Specific Aims

The identification of small molecules, each able to activate or block a distinct receptor underlying an addiction will provide a means to untangle the many pathways resulting in addictive behavior and create detailed pharmacological maps for designing novel targeted treatments. This project proposes screening a G protein-coupled receptor relevant to drug abuse and to the study and treatment of addiction, in a fashion that affords the unique opportunity to discriminate between G protein and arrestin-based signaling modalities. This project will hopefully contribute to understanding and treating addiction by providing chemical probes for dissecting the individual brain pathways that underlie addictive behavior thus enabling improved understanding of the molecular basis of addiction and potentially providing targeted therapeutics for this affliction.

The specific aims of this project are to identify subtype specific small molecule agonists of the human kappa opioid (KOP) receptor. Such agonists may be useful in elucidating different signaling modalities for kappa receptors, but more importantly may be partial agonists that will facilitate the discovery of new molecular scaffolds for kappa antagonists. Subtype selectivity is defined as selective for KOR but not active against μ (MOR) or δ (DOR) subtypes

Background and Significance

For normal activities that produce rewards, there is a rapid habituation of the circuits involved and the behaviors will wane. However, for addictive drugs habituation does not occur and dopamine release persists despite repetitive trials. Upon withdrawal of the drug, a decrease of dopamine levels in the nucleus accumbens results, and this has been observed for opioids, cannabinoids, alcohol, amphetamines, and nicotine (1). This loss of dopamine accounts for the withdrawal syndromes observed with these drugs. The prototype opioid drug is morphine. It produces many effects typical of most opioids including analgesia, euphoria, nausea, and respiratory depression. Repeated use of opioids produces physical dependence and tolerance. These manifestations of opioid use are due to the three recognized types of opioid receptors that are members of the GPCR family, the mu (μ), delta (δ), and kappa (κ) subtype receptors. While stimulation of the mu and delta receptors increases dopamine release in the nucleus accumbens, κ receptor activation by its endogenous ligand dynorphin-A reduces extracellular dopamine. It has been suggested that stimulation of κ receptors by endogenous opioids like dynorphins should produce an aversive state that can antagonize the effects of other addictive compounds like alcohol, cocaine and nicotine. Moreover, exogenous κ agonists have also been observed to attenuate drug-taking behavior (2–6). Interestingly, kappa opioid antagonists have also been considered for use in treatment of cocaine abuse and appetite suppression (6).

The κ opioid receptors provide desirable substrates for targeted therapy and unraveling pathways responsible for mediating addictive behavior. Kappa opioid agonists offer a means to modulate the effects of stimulant drugs. However, there are currently no approved agents or compounds for these purposes (2).

In this probe report, we describe the discovery and optimization of novel agonists for the kappa-opioid (κ-opioid) receptor [KOP receptor or KOR] that is greater than 100-fold selective over the mu- (μ) [MOP receptor or MOR] and delta- (δ) [DOP receptor or DOR] opioid receptors (as measurable within the limits of solubility). We sent the probe for broad GPCR paneling and these data are discussed below (see Table 9). Furthermore, these probes and their analogs represent a novel chemical classes compared to current literature agonists with potentially novel pharmacology that may serve as interesting tools to advance addiction research.

Table 9

Comparative summary of two current probes CID5236771 & CID44601470 to literature examples.

Follow up studies subsequent to the probe report to investigate the properties of the new probes will be required and will employ confirmatory assays to validate that the compounds are interacting directly with the KOR and to characterize compound signaling. To demonstrate subtype specific binding in competitive assays a cohort of radiolabeled compounds are available commercially. [³H]-Diprenorphine has been used to demonstrate high affinity and selectivity (K_d = 0.1 nM) toward KOR and MOR. [³H]-U-69,593, [Phenyl-3,4-^–H] can be used to demonstrate high affinity and selectivity (Kd = 1 nM) toward KOR. Likewise, [³H]-DAMGO, [Tyrosyl-3,5–³H(N)] is also available for MOR displacement binding and Naltrindole [5′,7′-³H] or [³H]-Enkephalin are available for DOR binding studies. Additionally, these receptors couple through a multiplicity of G proteins and signaling pathways that can be studied by available assays including determination of the changes in cAMP, calcium, and certain phospho-kinase levels in cells.

