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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Antagonists of IAP-family anti-apoptotic proteins - Probe 2

, , , , , , , , , , , , , , , , and .

Received: ; Last Update: February 10, 2011.

Various Inhibitors of Apoptosis Proteins (IAPs) are over-expressed in specific types of cancer, and some tumors over-express more than one member of this family of proteins simultaneously. Given that multiple IAPs are simultaneously over-expressed in many cancers, unfortunately antisense or RNA interference (RNAi) methods for assessing function of these proteins are presently often lacking. Therefore, probing the competition of small molecule compounds against either the BIR2 or BIR3 SMAC-binding sites on IAPs, such as XIAP, cIAP1, cIAP2 or other IAPs from human and other organisms will serve as useful tools for elucidating their roles in cancer biology. In this study, we investigated small molecule compounds that selectively mimic the effects of SMAC (second mitochondria-derived activator of caspase) in antagonizing IAPs by causing them to release Caspases. These non-peptidyl chemical inhibitors would have advantages over SMAC peptides, in terms of their greater cell permeability, stability, and in vivo pharmacology. As such, the probe identified in this report ML183 (CID 44176340) met the project’s goal of displaying potent binding affinity and selectivity for the BIR2 domain of XIAP. Moreover, in that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a potent inducer of apoptosis in a wide variety of tumor cells, another valuable finding of this project is that it also promotes cell survival against a challenge by rhTRAIL, an inducer of cell death.

Assigned Assay Grant #: R03 MH081277-01

Screening Center Name & PI: Sanford-Burnham Center for Chemical Genomics & Dr John C Reed

Chemistry Center Name & PI: Sanford-Burnham Center for Chemical Genomics & Dr John C Reed

Assay Submitter & Institution: Dr John C Reed & Burnham Institute for Medical Research

PubChem Summary Bioassay Identifier (AID): AID 1638

Probe Structure & Characteristics

(This report describes a 2nd probe for XIAP elucidating another structurally distinct chemical scaffold with improved potency and selectivity for the BIR1/2 domain over the BIR3 domain of XIAP compared to the 1st probe CID 25241665, submitted in May of 2009)

Image ml183fu1
CIDTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 44176340/ML183 (Formic acid salt)BIR1/2
XIAP		IC50 2200 (nM)
SID 85164169/AID 1722		BIR3IC50 108.6 (µM)
SID 85164169/AID 1750		49.4XN/A
CID 46916243
ML183 (TFA salt)		BIR1/2
XIAP		IC50 4617 (nM)
SID 99380920
AID 488957		BIR3IC50 137.2 (µM)
SID 899380920
AID 488963		29.7XN/A

Recommendations for the scientific use of this probe

Various IAP-family proteins are over-expressed in specific types of cancer and some tumors over-express more than one member of the family simultaneously. Given that multiple IAPs are simultaneously over-expressed in many cancers, antisense or RNA interference (RNAi) methods for assessing function of these proteins are often lacking. Therefore, probing the competition of small molecule compounds against either the BIR2 or BIR3 SMAC-binding sites on Inhibitor of Apoptosis Protein (IAP) such as XIAP, cIAP1, cIAP2 or other IAPs from human and other organisms will serve as useful tools for elucidating their roles in cancer biology.

1. Scientific Rationale for Project

Specific aims

The goal of this project was to generate small molecule compounds that mimic the effects of SMAC peptides, inhibiting the function of Inhibitor of Apoptosis Proteins (IAPs).

Background and Significance

Apoptosis, or programmed cell death, is a critical cellular process in normal development and homeostasis of multicellular organisms (1). Caspases are the executioners in both intrinsic and extrinsic pathways of apoptosis by cleaving a plethora of cellular components (2). These intracellular proteases are suppressed by Inhibitor of Apoptosis Proteins (IAPs), a family of evolutionarily conserved anti-apoptotic proteins (3). The X-linked inhibitor of apoptosis, XIAP, is a key member of the family of intrinsic inhibitors of apoptosis proteins (IAP), which block cell death both in vitro and in vivo by virtue of inhibition of two effector caspases (caspase-3 and -7) and an initiator caspase (caspase-9). XIAP contains three baculoviral IAP repeat (BIR) domains, defined by a novel ~80 amino acid motif. The third domain (BIR3) of XIAP selectively binds to the amino terminus of the caspase-9 linker peptide while the BIR2 domain inhibits both caspase-3 and caspase-7. This inhibiting activity of XIAP is negatively regulated by at least two XIAP interacting proteins, XAF1 and SMAC/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low pl). As a dimer, SMAC targets both the BIR2 and BIR3 domains of XIAP. Proteins released from mitochondria (SMAC and HtrA2) (4–6) can competitively displace IAPs from the Caspases, thus helping to drive apoptosis. It has been shown that only a few residues at the N-terminus of activated SMAC protein (4′mer) are sufficient to affect the release of IAPs from Caspases (7).

