) patients, is a promising therapeutic target.99 The SMAC mimetics LCL161 and LBW242 enhance the proapoptotic effects of a FLT3 inhibitor, PKC412, against leukemia both in vitro and in vivo.100, 101 In addition, SMAC mimetics have been shown to ACS Paragon Plus Environment


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exhibit enhanced effects in combination with BCR-ABL inhibitors, such as nilotinib and imatinib, and epidermal growth factor receptor (EGFR) inhibitors, such as trastuzumab, lapatinib and gefitinib.92 TNF-related apoptosis-inducing ligand (TRAIL), a death receptor ligand, is used to treat various cancers. Debatin et al. first reported a proof-of-concept study to show that a SMAC peptide could strongly enhance the antitumor activity of TRAIL in an intracranial malignant glioma xenograft model.102 The bivalent SMAC mimetic (32, SM-164) exhibits a highly synergistic effect with TRAIL in both TRAIL-sensitive and TRAILresistant breast, prostate, and colon cancer cell lines. In a breast cancer (cell name: 2LMP) xenograft model, the agents are ineffective alone, but a combination of SM-164 with TRAIL induces rapid and consistent tumor regression.58 The use of SMAC mimetics in combination with radiation is also an effective strategy,103, 104 such as the treatment of pancreatic cancer,105 prostate cancer,106, 107 gliblastoma,108, 109 head and neck squamous cell carcinoma,110, 111 and breast cancer.112

6.4 Future directions and new applications of IAP antagonists. 6.4.1 Selective IAP inhibitors. As presented in Sections 3 and 4, most of the SMAC mimetics exhibit non-selective binding to XIAP, c-IAP1, and c-IAP2, with a few exceptions. Genentech reported the design of SMAC mimetics, such as compound 5, that selectively target c-IAP1 and c-IAP2 over XIAP.10 Compound 8 forms a covalent bond with a lysine residue of XIAP, exhibiting promising selectivity for XIAP over c-IAP1/2.37 The bicyclic (compound 9) and tricyclic (10) mimetics selectively bind to c-IAP1 BIR3 over XIAP BIR3.38, 39 Second-generation monovalent mimetics 47-53 exhibit selectivity for BIR2 over BIR3, disrupting the interaction with the terminal caspase-3 and caspase-7 and exhibiting different PD properties in mice.74 In addition, selective targeting of other family members, such as ML-IAP and survivin, has also demonstrated promising therapeutic potential.113,

114

Notably, most of the activity of the current IAP antagonists are likely attributed to their antagonism of cIAP1. ACS Paragon Plus Environment

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A study showed that monovalent and bivalent IAP antagonists activate the E3 ligase activity of cIAP1 by promoting RING domain dimerization, leading to caspase-8-mediated apoptosis.115 In another words, XIAP is not required. However, most IAP antagonists have been designed based on the XIAP-BIR3/SMAC interaction and the homology between XIAP and cIAP1 BIR3 domains. Therefore, what is the true role of cIAPs versus XIAP in the activity of pan-IAP or “selective” IAP antagonists? The development of truly selective, i.e., not BIR-directed, IAP antagonists will help address this issue. Given the sufficient structural differences of the IAP family, selective IAP antagonists can be developed. These selective agents at least could be applied as useful tool compounds or chemical probes to characterize the biological functions of individual IAP members and may be further evaluated as therapeutic agents if they happen to have clinical advantages.

6.4.2 Induction of protein degradation by incorporating IAP ligands into conjugates. The occupation of a binding site is a mainstream strategy for small-molecule drug discovery. Alternatively, the target protein can be tagged for elimination by protein degradation.116 Proteolysis-targeting chimeras (PROTACs) and specific and non-genetic IAP-dependent protein erasers (SNIPERs) are two protein degradation strategies.116-131 These strategies share a mechanism for degradation of the protein of interest (POI) using hybrid molecules or conjugates with of two different ligands and a linker (three examples are shown in Figure 15). These molecules contain one ligand for the POI and another for the ubiquitinproteasome system (UPS) to degrade the protein. Using this mechanism, several protein degradation inducers have been designed for several POIs,116,

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such as androgen receptor (AR),117 BCR-ABL

protein,118 and estrogen receptor α (ERα).131 For instance, compound 60, with a LCL161 moiety, is developed with effective proteasomal degradation of AR at 3 μM, and AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells were suppressed. Caspase activation and apoptosis are also observed, but not in cells treated with AR antagonists alone.117 BCR-ABL allosteric ligands are incorporated in the SNIPER using LCL161 as an IAP antagonist moiety to obtain compound 61, which exhibits potent BCRACS Paragon Plus Environment