a. Original goal for probe characteristics

The original goal from the CPDP was to find agonists different from current literature probes, with potencies of less than 1 µM for the kappa(κ)-opioid receptor (KOR), but with 100X selectivity against the mu(μ)-opioid receptor (MOR) and 100X selectivity against the delta(δ)-opioid receptor (DOR).

b. Information for each Assay Implemented and Screening Run

iii. Summary of Results

The following flowchart summarizes the compound triage and decision tree for advancement of compounds:

A library of approximately 290,000 compounds was tested in the KOR1 DiscoveRx β-Arrestin primary screen. Upon data analysis, 292 hits with activity >50% at a single concentration point of 10 µM were identified. Liquid samples were then ordered through DPI and 276 compounds were received.

The compound solutions resupplied by the MLSMR were first confirmed in 10 µM single-point duplicate in the KOR1 DiscoveRx β-Arrestin primary assay. Of these, 37 compounds were confirmed to have at least 50% activity at a 10 µM assay concentration. The confirmed compounds were further tested in dose response in the KOR1 DiscoveRx β-Arrestin primary assay to obtain EC50 values and were also tested in a β-galactosidase Counterscreen assay to assess the possibility that these compounds might somehow activate the enzyme. All compounds were confirmed as active in the KOR1 β-Arrestin dose response assay and none of the compounds were found to have activated β-galactosidase.

The active, confirmed compounds were then tested in the KOR1 High-Content Transfluor Agonist assay for further confirmation, then in the MOR and DOR High-Content Transfluor Agonist assays to determine subtype selectivity.

Chemistry and cheminformatics resources were then employed in the selection of both novel and chemically tractable molecules to pursue for a KOR1 selective probe. Structures of interest and analogs thereof were either purchased as commercial dry powders. In total, 30 structures were ordered though commercial vendors. From these experiments, a diverse SAR emerged and several compounds were discovered that met our established probe criteria.

Additional medicinal chemistry/SAR testing of re-constituted powders encompassed dose response testing of compounds in the four assays: KOR1 DiscoveRx β-Arrestin assay, the KOR1 High-Content Transfluor Agonist assay, and the MOR and DOR High-Content Transfluor Agonist assays.

c. Probe Optimization

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

The screening of the MLPCN compound collection at the Sanford-Burnham Center for Chemical Genomics identified several promising chemotypes (Figure 1) with potencies of the lead exemplar noted from solution reconfirmation. This probe report describes the optimization of probes from the first two chemotypes, bisamides (series 1) and triazoles (series 2). The third series did not yield sufficiently compelling SAR and was set aside.

Figure 1

Cheminformatics and Medicinal Chemistry Analysis

Probe optimization/SAR

Bisamide series

Compound 1 CID5236771 (MLS-0254632) (entry 1, Table 3) was identified through a high-throughput screening campaign on 284,076 compounds as an active and selective scaffold. After confirmation of the initial results, the hit-to-probe process was initiated by both analog-by-catalog approach and collaborative medicinal chemistry effort with KSCC. Probe candidate CID5236771 (MLS-0254632, entry 1) and analogs (entries 2–9) were obtained from commercial sources. Several additional analogs were prepared using a protocol similar to the route shown in section 4h (entries 10 – 14 in Table 3). The structure-activity data are presented Table 3 below:

Table 3

SAR Analysis for Selective κ–opioid receptor agonist for the Bisamide Scaffold.