In this study we investigated small molecule compounds that mimicked the effect of SMAC in antagonizing IAPs by causing them to release Caspases. Non-peptidyl chemical inhibitors would have advantages over SMAC peptides, in terms of cell permeability, stability, and in vivo pharmacology. Several strategies were employed for the design of small-molecular inhibitors of XIAP, including the structure-based rational design and synthesis of small molecules that mimic the binding interactions between this protein and SMAC.

2. Project Description

a. The original goal for probe characteristics

All current probes are polypeptides derived from natural IAP inhibitors (e.g. SMAC or HtrA2), with the 4-mer being the smallest that can displace IAPs from caspases. This project looked for non-peptidyl small molecule chemical inhibitors that would have advantages over SMAC peptides, in terms of cell permeability, stability, in vivo pharmacology and ideally having potency of 1 μM or better. In particular, the goal was to identify compounds that inhibit the BIR2 domain of XIAP, either selectively or in addition to inhibiting the BIR3 domain.

b. Assay implementation and screening

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

Table 1PubChem Assay Summaries for Probe project

PubChemBioAssay NameAIDsProbe TypeAssay TypeAssay FormatAssay Detection & well format
Chemical Antagonists IAP-family anti-apoptotic proteins—primary screen and HTS/DPI liquid hit confirms1018InhibitorPrimaryBiochemicalFluorescence Polarization (384)
XIAP-Bir3 for Chemical Antagonists of Bir1/2 domains of IAP-family anti-apoptotic proteins—liquid hit confirmation from DPI1513InhibitorCounter screenBiochemicalFluorescence Polarization (384)
SAR analysis of Antagonists of IAP-family anti-apoptotic proteins –Dry powder [Confirmatory]1749InhibitorCounter screenBiochemicalFluorescence Polarization (96)
SAR analysis of Antagonists of XIAP-Bir3 domain of IAP-family anti-apoptotic-dry powder.1750InhibitorCounter screenBiochemicalFluorescence Polarization (96)

ii. Assay Rationale & Description

This XIAP dose response assay was developed and performed to confirm hits originally identified in the XIAP HTS binding assay (AID 1018) and to study the structure-activity relationship of analogues of the confirmed hits. Compounds were acquired from commercial sources or synthesized in-house.

The rationale for the assay was based on the disruption of fluorescence polarization resulting from binding of a his-tagged-BIR1-BIR2 (baculoviral IAP repeat, “Bir1/2”) domain protein derived from two of the three conserved caspase binding “BIR” domains of XIAP to a rhodamine tagged 7-mer N-terminal SMAC peptide.

