References for Summary

1.

Roberts AW, Kim C, Zhen L, et al. Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity. 1999;10:183–196. [PubMed: 10072071]

2.

Kim C, Williams DA, Dinauer MC. Rac2 is an essential regulator of specific signaling pathways which activate neutrophil NADPH oxidase. Blood. 1999;94:615a.

3.

Ambruso DR, Knall C, Abell AN, et al. Human neutrophil immunodeficiecy syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci U S A. 2000;97:4654–4659. [PMC free article: PMC18288] [PubMed: 10758162]

4.

Chrostek A, Wu X, Quondamatteo F, Hu R, Sanecka A, Niemann C, Langbein L, Haase I, Brakebusch C. Rac1 Is Crucial for Hair Follicle Integrity but Is Not Essential for Maintenance of the Epidermis. Mol Cell Biol . 2006 September;26:6957–6970. [PMC free article: PMC1592871] [PubMed: 16943436]

5.

Hirano K, Ikegami C, Zhang Z. Contribution of Cdc42 to cholesterol efflux in fibroblasts from Tangier disease and Werner syndrome. Methods Enzymol. 2008;439:159–69. [PubMed: 18374163]

6.

Heasman SJ, Ridley AJ. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol. 2008 Sep;9(9):690–701. [PubMed: 18719708]

7.

Spinosa MR, Progida C, De Luca A, Colucci AM, Alifano P, Bucci C. J Neurosci. 2008 Feb 13;28(7):1640–8. Functional characterization of Rab7 mutant proteins associated with Charcot-Marie-Tooth type 2B disease. [PMC free article: PMC6671532] [PubMed: 18272684]

8.

Williams DA, Tao W, Yang F, Kim C, Gu Y, Mansfield P, Levine JE, Petryniak B, Derrow CW, Harris C, Jia B, Zheng Y, Ambruso DR, Lowe JB, Atkinson SJ, Dinauer MC, Boxer L. Blood. 2000 Sep 1;96(5):1646–54. [PubMed: 10961859]

9.

Chrostek A, Wu X, Quondamatteo F, Hu R, Sanecka A, Niemann C, Langbein L, Haase I, Brakebusch C. Mol Cell Biol. 2006 Sep;26(18):6957–70. [PMC free article: PMC1592871] [PubMed: 16943436]

10.

Heasman SJ, Ridley AJ. Nat Rev Mol Cell Biol. 2008 Sep;9(9):690–701. [PubMed: 18719708]

11.

Karlsson R, Pedersen ED, Wang Z, Brakebusch C. Biochim Biophys Acta. 2009 [PubMed: 19327386]

12.

Spinosa MR, Progida C, De Luca A, Colucci AM, Alifano P, Bucci C. J Neurosci. 2008 Feb 13;28(7):1640–8. [PMC free article: PMC6671532] [PubMed: 18272684]

13.

Rezaie T, Child A, Hitchings R, Brice G, Miller L, Coca-Prados M, Héon E, Krupin T, Ritch R, Kreutzer D, Crick RP, Sarfarazi M. Science. 2002 Feb 8;295(5557):1077–9. [PubMed: 11834836]

14.

Yang S, Rarias M, Kapfhamer D, Tobias J, Grant G, Abel T, Bucan M. Genes Brain Behav . 2007;6:77–96. [PMC free article: PMC2914309] [PubMed: 16734774]

15.

Potenza N, Vecchione C, Notte A, De Rienzo A, Rosica A, Bauer L, Affuso A, De Felice M, Russo T, Poulet R, Cifelli G, De Vita G, Lembo G, Di Lauro R. EMBO Rep. 2005 May;6(5):432–7. [PMC free article: PMC1299307] [PubMed: 15864294]

Overview

This target, which grow out of a collaboration between the Center and the target provider was assigned in MLSCN Cycle 5, prior to the time when a CPDP was required. As the first large multiplex performed by our Center on the complete MLSMR available at that time, it provided an extraordinarily rich data set which has continued to be evaluated through the so-called MLSCN Pilot Phase “Phase-out” period. A multiplex target has the potential to yield small molecules that are activators or inhibitors of individual targets, the entire set of targets, or a subset of targets. The primary screen and dose-response yielded apparently non-selective activators, and inhibitors with varying degrees of selectivity. Unfortunately, convenient cell-based secondary assays were not part of the R03 submission and the Center and the target provider worked hard to develop novel secondary assays with variable, but low throughput once the screen had been performed.

The characteristics of the novel small molecule activators of GTPases include the ability to modulate GTP-binding and protein-protein interaction activities of members of the GTPase families represented by the series of targets included in this multiplex, with a potency of at least 100 nM. While 3 families of probe scaffolds have been identified, we chose to characterize in extensive detail the probe identified for the first family in the extended project description below. (Figures, legends, and detailed methods are provided at the end of this section. The compound referred to in this section as MLS00088004 is Probe 1 also known by Compound ID 888706 or Substance ID 57578335.)