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ABL degradation activity.118 Compound 62 is an example associated with ERα degradation and is synthesized by connecting the ERα ligand 4-hydroxy-tamoxifen with an analog of compound 4 by a PEG linker. This compound exhibits high binding affinity for IAPs and potently induces the degradation of ERα at concentrations lower than 3 nM with the simultaneous degradation of c-IAP1 and XIAP.131 Collectively, instead of traditional small-molecule-based strategies, emerging technologies based on IAP degradation, such as SNIPER technology, are attracting considerable attention in drug discovery. Highly potent IAP antagonists, monovalent and bivalent SMAC mimetics, might be used as tool compounds for assembly into conjugates for degradation as an alternative strategy for drug development.

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6.4.3 Beyond apoptosis: inflammation, cell migration, immunity, necroptosis and pyroptosis. In addition to apoptosis, XIAP and c-IAP1/2 proteins play critical roles in many other cellular processes.132 For instance, the activation of NF-κB is regulated by IAPs. The NF-κB pathway controls the expression of genes in regulating inflammation, cell migration and immunity as well as cell survival. c-IAP1 and c-IAP2 cause cancer cells resistant to apoptosis induced by TNFα, which is one key mediator of cancer-related inflammation.132, 133 IAPs are shown to interact with other substrates, leading to either a positive or negative influence on cell movement.134 XIAP can negatively regulate the activity of the Rho GDP dissociation inhibitor by modulating the SUMOylation of this protein to increase actin polymerization and cell motility.135 Ubiquitin-dependent proteasomal degradation of XIAP and c-IAP1 limits cell migration.136 IAP antagonists can also modulate the immune system.137 A recent work indicates that IAP antagonists induces antitumor activity by modulating innate and adaptive immunity via activation of the NF-κB pathway.19, 137-139 Mouse models exhibit synergism between IAP antagonists, such as LCL161, and anti-PD-1 immunotherapy.139 Another report show that IAP antagonists could enhance cytokine production in iNKT cells.140 These results suggest that IAP antagonists may also be useful as novel immunomodulation therapies for the treatment of human cancers.33 In addition to the above roles of IAPs, SMAC mimetics are also widely applied to promote necroptosis, another important form of PCD.141-144 A necroptosis model has been established by Yuan et al. A standard stimulus, such as TNF or FasL, is used to activate TNFR1 signaling resulting in both apoptosis and necroptosis. Then, IAPs are inhibited by a SMAC mimetic (such as SM-164, BV6, or Birinapant) to further promote TNFinduced cell death.144-146 Finally, a caspase inhibitor, usually Z-VAD-FMK, is used to block caspase-8 to inhibit the apoptotic pathway and stimuli the necroptotic signaling. SMAC mimetics here are used as tool compounds to consume/inhibit IAPs and facilitate the formation of a necrosome. Therefore, it is possible to use SMAC mimetics to treat apoptosis-resistant cancers by inducing necroptosis.146-154 IAP inhibition using SMAC mimetics or genetic deletion triggers cleavage of IL-1β mediated by the NLRP3ACS Paragon Plus Environment


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caspase-1 inflammasome and caspase-8 in a caspase-1-independent manner.155 A mouse model study shows that c-IAP1 is tightly linked to caspase-11.156, 157 IAPs, in theory, may be associated with pyroptosis, a lytic form of PCD induced by the inflammatory caspase-1 and caspase-11.158 Elucidation of the mechanism by which IAP family proteins are involved in pyroptosis requires further research.

6.4.4 Beyond cancer: hepatitis B virus (HBV) A recent study shows that antagonizing the function of c-IAPs might promote the clearance of HBV infection by preventing TNF-mediated killing/death of infected cells.159, 160 The SMAC mimetic birinapant (34) rapidly reduces serum HBV DNA levels and hepatitis B surface and core antigens in animal models.159 In addition, compound 59 (APG-1387) is in phase I trials in patients with chronic hepatitis B (NCT03585322). This is a new strategy for the treatment of HBV, and the effectiveness of this strategy in the clearance of HBV in patients requires further clinical research.