Assay materials

Table 2Reagents used for the uHTS experiments

ReagentsVendor
Expression plasmid for BIR12 of XIAP clonedDr. Reed’s laboratory
BIR1/2 proteinPurified in Dr. Reed’s laboratory
1 M Hepes bufferQmega Scientific, Inc
TCEPSigma
0.5 M Tween 20BIO-RAD
SMAC-rhodamineSynthesized in Dr. Houghten’s laboratory at The Torrey Pines Institute for Molecular Studies (TPIMS)
SMAC-7merSynthesized in Dr. Houghten’s laboratory at The Torrey Pines Institute for Molecular Studies (TPIMS)
Bir1/2 assay materials
  1. Bir1/2 protein and rhodamine-SMAC peptide (AVPIAQK-rhodamine) were provided by Prof. John Reed (Burnham Institute for Medical Research, San Diego, CA)
  2. Assay buffer: 31.25 mM HEPES-NaOH, pH 7.5, 1.25 mM TCEP, 0.00625% Tween 20.
  3. Bir1/2 working solution contained 2.5 μM Bir1/2 in the assay buffer.
  4. Rhodamine-SMAC working solution contained 50 nM FITC-Tr3-R8 peptide in the assay buffer.
Bir1/2 protocol
  1. 4 µL of serially diluted compounds in DMSO were dispensed in columns 3–22 of Greiner 384-well black small-volume plates (784076). Compounds were serially diluted in DMSO to have duplicate 10-point curves with 2-fold dilution between concentrations. Columns 1–2 and 23–24 were added with 4 µL of 10% DMSO.
  2. Positive control wells, that contained no Bir1/2, were assigned to columns 1–2 and were added 8 µL of assay buffer using WellMate bulk dispenser (Matrix).
  3. 8 µL of Bir1/2 working solution was added to columns 3–24 using WellMate bulk dispenser (Matrix). Negative control wells that contained DMSO were assigned to columns 23–24.
  4. Plates were briefly spun down and incubated for 1h at room temperature.
  5. 8 µL of Rhodamine-SMAC working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
  6. Final concentrations of the components in the assay were as follows:
    1. 25 mM HEPES-NaOH, pH 7.5, 1 mM TCEP, 0.005% Tween 20.
    2. 20 nM Rhodamine-SMAC (columns 1–24)
    3. 1 μM Bir1/2 (columns 2–24)
    4. 2 % DMSO (columns 1–24
    5. 20 μM compounds (columns 3–24)
  7. Plates were incubated for 30 min at room temperature protected from direct light.
  8. Fluorescence polarization was measured on an EnVision plate reader (Perkin Elmer) using rhodamine filters: excitation filter - 540 nm, emission filter 590 nm, dichroic mirror - 560 nm. The signal for each well was acquired for 100 ms.
  9. Data analysis was performed using CBIS software (ChemInnovations, Inc).
  10. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

195,576 compounds were tested in this assay. The average Z′ for this assay was 0.85, the signal to background ratio was 3.8, the signal to noise ratio was 46.6 and the signal window was 34.7.

Rationale for confirmatory, counter and selectivity assays

SMAC is synthesized and enters the inner membrane space of mitochondria (4,5). The N-terminal of SMAC is cleaved off generating a new N-terminal AVPI sequence (4,5). On release of the activated SMAC from the mitochondria, it binds to a SMAC binding pocket on BIR2 and BIR3 competing off Caspase-3/7 and Caspase-9, respectively, and thus driving the activation of the executioner and initiator caspases by releasing the caspase from the BIR2 or BIR3 SMAC binding domains (7,8). It is established that the binding site of BIR3 for SMAC requires only the first 4 amino acids of activated SMAC (7). The first 7 amino acids of activated SMAC are AVPIAQK (7). We utilized the ɛ-amino group of lysine to couple the rhodamine to the peptide at the C-terminal. This spaced the rhodamine away from the SMAC binding pocket allowing the AVPI portion of the fluorescent probe to bind without hinderance to the BIR2 pocket yet still having the rhodamine probe tumbling with the rate of the protein.

We chose to target Caspase-3 and -7 and looked at the competition of compounds against SMAC-rhodamine in the presence of BIR1/2 where only the BIR2 domain was capable of binding SMAC-rhodamine. Development of the fluorescence polarization assay used the SMAC-7mer as a competitor for the SMAC-rhodamine (10). A similar assay was developed for BIR3 and was utilized as a counter screen.

iii. Summary of Results

We developed a binding assay based upon fluorescence polarization, using a short peptide representing residues from the N-terminus of activated SMAC with an attached fluorochrome. This fluorescence polarization assay (FPA) formed the basis for a high-throughput competitive displacement assay that was optimized for screening. We screened the NIH compound library using this FPA to identify chemical compounds that compete with the SMAC peptide for binding to IAPs. A secondary assay was also devised, so the hits would be independently confirmed. However, the single hit from the conventional HTS screen was very weak (> 25 μM IC50), we therefore decided to take a structure-based rational design approach to develop novel chemical probes of XIAP. Chemistry has made additional significant progress towards this goal and the results are described in section 2b-3 below.