However, in the MLSCN Phase-out period, a CPDP for a selective GTPase inhibitor probe has also been developed with the Kansas University Chemistry Specialty Center. KUSCC is currently evaluating the results presented in this probe report for the possibility of synthetically creating selective activators. We have retained the lead probes from all three scaffold families to provide an optimal opportunity should we agree to proceed to selectivity.

Compound identification and verification using multiplex screen

For detection of GTP binding to small G-proteins we used a flow cytometry-based assay 13 that allowed us to measure a dose response of GTP binding to individual GST-tagged small G-proteins.

It was observed that different conditions were required for the optimal activity of different GTPases. BODIPY-GTPγNH was identified as a better substrate for some of the small GTPases such as RhoA wt, Rac1wt; Cdc42, while others (Rab proteins and Ras) prefer binding to BODIPY-FL-GTP. Mg²⁺ ions were crucial for the Rho enzyme activity, while Rab proteins and H-Ras more effectively bound fluorescent GTP in the presence of EDTA. Activity of H-Ras was completely inhibited in the presence of 0.01% dodecyl maltoside in the assay, while this detergent had negligible effect on GTP binding by other GTPases.

Based on these preliminary results, we had chosen specific conditions which are not optimal for all GTPases, but allow us to simultaneously measure the GTP binding of several small GTPases. This approach was used for multiplex analysis of GTPase activity and, therefore, HTS of the MLSMR. This is a low-cost, time saving and highly efficient method for the simultaneous measurements of the activity for the number of proteins. Moreover, it simplifies analysis, comparison and detection of the specific activators/inhibitors of individual GTPases.

In primary screening, each well contained beads of seven different red fluorescent intensities, coupled with individual GST-GTPases. This mixture was incubated for 45 min with 100 nM fluorescent GTP in the presence of 1μM compound, and protein binding fluorescence was measured using a flow cytometer with the beads delivered by a HyperCyt system20. Briefly, HyperCyt consists of an autosampler connected to a peristaltic pump: the autosampler sips ~2 μl of suspension from each of the wells of a multi-well plate, leaving an air gap between samples.

The primary screening of one plate of the MLSMR library using bead-based multiplex method is presented in Fig. 1. Fig. 1B demonstrates that only one compound out of 320, had an effect on BODIPY-FL-GTP binding. Analysis of the row data with IDLeQuery software, GraphPad Prism and Excel template revealed that this activator was MLS000088004 (2-[(2-phenylethyl)thio]nicotinic acid. IUPAC Name: 2-(2-phenylethylsulfanyl)pyridine-3-carboxylic acid) which non-specifically activated small G-proteins (Fig. 1D).

Figure 1

High throughput screen identifies small molecule activators and inhibitors of Ras GTPase superfamily. (A) Left panel shows a plot of forward scatter versus a logarithm of side scatter. Singlet beads are selected by drawing a gate as shown. Middle panel (more...)

The MLSCN library of 194,738 compounds was screened in a multiplex for potential activators and inhibitors of fluorescent GTP binding to six different GTPase proteins: Cdc42, Rac1, Rac1 activated mutant, Rab2, Rab7, and Ras. The results from the multiplex screen were published on PubChem (http://pubchem.ncbi.nlm.nih.gov/) with PubChem Bioassay identification numbers of 761, 757, 764, 760, 758, 759, respectively. The quality control Z′ statistics were very good for each target. In particular, the average Z′ was 0.87 +/−0.04 for Cdc42, 0.85 +/− 0.04 for Rac1, and 0.90 +/− 0.03 for Rac1 activated mutant (Fig. 1C).

Dose response analysis

To verify the primary screen results, we used dose response assays with one multiplex (Rab7 wt, Rab2 wt, H-Ras wt, constitutively active mutant H-RasG12V, Cdc42 wt, and constitutively active mutant Cdc42Q61L) and 3 single-plexes (for Rac1 wt, constitutively active mutant Rac1Q61L and GST-GFP). GST-GFP single-plex was used to eliminate the possibility that compound affects GST-protein binding to beads.

Fig. 1 shows that MLS00088004 behaved as a non-selective, high affinity activator of eight different GTPases (Fig. 1E; EC50 1.6 x 10⁻⁸ – 1.4 x 10⁻⁷ M), though Rho-family GTPases (H-Rac1 and Cdc42) showed the greatest sensitivity to the compound. Table 1 shows that EC50, we have identified a novel compound with nanomolar affinity for the Ras-family GTPases and particular affinity for Rac1 and Cdc42 relative to members of the Ras and Rab subfamilies.