SAR Analyses for CID5236771 (bisamide series)

The above data set illustrate several emerging SAR trends for this chemotype. Foremost, the requirement for substitution on the methylene bridge between amides is demonstrated from the inactive glycine-derived analogs CID1458208 (MLS-0435478), CID44483222 (MLS-0435480), CID44483224 (MLS-0435479), CID44483221 (MLS-0435482) and CID44483223 (MLS-0435481) (entries 10–14). While the necessity for alkyl substitution is clear, the ideal composition of the R²/R³ groups has not been completely optimized for this chemotype The exact nature of the side group on the secondary amide on the left hand side of the general structure appears to have a less dramatic effect on potency than the R²/R³ substitution. While a range of hydrophobic groups afforded highly potent analogs, the most potent analog was obtained when R¹ was cyclohexyl (CID5236771 (MLS-0254632), entry 1). The single case where a direct comparison is possible suggests the thiophene-containing side chain to be advantageous over the furan-containing side chain (CID5236771 and CID3950926, entries 1 and 3). Finally, a clear advantage was observed for the 2-pyridyl amides over the 2-pyrazinyl amides, as exemplified by CID5236771 compared to CID4014737 (entries 1 and 2 ) compared to CID3242935 (entries 9 and 8).

Triazole series

The triazole (Series 2) chemotype originally contained four compounds with potency around 2 μM and one example at 6.7 μM. This scaffold was pursued in a short and intense analog campaign to increase potency by at least two fold. The synthesis and purchase of a total of 43 analog compounds provided not only a compound exceeding the necessary increase in potency, but also less potent compounds possessing super agonist efficacies (Emax >100%): cmps 1 – 3, 5 – 11 in Table 4 below, which also details the SAR.

Table 4

SAR Analysis for Selective κ–opioid receptor agonist for the Triazole Scaffold.

While a limited selection of analogs for the series 2 chemotype are commercially available, limited substitution on the phenyl ring and no available thiophene-containing analogs led us to adopt an entirely synthetic approach. Beginning with the appropriate isothiocyanate and 2-picolynyl hydrazide, we synthesized the 1,2,4-triazole-3-thione scaffolds in two steps and excellent yields (77–82% overall yields) without chromatographic separations (section 4 h). The coupling with a wide range of benzyl halides proceeded smoothly in acetone facilitated by K₂CO₃ to furnish the 43 analogs described in Table 4.

In conjunction with canonical SAR substitution strategies (7,8), our approach toward the optimization of the Series 2 compounds benefited from the observation in the Series 1 work that in some cases a switch from the furan to thiophene moiety afforded at least a two-fold increase in potency. For example see: CID5236771 (MLS-0254632)/CID3964693 (MLS-0080731) (earlier section). For this chemotype, the thiophene and furan moieties afforded roughly equipotent analogs within the experimental standard deviation. A more productive approach was varying the substitution on the phenyl ring, producing the two most potent analogs CID44601470 (MLS-0435589) and CID44601475 (MLS-0435599) (entries 1 and 11), both bearing a 3,4-dichlorophenyl moiety. Other phenyl ring substitution also bore interesting results not in potency, but in the E_max values. Six compounds (entries 2, 3, 5, 6, 7 and 9 in Table 4) synthesized displayed 150% or greater activation at 20 μM. This unexpected (and to our knowledge unprecedented) super agonistic activity provides a strong rationale for further explorations into this chemotype.

a. Chemical name of probe compound (s) (Chemical IUPAC)

Probe 1 (bisamide - series 1): N-[1-(cyclohexylcarbamoyl)cyclohexyl]-N-(thiophen-2-ylmethyl)pyridine-2-carboxamide [ML139]

Probe 2 (triazole – series 2): 2-(5-(3,4-dichlorobenzylthio)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)pyridine [ML138]

b. Probe chemical structure(s) including stereochemistry

c. Structural Verification Information of probe SID

The probe SIDs are 87218782 (corresponding to CID5236771) and 87334039 (corresponding to CID44601470)

Purity

SID-87218782 >98% (HPLC);

SID-87334039 >99% (HPLC)

(1) SID-87218782:

(2) SID-87334039:

NMR Purity

(1) SID-87218782: >95% (¹H-NMR): ¹H NMR (400 MHz, CDCl₃) δ 1.06–1.22 (m, 3 H), 1.28–1.38 (m, 2 H), 1.42–1.83 (complex, 11 H), 2.14–2.26 (m, 4 H), 3.62–3.71 (m, 1 H), 4.98 (s, 2 H), 6.78 (d, J = 2.8 Hz, 1 H), 6.83 (t, J = 4.4 Hz, 1 H), 6.95 (br s, 1 H), 7.14 (d, J = 5.2 Hz, 1 H), 7.33 (dd, J = 0.8, 4.8 Hz, 1 H), 7.59 (d, J = 8.0 Hz, 1 H), 7.75 (dt, J = 1.6, 7.6 Hz, 1 H), 8.57 (d, J = 4.8 Hz, 1 H). ¹³C (100 MHz, CDCl₃, APT pulse sequence) δ d (CH, CH₃) 48.0, 124.3, 124.7, 125.2, 126.5, 126.6, 137.1, 148.0; u (C, CH₂) 22.4, 24.7, 25.6, 25.7, 32.8, 33.1, 44.9, 66.9, 141.8, 155.1, 171.7, 172.7.

(2) SID-87334039: >90% pure. ¹H NMR (acetone-d6) δ 4.54 (s, 2 H), 5.91 (s, 2 H), 6.24 (d, J = 2.8 Hz, 1 H), 6.29 (dd, J = 2.0, 3.2 Hz, 1 H), 7.40–7.51 (complex, 4 H), 7.70 (d, J = 2.0 Hz, 1 H), 7.96 (dt, J = 1.6, 7.6 Hz, 1 H), 8.24 (d, J = 8.0 Hz, 1 H), 8.72 (d, J = 4.4 Hz, 1 H); ¹³C NMR (acetone-d6, APT pulse sequence) δ d (CH, CH₃) 109.7, 111.3, 123.7, 125.2, 130.1, 131.4, 132.0, 138.2, 143.8, 149.7; u (C, CH₂) 36.7, 42.7, 131.7, 132.5, 139.7, 148.8, 150.3, 152.8, 153.4; (neat) 1701, 1589, 1463, 1446 cm⁻¹

d. PubChem CID(s) (corresponding to the SID)

PubChem CIDs are CID5236771 (corresponding to SID-87218782) and CID44601470 (corresponding to SID-87334039)

e. Availability from a vendor

1. The bisamide probe, CID5236771 is commercially available in milligram quantities from Chem Div (catalog # C094-1951), Aurora Screening Library (catalog # kcd-303814), Ambinter Screening Collection (catalog # STK142410), New Chemistry Horizons Laboratories Screening Library (catalog # NCHSC1-21611), Princeton Gold Collection I (catalog # OSSK_529711) and AKOS Screening Library (catalog # AKG-C094-1951). However, KSCC is depositing 50 mg of newly synthesized material with DPI.
2. The triazole probe, CID44601470 is not commercially available. However, KU SCC has deposited 50 mg of synthesized material with the MLSMR.

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

(see Table 5)

Table 5

Probe and Analog Submissions to the MLMR (BioFocus DPI).

g. Mode of action for biological activity of probe

Both probes were submitted to Bryan Roth’s Psychoactive Drug Screening Program (PDSP) GPCR Panels and the results are summarized in Table 6 below. The first bisamide probe CID5236771 [ML139] was indeed very clean with no activity < 10,000 nM except for the KOP, MOP and DOP receptors, with values of 0.79, 671.1, and 1,315 nM, respectively. These yield selectivity values of 850-fold (MOR/KOR) and 1,660-fold (DOR/KOR) in the PDSP assays which compare favorably with those obtained by our DiscoveRx (>270-fold for MOR & DOR) and HCS (>530-fold for MOR & DOR) assays (see “Probe(s) Structure & Characteristics:” table on p.1).

Table 6

Receptor Paneling of KOP agonist Probes 1 [ML139] CID5236771 & 2 [ML138] CID44601470 through the PDSP (Bryan Roth, PI).