c. Probe Optimization

i. SAR & chemistry strategy (including structure and data) that led to the probe

Structure based approaches to synthesis of BIR3 XIAP inhibitors are described in the literature. Novartis (ref 1) and Genetech (ref 2) have compounds that are advancing through clinical trials. There are no reports of compounds designed to inhibit the BIR2 domain of XIAP selectively over the BIR3 domain. Selective inhibition of BIR2 over BIR3 could yield valuable information about the XIAP signaling pathway. In addition, selective inhibition of BIR2 vs. BIR3 could lead to clinical candidates that have significantly different properties compared to the BIR3 selective Norvatis and Genetech clinical candidates. Our initial research efforts on the design, synthesis and evaluation of novel BIR2 selective Smac mimetics as chemical antagonists of the anti-apoptotic protein XIAP were summarized in our previous probe report for CID 25241665. Compound CID 25241665 (MLS-0391005) was selected as a Probe at that time due to its relatively good selectivity (8-fold) for BIR2 over the BIR3 binding domain of XIAP. These initial findings are the first report in the literature that selectivity of the BIR2 domain over the BIR3 domain is achievable. In the previous report, we provided a model of the key differences between the BIR2 and BIR3 protein structures at the SMAC binding site that provided a rational basis for designing BIR2 selective compounds by substituent variation of the key positions noted below in Figure 1. Comparative docking into the BIR2 and BIR3 domains rationalized the increased selectivity of this probe. Modeling studies also suggested the proline motif in Figure 1 could be amenable for specificity design. As an extension of these studies, we undertook the synthesis of a second library composed of 80 new Smac mimetics which lack the conformational restriction of the prolyl group, yet probes the interaction at the differential binding pocket of BIR2 vs. BIR3 at position 3 (Figure 2). As a consequence of significantly improved potency and BIR2 selectivity of compounds from this second library, we are now able to nominate a second highly selective Probe, CID 44176346 (MLS-0412114).

Figure 1. Key SAR for our first library.

Figure 1

Key SAR for our first library.

Figure 2. Key SAR for our second library.

Figure 2

Key SAR for our second library.

Figure 1 summarizes the evolution of the SAR studies for the synthesis of our first library of Smac mimetics. In addition to the chemical modifications at positions P1, P2, N-terminus and C-terminus, we decided to extend our SAR studies to the P3 position of the parent tripeptide (3-mer) (Figure 2).

Specific analogues were synthesized and the resulting new candidates were evaluated for binding to the BIR2 and BIR3 domains of XIAP using fluorescence polarization (FP) assays. The IC50 values obtained are summarized in Table 3. Compound CID 44176336 (MLS-0391057), which contains an indole group at the C-terminus (R4) showed improved binding affinity (IC50 = 2.0 μM) and 7-fold selectivity for BIR2. Substitution of the proline group at P3 for a valine residue [e.g. CID 44176342, CID 44176338, and CID 44176344 (MLS-0412059, MLS-0391058 and MLS-0412062, respectively)] showed improved selectivity for the BIR2 domain. We found that hydrazine [e.g. CID 44176338, CID 44176344, and CID 44176334 (MLS-0391058, MLS-0412062 and MLS-0391056, respectively)] and alpha-naphthyl [e.g. CID 44176342 (MLS-0412059), CID 44176340 (MLS-0412053)] C-terminus capping groups were optimal for BIR2 potency and selectivity. Incorporation of an alanine residue at P3 [e.g. CID 44176334 (MLS-0391056), CID 44176340 (MLS-0412053)] also resulted in similar or improved selectivity for the BIR2 domain, while maintaining the potency observed with the aforementioned candidates. We also studied the effect of substitution at the N-terminus on potency and selectivity. Compound CID 44176346 (MLS-0412114), which lacks a substituent at the N-termimus, showed extraordinary binding affinity (IC50= 1.8 μM) and selectivity (57-fold) for the BIR2 domain. However in our cellular based assay this compound is considerably less active than MLS-0412053. MLS-0412053 was selected on the basis of its potency, greatly improved selectivity (49.4 fold) for the BIR2 domain and cellular based activity as our new XIAP Probe. MLS-0412053 (15.0 mg) was submitted to the NCI 60 in vitro screen on September 21, 2010.

Table 3. Binding affinity to the XIAP-BIR1/2 and BIR3 domains for representative examples.

Table 3

Binding affinity to the XIAP-BIR1/2 and BIR3 domains for representative examples.

Figure 3. A docking model of new probe molecule CID 44176340, that provides a rational explanation of the improved selectivity of this new probe.

Figure 3A docking model of new probe molecule CID 44176340, that provides a rational explanation of the improved selectivity of this new probe

The Glide program (Schrodinger LLC) was used for flexible protein-ligand docking of the probe molecule. The Bir2 and Bir3 protein structures are rendered as molecular surfaces colored according to cavity depth (red – deep, blue – shallow)

The modeling results suggest that the probe compound CID 44176340 (MLS-44176340) adopts two different binding modes in BIR2 and BIR3 due to the differences of the two binding sites. The rigid naphthalene group of the compound cannot fit as well as the Ile side chain of the AVPI peptide into the right sub-pocket in BIR3. The replacement of the proline with alanine also eliminates the favorable van der Waals interaction between the proline of AVPI and the protein residue Trp323 in BIR3. Taken together, these two structure-based modifications make the compound selective towards BIR2.