Small molecules modulate Rho-family GTP-binding kinetics and in vitro activation

When assays were performed at a fixed concentration of fluorescent GTP and optimal activator concentration, the magnitude of activation appeared to vary widely among the GTPase families, potentially because the Kd of the interaction between fluorescent GTP and/or the activator varies for individual GTPases. To explore this possibility, we varied the concentration of the fluorescent GTP as shown in Fig. 2A, B and quantified in the Table (Fig. 2C) where the apparent Bmax is shown to increase (1.2 to 1.8 fold) and the apparent Kd in the presence or absence of MLS000088004 were performed using wild-type Cdc42 and our established flow cytometry based assay 28 (Fig. 2D). The results are consistent with the activator increasing the affinity of nucleotide binding. All of the families in this report have been shown to exhibit an increase in affinity (data not shown). Real-time assays of GTP-binding kinetics of dissociation typically exhibits a corresponding decrease (1.2–3.1 fold).

Figure 2

Effects of small molecule activator and inhibitor on Cdc42 GTP-binding, kinetics and in vitro Rac1 activation. (A) Simultaneous binding analysis of six G proteins and one control as a function of the concentration of fluorescent GTP in the presence of (more...)

In vitro detection of active Rac 1. Two different approaches were used for detection of Rac 1 activation in vitro. Both of these methods are based on so-called pull down assay of Rac. This method is well known and widely used for detection of active Rac1 and Cdc42. It is based on the fact that only active, GTP bound form of Rac is capable of binding to protein binding domain of PBD of the serine/threonine kinase PAK, and activating this enzyme. To measure the in vitro activation of Rac1 by MLS000088004, His-Rac1 was incubated with increasing concentrations of the compound (0–12.5 μM) in the presence of GTPγS to prevent hydrolysis during the assay. The fraction of active His-Rac1 was identified by binding to GST-PAK-PBD immobilized on glutathione beads and immunoblot analysis of bound Rac1. The precipitated GTPase was then quantified by Western blotting, using specific antibodies 16. A dose dependent increase in Rac1 activation in the presence of increasing amounts of MLS000088004 was clearly evident (Fig. 3A). In second set of experiments, Swiss 3T3 cells were used to analyze the effect of MLS000088004 on Rac1 activation. For positive control, the cells were treated with 10 ng/ml EGF for 2 min. As shown in Fig. 3B, resting starved 3T3 cells treated with DMSO had low levels of active Rac1 (lane 1). Active Rac1 was dramatically elevated following treatment with MLS000088004 (lane 2).

Figure 3

A) Purified His-Rac1 (80 ng) was briefly preincubated with MLS000088004 (0–12.5 μM), 125 nM GTPγS was added and active Rac1 was isolated by binding to 5 μg GST-PAK-PBD immobilized on GSH-beads. Bound active Rac1 was detected (more...)

Cell-based assay

GTPases play a key role in actin re-organization and morphological transformations of the cell. Rac1 promotes polymerization at the leading edge, orchestrating the formation of lamellipodia and membrane ruffles.21, while Cdc42 is responsible for filopodia formation22. As was mentioned above, MLS000088004 behaved as a non-selective activator, but Rho-family GTPases, Rac1 and Cdc42 showed the greatest sensitivity to the compound. For this reason, the cell-based assay was used to examine the effect of the compound on cell morphology and actin re-organization in RBL 2H3 cells. This cell line has been widely used as a model for studying of hypersensitivity diseases that include allergy, asthma, and anaphylaxis.

RBL cells can be activated via several pathways (IgE cross-linking or aggregation of GPI-anchored proteins.23. Cross-linking of the high affinity IgE receptor (FcɛRI) activates the main pathway, which initiates cascades of biochemical events leading to degranulation, membrane ruffling and other physiological responses 24. Early stages of activation are characterized by an increase of tyrosine phosphorylation of proteins (key enzymes are Syk and Lyn), while secretion of inflammatory mediators is observed at later stages of activation. In addition to secretion, activated cells begin to flatten (within 2min), spread on their substratum, and extend lamellipodia which show active ruffling 24, 25.

Recent studies indicate that the same GTPases are involved in activation of the RBL cells. Stable expression of dominant negative Rac and Cdc42 in RBL cells inhibits FcɛRI-mediated degranulation 26. The lethal toxin of Clostridium sordellii which inactivates Rac, and possibly Cdc42 (but not Rho) inhibits FcɛRI-mediated tyrosine phosphorylation of phospholipase C, inositol phosphate formation, calcium mobilization and secretion of hexosaminidase suggesting a crucial role of Rac/Cdc42 in RBL cell signaling27.