The second triazole probe CID44601470 [ML138] was not as “clean” and showed some weak activity (1200 – 9600 nM K_is) against 5HT2b, 5HT5a, α2A, α2B, b3, D1, and D5 receptors, in addition to stronger activities against the KOR, MOR and DOR receptors, with values of 0.23, 773.3, and 484.1 nM, respectively. In light of this potent binding at the KOR, even the most potent binding to other receptors in this panel translates to an impressive selectivity of greater than 5,000 fold. These yield selectivity values of 2,100-fold (MOR/KOR) and 3,360-fold (DOR/KOR) in the PDSP assays which compare much more favorably with those obtained by our DiscoveRx (>37-fold for MOR & DOR) and HCS (>91-fold for MOR & DOR) assays.

Is should be noted the that the subnanomolar binding K_is of CID5236771 (0.79 nM) and CID44601470 (0.23 nM) for the KOR receptor are the markedly more potent than those determined by either functional assay (see table on p. 1) for CID5236771 (120 nM DiscoveRx; <60 nM HCS) and CID44601470 (870 nM DiscoveRx; 350 nM HCS) and the rank order is inverted. Although corrrelation of the functional EC₅₀ data to equilibrium ligand binding values (K_i) is not straightforward, these potencies and selectivites compare very favorably with even the most potent previously reported compounds in the literature and augue favorably for the utility of the presently reported probes or their analogs in the future. For example, the comparative K_i data for salvinorin A (also obtained in the PDSP under analogous conditions), a KOR agonist of extremely high contemporary interest, are 7.4 nM for KOR, 1370 nM for the MOR, and >10,000 for the DOR.

G protein coupling assays using the ³⁵S-GTPγS binding assay were undertaken to demonstrate single point efficacy for the probes and select derivatives. Full concentration response curves were pursued to determine agonist potencies, which are shown in Figure 2. Interestingly, the efficacy and potency of the current compounds in the G protein coupling assay was highly correlative with the observations made in the beta-arrestin translocation assay further reinforcing this assay as a sensitive and selective means to identify novel agonists.

Figure 2

³⁵S-GTPγS binding assay – Secondary functional assays for KOR agonist probes and selected analogs.

Since agonists can differentially activate GPCRs to engage diverse signaling cascades, a secondary, downstream Map kinase assay (Erk1/2 activation) was used to evaluate agonist activity in Figure 3. Single point efficacy curves reveal a tendency for CID44601470 to be more efficacious in this assay, which was confirmed by full concentration response curve analysis.

Figure 3

Downstream MAP Kinase Activation - Secondary functional assays for KOR agonist probes and selected analogs.

The probe appears to bind reversibly to the opiate agonist binding site of the receptor on the basis of the beta-arrestin primary and secondary assays. As described in the introduction to the report, the site specificity will be tested through competitive assays with known KOR ligands. It is clear from these report findings that the probes are able to activate beta-arrestin. It will be of interest in the follow up studies whether they will demonstrate a similar efficacy for G protein signaling, since it is possible that biased agonism towards one or the other pathway produces a distinctive physiological response profile.

h. Detailed synthetic pathway for making probe

Bisamide Probe 1

N-(1-(cyclohexylcarbamoyl)cyclohexyl)-N-(thiophen-2-ylmethyl) picolinamide (SID-87218782). The carboxylici acid precursor [1-(N-(thiophen-2-ylmethyl) picolinamido)cyclohexanecarboxylic acid] (64 mg, 0.186 mmol) was combined in a Biotage microwave process vial (2–5 mL size) with HOBt (30 mg, 0.223 mmol), DCC (46 mg, 0.223 mmol) and cyclohexylamine (55 mg, 0.558 mmol) in MeCN (2 mL). The reaction was heat at 100 °C for 6 min using microwave irradiation. After cooling, the reaction mixture was adsorbed on Celite and chromatographed on silica to afford the bisamide product SID-87218782 (27 mg, 0.063 mmol, 34% yield).