In summary, the SAR studies undertaken around our standard tripeptide (Figure 2) have established that the presence of an N-methylalanine residue at P3 is a key structural element to improve the BIR1/2 selectivity and cellular based activity of the resulting Smac mimetics.

3. Probe

a. Chemical name of probe compound

Formic Acid; (2S)-3-methyl-2-[[(2S)-2-(methylamino)propanoyl]amino]-N-[(2S)-1-(naphthalen-1-ylamino)-1-oxopropan-2-yl]butanamide [ML183]

b. Probe chemical structure

Image ml183fu9

c. Structural verification information of probe SID

Probe SID number is SID 85164169

Purity: >95% (HPLC-MS)

Image ml183fu10
Image ml183fu11

d. PubChem CID (corresponding to the SID)

PubChem CID is 44176340 (corresponding to SID 85164169)

e. Availability from a vendor

This probe is not commercially available from vendors

f. MLS# of probe molecule and related samples that were submitted to the SMR collection

Table 5Submission of Probe and analogs

Probe/analogDPI ML#MLS-# (BCCG#)CIDSIDSource (vendor or BCCG syn)Amt (mg)Date ordered/submitted
Probe [ML183]MLS00247715604120534417634085164169BCCG syn19.209/01/09
Analog1MLS00247715403910574417633685164167BCCG syn21.009/01/09
Analog2MLS00247715504120594417634285164170BCCG syn18.609/01/09
Analog3MLS00247715703910584417633885164168BCCG syn19.309/01/09
Analog4MLS00247715904120624417634485164171BCCG syn21.309/01/09
Analog5MLS00247715803910564417633485164166BCCG syn20.809/01/09
Analog6MLS00247715304121144417634685164172BCCG syn22.609/01/09

g. Mode of action for biological activity of probe

The reported probe mimics the binding of a single AVPI binding motif to the BIR1/2 domain of XIAP.

Additionally the probe was found to promote cell survival against a challenge by rhTRAIL, an inducer of cell death (see two graphs below). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in a wide variety of tumor cells, while it has no toxicity for the majority of normal cells. MDA-MB-231 breast carcinoma cells (Fig. 4a: CC50>100 ng/mL) or HeLa cervical cancer cells (Fig. 4b: CC50 >100 ng/mL) or pre-treated with 5 μM of the probe CID 44176340 (MLS-0412053) for 4 hours showed a pronounced loss of cell viability upon 20 hour treatment with increasing concentrations of rhTRAIL for MDA-MB-231 (Fig. 4a: CC50 ~ 1 ng/ML) and HeLa (Fig. 4b: CC50 ~ 2 ng/mL)cells, a sensitization of these cells to TRAIL of > 100 and >50-fold, respectively. The same cells treated with vehicle before TRAIL as above were mostly refractory to the observed apoptosis induction showing only partial loss of viability, only at the highest concentrations of TRAIL tested. Indeed the use of relatively non-toxic agents as TRAIL sensitizers is envisaged to allow less toxic doses of TRAIL to be used in a clinical setting.

Figure 4. TRAIL Sensitization Studies of Cancer Cells Pre-Treated with Probe CID 44176340 (MLS-0412053) [ML183].

Figure 4

TRAIL Sensitization Studies of Cancer Cells Pre-Treated with Probe CID 44176340 (MLS-0412053) [ML183]. MDA-MB-231 breast carcinoma (A) or HeLa cervical cancer (B) cells were pre-treated with vehicle (blue curve) or probe ML183 (magenta curve) at 5 µM (more...)

h. Detailed synthetic pathway for making probe

Scheme 1. Synthesis of probe CID 44176340.

Scheme 1Synthesis of probe CID 44176340

i. Probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

Our probe has demonstrated potent binding affinity and selectivity for BIR2 domain of XIAP, as assessed by a BIR1/2 interaction assay and modeling studies.

In Vitro Pharmacology Profiles of Probe CID 44176340 (See Table 8 below). The probe molecule, CID 44176340 was evaluated in a detailed in vitro pharmacology screen and has excellent solubility at all pH’s tested.

Table 8. Summary of in vitro ADMET/PK Properties of West Nile Virus Inhibitor Probes.