Treatment of resting RBL cells with MLS000088004 induces ruffling, spreading and flattening

We examined the actin cytoskeleton structures of the resting, ligand stimulated and MLS000088004 treated RBL cells. Cells were grown overnight with or without 10μM MLS000088004. Immunofluorescence staining with rhodamine-phalloidin and confocal microscopy were used to detect the changes in cell shape, as well as reorganization of F-actin. Resting RBL cells were round and tall or modestly spread on the adherent growing surface (Fig. 4A). Activation of RBL cells by cross-linking of FcɛRI caused a remarkable transformation in cell surface topography, and the cell height decreased compared to DMSO controls. Cells lost their spherical shape and became spread and elongated on the substratum with actin becoming localized to the cell perimeter, leading edge and surface ruffles (Fig. 4). The same reorganization of actin and cell shape (dramatic increase in cell spreading and flattening) was observed after treatment of resting RBL cells with MLS000088004.

Figure 4

MLS000088004 modulate actin remodeling in mast cells. RBL-2H3 cells were treated as indicated, fixed, stained with rhodamine phalloidin and imaged on Zeiss LSM510. Individual panels show x-y and x-z views of resting cells, DNP-stimulated cells and cells (more...)

Live cell imaging

Live cell imaging studies revealed that whereas DMSO-treated control resting cells maintained a spherical shape with few protrusions (Fig. 5A), ligand-stimulated (cross-linking of IgE receptors) cells began flattening, ruffling and forming lamellipodia and filopodia within a minute after stimulation, and surface ridges were enhanced after 15–20 min (Fig. 5B). Addition of 10 μM activator to resting RBL cells induced formation of lamellipodia and microspikes within 10 min, and by 25 min cells were flattened and surface area was significantly increased (Fig. 5C).

Figure 5

MLS000088004 induces cell morphology changes comparable to ligand-stimulated mast cells. Live RBL-2H3 cells were monitored by DIC microscopy on a Zeiss inverted microscope for up to 30 min. (A) Resting RBL-2H3 cells. (B) RBL-2H3 cells stimulated with (more...)

Taken together, our observations demonstrate that small GTPase activator MLS000088004 induced similar morphological changes as cell activation via IgE receptor cross-linking. The magnitudes of those reorganizations are comparable. Live cell image figures show that formation of lamellipodia and fillopodia after addition of GTPase activator occurred rapidly and on a similar timescale as receptor-mediated activation. All of the probe families have been shown to induce morphological changes (data not shown) and quantitative dose-response analysis is currently being undertaken.

Signaling pathways upregulated by MLS000088004 are different from those induced by IgE receptor activation

To determine if morphological changes induced by MLS000088004 involve the same signaling pathway as IgE receptor cross-linked stimulation, we examined the effect of this compound on Syk phosphorylation and β-hexosaminidase release (early and late phase activation markers, respectively). Syk tyrosine kinase is tyrosine-phosphorylated in the early stages of RBL cell activation via several pathways (IgE cross-linking, or aggregation of GPI-anchored proteins)18, 28, 29 and plays key role in activation of RBL cells. To determine the effect of MLS000088004 on Syk phosphorylation, resting RBL cells were treated with indicated concentration of the activator (Fig. 6A). Immunoprecipitation with anti-Syk polyclonal antibody and immunoblot with phosphotyrosine-specific antibody indicated that treatment of RBL cells with MLS000088004 did not initiate Syk tyrosine-phosphorylation (Fig. 6A).

Figure 6

MLS000088004 has no effect tyrosine phosphorylation of Syk and ligand-stimulated β-hexosaminidase secretion of RBL cells. A) RBL cells after treatment of indicated concentrations of MLS000088004 were lysed, and Syk kinase was immunoprecipitated (more...)

Last, we examined the effect of MLS000088004 on RBL cell activation, characterized by β-hexosamindase secretion. Fig. 6B (black bars) shows that in the absence of ligand stimulation there was no β-hexosamindase release in MLS000088004-treated cells, indicating that compound is not toxic. In RBL cells activated via IgE-receptor cross-linking, the presence of MLS000088004 had no further stimulatory effect on secretion (Fig. 6B open bars). These data indicate that, MLS000088004 compound induces morphological changes of RBL cells via activation of small GTPases Rac and Cdc42. However, these changes do not involve traditional, FcɛRI -induced signaling pathway.

Summary

A multiplex flow cytometric assay was used for HTS of MLSCN library. Nearly 200,000 compounds were screened for identification of potential activators and inhibitors of GTP binding to six different small GTPases. This approach allowed us to collect and analyze more than 1.2 million data points in the primary screen. 100–500 positive compounds for each member of multiplex were identified by primary screening. (The results from this multiplex screen were published on PubChem http://pubchem.ncbi.nlm.nih.gov/, with PubChem Bioassay identification numbers of 761, 757, 764, 760, 758, 759, respectively). False positives such as autofluorescent compounds and compounds affecting GST-protein binding to glutathione-beads were eliminated, and ~1000 compounds were selected and tested in secondary dose-response assays. Dose-response results were reported in PubChem (AID-1333–AID-1337, AID-1339–AID-1341) with compounds categorized as having activating or inhibitory effect on one or more of the GTPases tested, including Ras (wild-type and activated), Rab2, Rab7, Rac1 (wild-type and activated), Cdc42 (wild-type and activated), and RhoA. Flow cytometry data of primary screening were confirmed with dose-response analysis and various biochemical and cell-based assays. Pull-down assay of active Rac1 shows that MLS88004 increases Rac1activity in Swiss 3T3 cells. Live cell imaging and confocal microscopy studies revealed that the activator-induced actin reorganization and cell morphology changes characteristic of Rho GTPase activation.