Triazole Probe 2

2-(5-(3,4-dichlorobenzylthio)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)pyridine (SID-87334039). Following the general procedure listed below and silica gel chromatography, furan thione (93 mg, 0.36 mmol) and 3,4-dichlorobenzyl chloride (0.66 mL, 0.43 mmol) afforded the product as an off-white solid (116 mg, 0.278 mmol, 77% yield). R_f = 0.24 (1:1 Hexanes: EtOAc); mp = 99.0–99.5 °C; ¹H NMR (acetone-d6) δ 4.54 (s, 2 H), 5.91 (s, 2 H), 6.24 (d, J = 2.8 Hz, 1 H), 6.29 (dd, J = 2.0, 3.2 Hz, 1 H), 7.40–7.51 (complex, 4 H), 7.70 (d, J = 2.0 Hz, 1 H), 7.96 (dt, J = 1.6, 7.6 Hz, 1 H), 8.24 (d, J = 8.0 Hz, 1 H), 8.72 (d, J = 4.4 Hz, 1 H); ¹³C NMR (acetone-d6) δ d 109.7, 111.3, 123.7, 125.2, 130.1, 131.4, 132.0, 138.2, 143.8, 149.7; u 36.7, 42.7, 131.7, 132.5, 139.7, 148.8, 150.3, 152.8, 153.4; (neat) 1701, 1589, 1463, 1446 cm⁻¹; HRMS (ESI) m/z calcd for C₁₉H₁₅C_l2N₄OS ([M+H]⁺), 417.0344., found 417.0353.

N-(Furan-2-ylmethyl)-2-picolinoylhydrazinecarbothioamide

2-Picolynyl hydrazide (410 mg, 2.99 mmol) and furfuryl isothiocyanate (416 mg, 2.99 mmol) in MeCN (15 mL) were stirred for 16 h at rt. The reaction mixture was filtered, the precipitate washed with additional MeCN (3 × 10 mL) and dried under vacuum to afford the thioamide as an off-white solid (610 mg, 2.21 mmol, 74% yield), which was used without further purification. IR (neat) 3135, 1673, 1533, 1500, 1465 cm⁻¹; HRMS (ESI) m/z calcd for C₁₂H₁₃N₄O₂S ([M+H]⁺), 277.0759, found 277.0761.

4-(Furan-2-ylmethyl)-3-(pyridin-2-yl)-1H-1,2,4-triazole-5(4H)-thione

To a slurry of the above thioamide (530 mg, 1.92 mmol) in water (25 mL) was added NaOH (4.00 g, 100 mmol). The reaction was heated at reflux for 2 h, the starting thioamide dissolved promptly upon warming. The reaction was cooled to rt, diluted with aqueous HCl (1 N, 20 mL) and acidified to pH = 6 with concentrated HCl. The solid precipitate was filtered, washed with water (2 × 15 mL) and dried under vacuum to afford the thione as a white solid (478 mg, 1.85 mmol, 96% yield), which was used without further purification. IR (neat) 3021, 2894, 2766, 1585, 1550, 1502, 1463 cm⁻¹; HRMS (ESI) m/z calcd for C₁₂H₁₁N₄OS ([M+H]⁺), 259.0654, found 259.0642.

Synthesis of Series 2 thiophene scaffold:

N-(Thiophen-2-ylmethyl)-2-picolinoylhydrazinecarbothioamide

2-Picolynyl hydrazide (883 mg, 6.44 mmol) and thiophene isothiocyanate (1,000 mg, 6.44 mmol) in MeCN (20 mL) were stirred for 16 h at rt. The reaction mixture was filtered, the precipitate washed with additional MeCN (3 × 10 mL) and dried under vacuum to afford the thioamide as an off-white solid (1,642 mg, 5.62 mmol, 87% yield), which was used without further purification. IR (neat) 3141, 1672, 1527, 1499, 1466 cm⁻¹; HRMS (ESI) m/z calcd for C₁₂H₁₃N₄OS₂ ([M+H]⁺), 293.0531, found 293.0516.