In summary, evading apoptosis and other type of programmed cell death is a key hallmark for cancer development. It also introduces a major barrier to cancer treatment. Antagonizing IAPs is a promising and logical strategy to restore the defected death pathway in cancer cells to improve the outcome of therapeutic treatment. To some extent, the science of IAPs, SMAC, and IAP antagonists, as well as how these proteins and compounds interact to mediate inflammation, apoptosis, and necroptosis, remains in its infancy. With additional progress in the preclinical and clinical stages of IAP antagonists and better understanding of the biological functions of IAPs, the clinical future is promising for IAP antagonists.

Author information Corresponding authors: Email addresses: [email protected] (C. Zhuang), [email protected] (C. Xing). ACS Paragon Plus Environment

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Author Contributions ¶These

authors contributed equally to this work.

Notes The authors declare that they have no competing interests.

Biographies Hui Cong received her bachelor’s degree from Bengbu Medical College, China (2016). She is now a graduate student at Ningxia Medical University under the supervision of Prof. Chengguo Xing. In 2018, she joined Second Military Medical University as a joint graduate student. Her research interests focus on drug discovery for the treatment of anti-multidrug resistant cancers. Lijuan Xu received her bachelor’s degree from Bengbu Medical University, China (2017). She worked as a technician at the School of Pharmacy, Second Military Medical University (2017-2018). She is now a graduate student at Ningxia Medical University. Her research interests focus on inhibitors of programmed cell death. Yougen Wu received his bachelor’s degree from Jiangxi Agricultural University (2000) and a Ph.D. in medicinal plant from Nanjing Agricultural University (2009). He worked in the College of Pharmacy, University of Hawaii at Hilo as a postdoctoral researcher (2012-2013, 2015). He was sponsored by the China Scholarship Council to work under the supervision of Prof. Chengguo Xing at the University of Florida (2018). Currently, he is a professor in the College of Tropical Agriculture and Forestry, Hainan University. His research interests focus on the isolation and identification of active ingredients from traditional Chinese medicine and their formation mechanisms. Zhuo Qu received her bachelor’s degree from Tianjin Medical University (2012) and a Ph.D. in applied chemistry from Tianjin University (2017). Currently, she is an associate professor at the School of Pharmacy, Ningxia Medical University. Her research interests focus on the neuropharmacology of traditional Chinese medicine, especially active ingredients and their mechanisms of action in the treatment of Alzheimer's ACS Paragon Plus Environment


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disease. Tengfei Bian received his bachelor’s degree from Harbin Engineering University, China (2008) and a Ph.D. in chemical engineering from Dalian University of Technology (2016). He was sponsored by the China Scholarship Council’s Ph.D. Abroad Training Plan to work under the supervision of Prof. Chengguo Xing at University of Minnesota (2013-2014). He also worked as a visiting scholar in Prof. Chengguo Xing’s lab at University of Minnesota (2016.1-2016.3). He is currently a postdoctoral associate in Prof. Chengguo Xing’s lab in the Department of Medicinal Chemistry, University of Florida. His research interests focus on the effect of carcinogens and co-carcinogens from cigarette smoking on the development of lung cancer. Wannian Zhang received his bachelor’s degree in pharmacy (1968) and M.S. in medicinal chemistry (1981) from Second Military Medical University. He has been a professor of Medicinal Chemistry since 1992 and served as the Dean of the School of Pharmacy from 1994 to 2001. Currently, Prof. Zhang is the Chief of the State’s Key Discipline of Medicinal Chemistry, Second Military Medical University. He has held a joint professorship with Ningxia Medical University and served as Dean of the School of Pharmacy since 2011. His research interests focus on antifungal and antitumor drug design and development. He has published more than 100 scientific articles and has developed four compounds that have been certificated by the CFDA. Chengguo Xing received his bachelor’s degree from Dalian University of Technology (1996) and his Ph.D. in organic chemistry from Arizona State University under the supervision of Prof. Edward Skibo (2001). After two years of post-doctoral training with Prof. Andrew G. Myers at Harvard University, he joined the University of Minnesota as a faculty member in 2003. Currently, he is a professor and the Frank A. Duckworth Eminent Scholar Chair in Drug Research and Development at the University of Florida. His research interests focus on the isolation, identification, design, and synthesis of biologically active small molecules, employing candidates such as probes to tackle health-related biological questions and evaluating the clinical potential of these compounds in relevant animal models. Chunlin Zhuang received his bachelor’s degree from Nanjing Medical University, China (2009) and a Ph.D. in ACS Paragon Plus Environment

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medicinal chemistry from Second Military Medical University (2014). He was sponsored by the China Scholarship Council’s Ph.D. Abroad Training Plan to work under the supervision of Prof. Chengguo Xing at the University of Minnesota (2012). He is currently a faculty member at the School of Pharmacy, Second Military Medical University, China. He has held a joint professorship with Ningxia Medical University. His research interests focus on structure-based drug design of inhibitors of protein-protein interactions.