On the following pages are Figures, Legends, and detailed Materials and Methods for Section 2a.

Materials and Methods for Section 2b

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

ii. Assay rationale and descriptions, table of reagents and source

iii. Summary of Results

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

This information is provided for each probe scaffold in Section 3.

a. Chemical name of probe compound

2-(2-phenylethylsulfanyl)pyridine-3-carboxylic acid [ML099]

b. Probe chemical structure including stereochemistry

Molecular Weight 259.32352 [g/mol]

Molecular Formula C₁₄H₁₃NO₂S

XLogP3-AA 3.2

H-Bond Donor 1

H-Bond Acceptor 3

Rotatable Bond Count 5

Exact Mass 259.066699

Topological Polar Surface Area 50.2

Heavy Atom Count 18

Solubility Sparingly Soluble (0.080 g/L calc in water RT)

Molar Volume 200.6±5.0 cm³/mol

pKa 3.01±0.36

c. Structural Verification Information of probe SID

HRMS analysis of 57578335. The results are consistent with the presence of the [M-H]- of C₁₄H₁₃NO₂S with a -1.5 ppm difference from the expected m/z of 258.0589 (observed mass = 258.0585)

d. PubChem CID (corresponding to the SID)

Compound ID CID-888706; Substance ID SID-57578335

e. Availability from a vendor

ChemBridge ID 7360080

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

Compound is in the MLSMR; not submitted

g. Mode of action for biological activity of probe

As shown in Fig. 2 above, the activators appear to increase the affinity of binding of the fluorescent GTP analogs to the GTPases. To understand the action and binding mode of these small molecule activators against the GTPase proteins, docking simulations were performed on Rac1 and Cdc42 using the Surflex-dock program, as implemented in Sybyl 8.0. On the basis of the mechanism of recruitment and activation of GTPases by GEFs (guanine nucleotide exchange factors are GTPase binding regulatosr) a working hypothesis has been developed. The assumption is that activators interact with the GTPases in their inactive form (GDP-bound state) and bind to an allosteric binding site localized between switch regions I and II (Fig 7).

Figure 7

Common architecture of GTPase: green circles indicate the two flexible regions switch I and II. Red and yellow cartoons represent GTPases in free and GDP-bound states, respectively. The GTP-active form is rendered as grey cartoon with the Tyr32 involved (more...)

The sequence identity and similarity between Rac1 and Cdc42 is about 70% and 81% respectively, and the sequence alignment indicated only one difference in the binding site of these proteins. At position 56 there is a Trp in the binding site of Rac1 and a Phe for the corresponding position from Cdc42 (Figure 8). This similarity between the binding sites could explain the lack of selectivity of these activators with respect to Rac1 and Cdc42.

Figure 8

Sequence alignment of human Rac1 and Cdc42. The cyan boxes emphasize residues involved in the interaction with GEF regulator while grey boxes show the amino acids selected to define the binding cavity in docking calculations

This binding mode of these compounds involves favorable hydrophobic interactions with surrounding amino acids, specifically the residues from position 56, Trp for Rac1 and Phe for Cdc42. The views obtained from the docking of MLS00088004 at Rac1 and Cdc42 are depicted in Fig. 9. The binding mode of this compound in Rac1 binding cavity (Fig. 9A) shows the carboxylic group oriented towards the NH group of Asn39 backbone and involves in H-bond interactions. Additional hydrophobic contacts are favored by the close contact between the pyridine moiety of the compound and the indole side chain of Trp56, while the peripheral phenyl group is oriented towards the hydrophobic region delimited by the side chain of Leu67. In the Cdc42 active site (Fig. (B), the binding mode is different. The terminal phenyl group of the compound is oriented towards a hydrophobic favourable region delimited by the benzyl ring of Phe37, while the carboxylic group is exposed to the solvent and the H-bond interactions are lost. The presence of Phe at position 56 instead of Trp triggers a different orientation of the amino acids in the binding site which result in a shallower cavity compared with the binding cavity of Rac1. The consequent loss of the favorable interactions with Asn39 and Leu67 could explain the decrease of biological activity of MLS00088004 for Cdc42 in comparison with Rac1.