4-(Thiophene-2-ylmethyl)-3-(pyridin-2-yl)-1H-1,2,4-triazole-5(4H)-thione

To a slurry of the above thioamide (602 mg, 2.06 mmol) in water (25 mL) was added NaOH (4.00 g, 100 mmol). The reaction was heated at reflux for 2 h, the starting thioamide dissolved promptly upon warming. The reaction was cooled to rt, diluted with aqueous HCl (1 N, 20 mL) and acidified to pH = 6 with concentrated HCl. The solid precipitate was filtered, washed with water (2 × 15 mL) and dried under vacuum to afford the thione as a white solid (530 mg, 1.93 mmol, 94% yield), which was used without further purification. IR (neat) 3019, 2896, 1584, 1549, 1501, 1462 cm⁻¹; HRMS (ESI) m/z calcd for C₁₂H₁₁N₄S₂ ([M+H]⁺), 275.0475, found 275.0412.

General procedure for the synthesis of series 2 analogs from thiones and benzyl halides

The thione scaffold (0.1 to 0.3 mmol), K₂CO₃ (2 equiv) and the benzyl halide (1.2 equiv) were combined in acetone (15 mL/mmol substrate) and stirred in a sealed vial. After 15 h, the solvent was removed and the residue washed with CH₂Cl₂ (2 × 3 mL) and filtered. The combined filtrates were evaporated down and either chromatographed on silica or subjected to mass-directed, reverse phase preparative HPLC purification.

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

Both probes are novel chemical scaffolds and have demonstrated potent (< 1µM EC50) agonist activity in both the primary enzyme complementation assay and the image base orthogonal assay for β-arrestin mediated signaling and activation of the kappa opioid receptors, with selectivity over for activation of both the mu- and delta opioid receptors. The bisamide probe, CID5236771, has exceeded the criteria for probe as defined in the CPDP Chem Update document filed on October 28, 2009 and revised and refilled on November 21, 2009, and finally refilled after corrections requested by NIH on January 12, 2010 with the NIH PT: KOP receptors of less than 1 µM, and at least 100-fold selective over MOP and DOP receptors or as obtained by the achievable test concentrations, (e.g. solubility limited), regardless of whether this selectivity is calculated between the enzyme complementation/HCS or the HCS/HCS assays.

The triazole probe, CID44601470, formally misses the 100-fold selectivity of against MOR and DOR, though it approaches the desired selectivity when the HCS/HCS ratios are considered. However, all these compounds may not be able to achieve a higher than 30–40 µM test concentrations without some precipitation, and in most cases the dose-response curve is completely flat so the >32 µM is an under estimate of the true IC₅₀, which is certainly >>32 µM, so well within a factor of 2–3 for an effective >100 –fold selectivity against MOR. The assay provider concurs that even at this pro forma lower ~40-fold estimate of selectivity this probe compound should be useful. Furthermore, we note the intriguing result that it is in the triazole scaffold series (Table 4) that the apparent super-agonism is experimentally seen, in contrast to the bisamide series (Table 3). While we do not currently have any mechanistic understanding of this difference, the clear pharmacologic difference between these two classes of compounds supports nomination of this a bonafide 2^nd agonist probe for the kappa-opioid receptor. KU, SBCCG and the assay provider all concur on this point.

In Vitro Pharmacology Profiles of Probes CID5236771 [ML139] and CID44601470 [ML138] (See Table 7 below). Both probes had poor solubility at all pH’s tested, with the 2^nd triazole probe (CID44601470) the poorest solubility.

Table 7

Summary of in vitro ADME/T Properties of Kappa Opioid Agonist probe(s).

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 are measured using UV spectroscopy. In this assay, both probes CID5236771 [ML139] and CID44601470 [ML138] have excellent permeability. CID5236771 [ML139] had moderate brain barrier permeabilty while CID44601470 [ML138] had almost 3-fold selectivity in the blood brain PAMPA assay.

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. CID5236771 [ML139] and CID44601470 [ML138] are both highly bound (90–99%) to both human and mouse plasma.

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. Both CID5236771 [ML139] and CID44601470 [ML138] shows excellent stability (>99%) 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. Both CID5236771 [ML139] and CID44601470 [ML138] are rapidly metabolized in either Human or mouse microsomes. CID5236771 [ML139] shows high levels of toxicity toward human hepatocyctes in our assay, whereas CID 44601470 does not.

j. Probe properties