Acknowledgments We thank Dr. Gunda I. Georg and Dr. Nicholas Meanwell for their kind invitation to write this Perspective. This work was funded by grants from the National Natural Science Foundation of China (81872791 and 81502978 to C.Z.); the Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (2017QNRC061 to C.Z.); the Shanghai Municipal Commission of Health and Family Planning (2017YQ052 to C.Z.); the Key Research and Development Program of Ningxia (2018BFH02001 to W. Z. and 2018BFH02001-01 to C.Z.); the Shanghai ‘‘ChenGuang’’ Project (16CG42 to C.Z.); the National Cancer Institute, National Institutes of Health, USA (R01CA193278 to C.X.), the College of Pharmacy, University of Florida (C.X.); the University of Florida Health Cancer Center (C.X.); and the Harry T. Mangurian, Jr. Foundation (C.X.). Abbreviations used AML, acute myelogenous leukemia; AUC, area under the curve; BIR, baculoviral IAP repeat; BRUCE, ubiquitinconjugating BIR domain enzyme; CARD, caspase recruitment domain; c-IAP1, cellular IAP1; c-IAP2, cellular IAP2; DELFIA, dissociation-enhanced lanthanide fluorescent immunoassay; DIABLO, direct IAP-binding protein with low pI; EGFR, epidermal growth factor receptor; ERα, estrogen receptor α; FBDD, fragmentbased drug discovery; FLT3, Fms-like tyrosine kinase-3; HBV, hepatitis B virus; IAP, inhibitor of apoptosis protein; IBM, IAP-binding motif; ILP2, IAP-like protein 2; LE, ligand efficiency; MDR1, multidrug resistance 1; ML-IAP, melanoma IAP; MM, multiple myeloma; MTD, maximum tolerated dose; NIAP, neuronal IAP; NIK, ACS Paragon Plus Environment


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NF-κB-inducing kinase; NSCLC, non-small cell lung cancer; PCD, programmed cell death; pCR, pathologic complete response; PD, pharmacodynamic; PI3K, phosphatidyl inositol-3 kinase; PK, pharmacokinetic; POI, protein of interest; PPI, protein-protein interaction; PROTAC, proteolysis-targeting chimera; RIP1, receptorinteracting protein 1; RP2D, recommended phase II dose; SAR, structure-activity relationship; SBDD, structure-based drug design; SMAC, second mitochondria-derived activator of caspase; SNIPER, specific and nongenetic IAP-dependent protein eraser; TDI, time-dependent inhibitor; TGI, tumor growth inhibition; TNFR1, TNF factor receptor 1; TRAF1, tumor necrosis factor receptor-associated factors 1; TRAF2, tumor necrosis factor receptor-associated factors 2; TRAIL, TNF-related apoptosis-inducing ligand; UBA, ubiquitinassociated; UPS, ubiquitin-proteasome system; XIAP, X chromosome-linked IAP.