Figure 9

The binding mode of MLS00088004 (Rac-pEC₅₀ = 7.70; Cdc42-pEC₅₀ = 7.23). The figure shows the docking views of this compound in A) the biding cavity of Rac1, depicted in light green and B) Cdc42 represented in grey. The relevant amino acids in both binding (more...)

h. Detailed synthetic pathway for making probe

The original hit compound (SID-57578335) was identified during the primary HTS campaign and confirmed in dose response screen to have a logEC₅₀=7.7 on Rac1 wild type, logEC₅₀=7.59 on Rac1 activated mutant, logEC₅₀=7.23 on cell division cycle 42 (GTP binding protein, 25kDa) activated mutant, logEC₅₀=7.00 on cell division cycle 42 (GTP binding protein, 25kDa) wild type, logEC₅₀=6.45 on Ras-related protein Rab-2A, logEC₅₀=6.74 on GTP-binding protein (rab7), logEC₅₀=7.02 on Ras protein activated mutant, logEC₅₀=6.85 on Ras protein wild type. The activity was confirmed by purchasing the compound from a commercial source (ChemBridge ID 7360080). The chemical optimization strategy involved modification of section A, B, and L (Fig. 10). Analogs of nicotinic acid were available commercially and were purchased. A chemical synthesis project was also done by ChemDiv, The following variations were investigated: linker section sulfur atom was substituted with nitrogen or oxygen atoms, linker length was varied from 1 to 3 atoms; section A ring small substituents were added and the carboxylic group was preserved in all variations because primary data in close analogs to SID-57578335 indicated that it is essential for biological activity; ring B section was varied by introduction of small substituents on phenyl ring. Based on these modifications, 9 compounds are synthetic derivatives, 8 compounds were commercially purchased, and 8 compounds were cherry picked from MLSMR. The synthetic analogs around SID-57578335 were synthesized by ChemDiv in an attempt to improve activity (Schema 1).

Fig. 10

SID-57578335.

Probe 1

Representative synthetic scheme. Reagents and conditions.

SAR analysis led to the following conclusions:

• Pyridine ring (section A): for active compounds at position 2 is carboxyl group, while R₁ through R₃ are hydrogen i.e. unsubstituted.
• Phenyl ring (section B): active compounds can have small non-polar substituents like: methyl, methoxy, halogen. Polar substituents like nitro or aldehyde groups render the compound inactive.
• Linker (section L): is between 1 and 3 atoms the first linker atom attached to pyridine ring can be sulfur, oxygen or nitrogen.

The detailed SAR table and biological activities are presented in the following:

View in own window

Supplier ID	PubChem SID	R1	R2	R3	R4	R5	R6	R7	R8	Linker
MLS000088004	57578335
Z494-0019	56431867					CH3
Z494-0017	56431866				CH3				CH3
MLS000522071	14733114					CH3
MLS000522069	14737743					CH3			CH3
K783-0843	56431812						OCH3
0412-0036	56431715
K781-5215	56431809					F
MLS000394214	22401197						CH3		CH3
Z494-0020	56431868							CH3
MLS000673633	24815349							Cl
MLS000715411	24801148								Cl
MLS000122880	14722433						CH3
8012-6709	56431758						Cl
Z494-0015	56431864				CH3				CH3
MLS000046237	4243562
K781-0014	56431808				CH3
Z494-0011	56431862			CH3		CH3
R052-0595	56431824				OCH3			CHO
8017-3981	56431763						CH3
Z494-0007	56431859			CH3	CH3				CH3
Z494-0006	56431858				CH3				CH3
2189-1384	56431719						NO2
Z494-0016	56431865			CH3	CH3				CH3
Z494-0010	56431861					CH3

View in own window

Active

Inactive

Inconclusive

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

See Section 3.1b above

j. A tabular presentation summarizing known probe properties

Section 3.1c above

a. Chemical name of probe compound

1-[2-(2,5-dimethylphenoxy)ethyl]-1H-indole-3-carboxylic acid [ML098]

b. Probe chemical structure including stereochemistry

Molecular Weight 309.35906 [g/mol]

Molecular Formula C19H19NO3

XLogP3-AA 3.9

H-Bond Donor 1

H-Bond Acceptor 3

Rotatable Bond Count 5

Topological Polar Surface Area 51.5

Heavy Atom Count 23

Solubility Sparingly Soluble (3.7E-3 g/L calc in water RT)

Molar Volume 263.0±7.0 cm³/mol

pKa 4.07±0.10

c. Structural Verification Information of probe SID

HRMS analysis of 57578337. The results are consistent with the presence of the [M-H]- of C₁₉H₁₉NO₃ with a 1.7 ppm difference from the expected m/z of 308.1287 (observed mass = 308.1292).

d. Pub chem. CID (corresponding to the SID)

Compound ID 7345532; Substance ID 57578337

e. Availability from a vendor

ChemDiv ID 7815-0263

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

Compound is in the MLSMR, not submitted.

g. Mode of action for biological activity of probe

Section 3.1g above.