Table 8

Summary of in vitro ADMET/PK Properties of West Nile Virus Inhibitor Probes.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. In this assay CID 44176340 has fair to excellent permeability at various pHs.

Plasma Protein Binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. CID 44176340 is highly bound (91–95 %) in human plasma and in mouse plasma it shows good binding (66 – 75 %).

Plasma Stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. CID 44176340 shows very good stability (64 – 71 %) in both human and mouse plasma.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. CID 4417634 shows excellent hepatic microsome stability (84 – 91 %) in both human and mouse liver homogenates.

CID 44176340 shows low levels of toxicity toward human hepatocyctes in our assay.

j. Properties Computed from Structure

Table 7Properties computed from Structure

CID 44176340 [ML183] (MLS-0412053)
Molecular Weight [g/mol]444.52398
Molecular formulaC23H32N4O5
H-Bond Donor5
H-Bond Acceptor6
Rotatable Bond Count8
Tautomer Count8
Exact Mass444.23727
MonoIsotopic Mass444.23727
Topological Polar Surface Area137
Heavy Atom Count32
Formal Charge0
Complexity591
Isotope Atom Count0
Defined Atom StereoCenter Count3
Undefined Atom StereoCenter Count0
Defined Bond StereoCenter Count0
Undefined Bond StereoCenter Count0
Covalently-Bonded Unit Count2

4. Appendices

a. Comparative data on (1) probe, (2) similar compound structures (establishing SAR) and (3) prior probes

Bir1/2 vs Bir3 potency and selectivity data for 153 additional compounds are shown in Table A1.

Table A1. Bir1/2 vs Bir3 potency and selectivity data for 153 additional compounds.

Table A1

Bir1/2 vs Bir3 potency and selectivity data for 153 additional compounds.

b. Comparative data showing probe specificity for target

A similar probe has been described in the literature (9,10,11). In this case, a fluorescein molecule is coupled to the SMAC peptide. In this study rhodamine was preferred since it was further red shifted than fluorescein allowing for less sensitivity in the assay to compounds, which are auto-fluorescent.

5. Bibliography

1.
Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science. 1995;267:1456. [PubMed: 7878464]
2.
Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nature Reviews. Molecular Cell Biology. 2004;5:897. [PubMed: 15520809]
3.
Deveraux QL, Reed JC. IAP family proteins: Suppressors of apoptosis. Genes Dev. 1999;13:239. [PubMed: 9990849]
4.
Du C, Fang M, Li Y, Li L, Wang X. SMAC, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell. 2000;102:33. [PubMed: 10929711]
5.
Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL. Identification of DIABLO, a Mammalian Protein that Promotes Apoptosis by Binding to and Antagonizing IAP Proteins. Cell. 2000;102:43. [PubMed: 10929712]
6.
Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Molecular Cell. 2001;8:613. [PubMed: 11583623]
7.
Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Hermann J, Wu JC, Fesik SW. Structural basis for binding of SMAC/DIABLO to the BIR3 domain. Nature. 2000;408:1004. [PubMed: 11140637]
8.
Huang Y, Rich RL, Myszka DG, Wu H. Requirement of both the second and third domains for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by SMAC. J. Biol. Chem. 2003;278:49517. [PubMed: 14512414]
9.
Glover CJ, Hite K, DeLosh R, Scudiero DA, Fivash MJ, Smith LR, Fisher RJ, Wu J-W, Shi Y, Kipp RA, McLendon GL, Sausville EA, Shoemaker RH. A high-throughput screen for identification of molecular mimics of SMAC-DIABLO utilizing a fluorescence polarization assay. Anal. Biochem. 2003;320:157. [PubMed: 12927820]
10.
Nikolovska-Coleska Z, Wang R, Fang X, Pan H, Tomita Y, Li P, Roller PP, Krajewski K, Saito NG, Stuckey JA, Wang S. Development and optimization of a binding assay for XIAP BIR3 domain using fluorescence polarization. Anal. Biochem. 2004;332:261. [PubMed: 15325294]
11.
Oost TK, Sun C, Armstrong RC, Al-Assaad A-S, Betz SF, Deckwerth TL, Ding H, Elmore SW, Meadows RP, Olejniczak ET, Oleksijew A, Olterdorf T, Rosenberg SH, Shoemaker AR, Tomaselli KJ, Zou H, Fesik SW. Discovery of potent antagonists of the antiapoptotic protein XIAP for the treatment of cancer. J. Med. Chem. 2004;47:4417. [PubMed: 15317454]

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