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153.Jin, G.; Lan, Y.; Han, F.; Sun, Y.; Liu, Z.; Zhang, M.; Liu, X.; Zhang, X.; Hu, J.; Liu, H.; Wang, B. SMAC mimetic-induced caspase-independent necroptosis requires RIP1 in breast cancer. Mol Med Rep 2016, 13, 359-366. 154.Chromik, J.; Safferthal, C.; Serve, H.; Fulda, S. SMAC mimetic primes apoptosis-resistant acute myeloid leukaemia cells for cytarabine-induced cell death by triggering necroptosis. Cancer Lett 2014, 344, 101109. 155.Vince, J. E.; Wong, W. W.; Gentle, I.; Lawlor, K. E.; Allam, R.; O'Reilly, L.; Mason, K.; Gross, O.; Ma, S.; Guarda, G.; Anderton, H.; Castillo, R.; Hacker, G.; Silke, J.; Tschopp, J. Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation. Immunity 2012, 36, 215-227. 156.Kenneth, N. S.; Younger, J. M.; Hughes, E. D.; Marcotte, D.; Barker, P. A.; Saunders, T. L.; Duckett, C. S. An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1. Biochem J 2012, 443, 355-359. 157.Rickert, R. C.; Salvesen, G. S.; Ware, C. F. Mousing around with caspases and IAPs. Biochem J 2012, 443, e1-2. 158.Ma, Y.; Jiang, J.; Gao, Y.; Shi, T.; Zhu, X.; Zhang, K.; Lu, K.; Xue, B. Research progress of the relationship between pyroptosis and disease. Am J Transl Res 2018, 10, 2213-2219. 159.Ebert, G.; Allison, C.; Preston, S.; Cooney, J.; Toe, J. G.; Stutz, M. D.; Ojaimi, S.; Baschuk, N.; Nachbur, U.; Torresi, J.; Silke, J.; Begley, C. G.; Pellegrini, M. Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis. Proc Natl Acad Sci U S A 2015, 112, 5803-5808. 160.Ebert, G.; Preston, S.; Allison, C.; Cooney, J.; Toe, J. G.; Stutz, M. D.; Ojaimi, S.; Scott, H. W.; Baschuk, N.; Nachbur, U.; Torresi, J.; Chin, R.; Colledge, D.; Li, X.; Warner, N.; Revill, P.; Bowden, S.; Silke, J.; Begley, C. G.; Pellegrini, M. Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus. Proc Natl Acad Sci U S A 2015, 112, 5797-5802.


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Figure Captions Figure 1. Functional domains of mammalian IAPs: typical IAP structure and the structures of XIAP, c-IAP1 and c-IAP2. Figure 2. Regulation of caspase-dependent apoptosis and NF-κB signaling by IAP and SMAC proteins. Figure 3. Crystal complex (PDB: 1G73) and detailed interactions of SMAC, XIAP-BIR3 (presented as a surface model and the carbons in gray), and the AVPI peptide (carbons in yellow, nitrogens in blue, and oxygens in red). Blue dashed lines represent hydrogen bonds. The figures were generated by PyMOL. Figure 4. Systematic summary for the different regions of the first-generation SMAC-based AVPI peptides and peptidomimetics. *: Structures with stars contain non-natural substituents. Chemical structures of representative potent and cell-permeable SMAC peptidomimetics as IAP antagonists. Substituents in different regions are colored as follows: alanine (blue), valine (pink), proline (black), and isoleucine (red). #: Compound has been advanced into clinical evaluations. Figure 5. Structure optimization workflow for the bicyclic monovalent IAP antagonists developed by Dr. Shaomeng Wang and colleagues. The bicyclic parts are colored in orange, and the side chains are colored in black. #: Compound has been advanced into clinical evaluations. The crystal structure of the complex is generated by PyMOL. Figure 6. Other representative bicyclic monovalent IAP antagonists obtained using different cyclization strategies. Figure 7. (A) First reported bivalent SMAC mimetic. The corresponding monovalent mimetic is highlighted in blue. (B) Common design strategies of the bivalent IAP antagonists, highlighted in different colors. Ala = P1, Val = P2, Pro = P3, Ile = P4. Linker 1: P4-P4 strategy; Linker 2: P2-P2 strategy; Linker 3: P3-P3 strategy. Figure 8. Bivalent antagonists linked using the P4-P4 strategy, highlighted in red. #: Compound has been advanced into clinical evaluations. Figure 9. Bivalent antagonists linked using the P2-P2 strategy, highlighted in pink. ACS Paragon Plus Environment


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Figure 10. Bivalent antagonists linked using the P3-P3 strategy, highlighted in orange. Figure 11. Structure optimization workflow of nonalanine IAP antagonists (A) and their cocrystal complexes with the XIAP BIR3 protein: (B) Fragment 43 (PDB: 5C7B); (C) AT-IAP (PDB: 5M6L); (D) ASTX660 (PDB: 5OQW). The red dots represent water molecules in the crystal complex. Figure 12. Structure optimization workflow for benzoxazepinone and benzodiazepinone synthesis (A) and the cocrystal complexes of these compounds with the XIAP BIR2 protein: (B) compound 50 (PDB: 4KJV); and (C) compound 52 (PDB: 4KJU). Figure 13. Macrocyclic molecules as IAP antagonists. Figure 14. IAP antagonists with disclosed structures that have been advanced into clinical trials. Figure 15. Representative conjugates with an IAP antagonist moiety for target protein degradation.


Inhibitor of Apoptosis Protein (IAP) Antagonists in Anticancer Agent

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