h. Detailed synthetic pathway for making probe

The original hit compound (SID-57578337) was identified during the primary HTS campaign and confirmed ina dose response screen to have a logEC₅₀=6.82 on Rac1 wild type, logEC₅₀=7.09 on Rac1 activated mutant, logEC₅₀=7.30 on cell division cycle 42 (GTP binding protein, 25kDa) activated mutant, logEC₅₀=6.99 on cell division cycle 42 (GTP binding protein, 25kDa) wild type, logEC₅₀=6.80 on Ras-related protein Rab-2A, logEC₅₀=6.74 on GTP-binding protein (rab7), logEC₅₀=6.71 on Ras protein activated mutant, logEC₅₀=6.46 on Ras protein wild type. The activity was confirmed by purchasing the compound from a commercial source (ChemDiv ID 7815-0263). The chemical optimization strategy involved modification of section A, B, C, and L (Figure 11). Analogs of substituted indole scaffold were available commercially and were purchased and chemical synthesis project was also done by ChemDiv. The following variations were investigated: linker section oxygen atom was substituted with carbon atom, linker length was varied from 1 to 4 atoms; section A ring small substituents were varied by introduction of methyl group; ring B section was varied by introduction of methyl, methoxy, halogen, tert-butyl, and iso-propyl substituents at various positions; section C ring was varied by introduction of methyl, methoxy, and halogen substituents at various positions. Based on these modifications a total of 14 compounds are synthetic derivatives and 14 compounds were commercially purchased, 4 compounds were cherry picked from MLSMR. The synthetic analogs around SID-57578337 were synthesized by ChemDiv in an attempt to improve activity (Schema 2).

Figure 11

Probe 2

Representative synthetic scheme. Reagents and conditions.

SAR analysis led to the following conclusions:

• Ring B: small substituents like methyl, methoxy, chlorine, bromine, fluorine are allowed. Larger hydrophobic substituents like tert-butyl, iso-propyl lead to inactive compounds.
• Ring A: no substituents allowed
• Ring C: no substituents allowed
• Linker: size between 1 to 3 atoms; one atom can be heteroatom like oxygen. Linker size larger than 3 leads to inactive compounds

Some compounds like 7815-0268, 7815-0267, 7815-0265 have noisy data or low response and were marked as inconclusive. The best active compound MLS000689786 has ring B substituted with methyl in position R₁ and R₄ and linker size 3

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Supplier ID	PubChem SID	R1	R2	R3	R4	R5	R6	R7	R8	R9	R10	Linker
7815-0253	56431743			F
7815-0252	56431742	F
MLS000689786	57578337	CH3			CH3
7815-0259	56431746			Br
MLS000689788	24814897	Cl
MLS000716591	24802779	OCH3
MLS000689785	24823826	CH3
7815-0255	56431744	Cl
7815-0260	56431747
7815-0256	56431745			Cl
MLS000689787	24814896			CH3
Z494-0049	56431894										CH3
Z494-0047	56431892	OCH3							Cl
Z494-0051	56431896	CH3								CH3
Z494-0050	56431895	CH3					CH3
Z494-0048	56431893	OCH3								Cl
Z494-0046	56431891	OCH3						Cl
Z494-0045	56431890	OCH3					Cl
Z494-0044	56431889	OCH3								CH3
Z494-0043	56431888	OCH3							CH3
Z494-0042	56431887	OCH3					CH3
Z494-0041	56431886	OCH3				CH3
Z494-0039	56431885	CH3						OCH3
Z494-0038	56431884	CH3					OCH3
Z494-0037	56431883	CH3				OCH3
7815-0272	56431752			t-Butyl
7815-0276	56431753	CH3
7815-0277	56431754	CH3			CH3
7815-0265	56431748		CH3	CH3
7815-0267	56431749			Cl
7815-0278	56431755		CH3	Cl
7815-0271	56431751	i-Propyl			CH3
7815-0268	56431750		CH3	Cl

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Active

Inactive

Inconclusive

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

See Section 3.2b above.

j. A tabular presentation summarizing known probe properties

See Section 3.2b above

a. Chemical name of probe compound

2-[(2-bromophenyl)methoxy]benzoic acid [ML097]

b. Probe chemical structure including stereochemistry

Molecular Weight 307.13934 [g/mol]

Molecular Formula C14H11BrO3

XLogP3-AA: 3.6

H-Bond Donor: 1

H-Bond Acceptor: 3

Rotatable Bond Count: 4

Topological Polar Surface Area: 46.5

Heavy Atom Count: 18

Solubility Sparingly Soluble (0.046 g/L calculated in water RT)

Molar Volume: 202.8±3.0 cm³/mol

pKa: 3.38±0.36

c. Structural Verification Information of probe SID

HRMS analysis of 57578338. The results are consistent with the presence of the [M-H]- of C₁₄H₁₁BrO₃ with a 1.9 ppm difference from the expected m/z of 304.9813 for the 79Br isotope (observed mass = 304.9819).

d. PubChem CID (corresponding to the SID)

Compound ID CID-2160985; Substance ID SID-57578338

e. Availability from a vendor

Ryan Scientific ID T5225985

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

Compound is in the MLSMR; not submitted.

g. Mode of action for biological activity of probe

See Section 3.1g above.

h. Detailed synthetic pathway for making probe

The original hit compound (SID-57578338) was identified during the primary HTS campaign and confirmed in dose response screen to have a logEC₅₀=6.82 on Rac1 wild type, logEC₅₀=7.09 on Rac1 activated mutant, logEC₅₀=6.60 on cell division cycle 42 (GTP binding protein, 25kDa) activated mutant, logEC₅₀=6.84 on cell division cycle 42 (GTP binding protein, 25kDa) wild type, logEC₅₀=6.84 on GTP-binding protein (rab7), logEC₅₀=6.53 on Ras protein activated mutant, logEC₅₀=6.47 on Ras protein wild type. The activity was confirmed by purchasing the compound from a commercial source (Ryan Scientific ID T5225985). The chemical optimization strategy involved modification of section A, B, and L (Fig. 12). Analogs of salicylic acid were available commercially and were purchased, chemical synthesis project was also done by ChemDiv, the following variations were investigated: linker section ether oxygen moiety was substituted with carboxylic, carbonyl amine, amide, and thioether moieties, linker length was varied from 1 to 4 atoms; section A ring small substituents were varied by introduction of methyl, methoxy, halogen, nitro, heteroalkyl, and amine substituents at various positions, group; ring B section was varied by introduction of methyl, methoxy, halogen, alkyl, and nitro substituents at various positions. Based on these modifications a total of 15 compounds are synthetic derivatives and 9 compounds were commercially purchased. 17 compounds were cherry picked from MLSMR. The synthetic analogs around SID-57578338 were synthesized by ChemDiv in an attempt to improve activity (Schema 3).

Figure 12

SID-57578338.

Probe 3

Representative synthetic scheme. Reagents and conditions.

SAR analysis led to the following conclusions:

• Ring A: No substitutions R₁–R₄ are allowed, any substitutions will render the compound inactive.
• Ring B: Small hydrophobic substituents are allowed like: methyl, methoxy, halogen, isopropyl.
• Linker: Length is between 1–4 atoms, with the following functional groups: amine, ether, carbonyl, carboxyl.
• Steric effects seems to play a role in the case of compounds: MLS000760373, MLS000714513 and R092-0019, the last compound (R092-0019) is not substituted at positions R₅ and R₉ whereas MLS000760373 and MLS000714513 are substituted with methyl. The R₅ and R₉ methyl induce a preferential out of ring B plane conformation which can explain the observed bioactivity for MLS000760373 and MLS000714513 and no activity for R092-0019.

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Supplier ID	PubChem SID	R1	R2	R3	R4	R5	R6	R7	R8	R9	Linker
MLS000761515	57578338									Br
MLS000104449	7975869
MLS000081810	3712189
MLS000081844	3713391							OCH3
0384-0034	56431714
4478-5409	56431724					F		F
R152-1213	56431826							Cl
MLS000760373	24837897						CH3	CH3
MLS000715316	24802136								OCH3
MLS000775662	24833685							OCH3
8524-6914	56431766							Cl
MLS000714513	24803424					CH3	CH3		CH3	CH3
MLS000111718	7974636									OCH3
8016-5752	56431762					C(CH3)2			CH3
K783-0963	56431813					Cl
MLS000043585	4243978	F	F	F	F				OCH3
K783-0702	56431811					Cl				Cl
MLS000689738	24814013	F	F	F	F			CH3		CH3
MLS000689734	24814024	F	F	F	F			CH3
MLS000775808	24831309		NO2							Cl
MLS000693348	24795918			Cl		NO2
MLS000663391	24797131			Cl				OCH3
Z494-0034	56431880		OCH3			CH3				CH3
Z494-0023	56431870	OCH3				Br
Z494-0022	56431869	CF3
Z494-0025	56431872			OCH3						Br
Z494-0028	56431875		CH3			CH3
8017-4247	56431764		NH2					Cl
Z494-0033	56431879	OCH3				CH3		CH3
Z494-0029	56431876			CH3				CH3
Z494-0031	56431878			OCH3				CH3
Z494-0024	56431871		OCH3			Br
MLS000760107	24835967		NO2
Z494-0036	56431882				OCH3	CH3		CH3
Z494-0027	56431874	CH3
Z494-0026	56431873				OCH3			Br
Z494-0035	56431881			OCH3		CH3		CH3
R092-0019	56431825						CH3
Z494-0030	56431877				CH3	CH3
Z494-0002	56431857	CF3								H
MLS000621480	24794372		NO2
MLS000621480	24794372		NO2

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Active

Inactive

Inconclusive

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

See Section 3.3c above.

j. A tabular presentation summarizing known probe properties

See Section 3.3c above.