Multitarget Profile Research Articles

Rational design, synthesis and biological screening of triazine-triazolopyrimidine hybrids as multitarget anti-Alzheimer agents

In our endeavor towards the development of potent multitarget ligands for the treatment of Alzheimer's disease, a series of triazine-triazolopyrimidine hybrids were designed, synthesized and characterized by various spectral techniques. Docking and scoring techniques were used to design the inhibitors and to display their interaction with key residues of active site. Organic synthesis relied upon convergent synthetic routes were mono and di-substituted triazines were connected with triazolopyrimidine using piperazine as a linker. In total, seventeen compounds were synthesized in which the di-substituted triazine-triazolopyrimidine derivatives 9a-d showed better acetylcholinesterase (AChE) inhibitory activity than the corresponding tri-substituted triazine-triazolopyrimidine derivatives 10a-f. Out of the disubstituted triazine-triazolopyrimidine based compounds, 9a and 9b showed encouraging inhibitory activity on AChE with IC50 values 0.065 and 0.092 μM, respectively. Interestingly, 9a and 9b also demonstrated good inhibition selectivity towards AChE over BuChE by ∼28 folds. Furthermore, kinetic analysis and molecular modeling studies showed that 9a and 9b target both catalytic active site as well as peripheral anionic site of AChE. In addition, these derivatives effectively modulated Aβ self-aggregation as investigated through CD spectroscopy, ThT fluorescence assay and electron microscopy. Besides, these compounds exhibited potential antioxidants (2.15 and 2.91 trolox equivalent by ORAC assay) and metal chelating properties. In silico ADMET profiling highlighted that, these novel triazine derivatives have appropriate drug like properties and possess very low toxic effects in the primarily pharmacokinetic study. Overall, the multitarget profile exerted by these novel triazine molecules qualified them as potential anti-Alzheimer drug candidates in AD therapy.

Indenocinnoline derivatives as G-quadruplex binders, topoisomerase IIα inhibitors and antiproliferative agents

DNA intercalating agents are a consolidated therapeutic option in the treatment of tumor diseases. Starting from previous findings in the antiproliferative efficacy of a series of indeno[1,2-c]cinnoline-11-one derivatives, we performed a suitable decoration of this scaffold by means of a simple and straightforward chemistry, aiming to a) enlarge the planar core to a pentacyclic benzo[h]indeno[1,2-c]cinnoline-13-one and b) introduce a basic head tethered through a simple polymethylene chain. In fluorescence melting and fluorescence intercalator displacement assays, these new compounds displayed fair to very good intercalating properties on different nucleic acid strands, with preference for G-quadruplex sequences. Inhibition of human topoisomerase IIα and antiproliferative assays on HeLa and MCF7 tumor cell lines outlined a multitarget antiproliferative profile for tetracyclic 6 and pentacyclic derivative 20, both bearing a N,N-dimethylamine as the protonatable moiety. Particularly, compound 6 displayed a very potent inhibition of tumor cell proliferation, while 20 returned the highest thermal stabilization in melting experiments. In summary, these results outlined a potential of such highly planar scaffolds for nucleic acid binding and antiproliferative effects.

ITH14001, a CGP37157-Nimodipine Hybrid Designed to Regulate Calcium Homeostasis and Oxidative Stress, Exerts Neuroprotection in Cerebral Ischemia.

During brain ischemia, oxygen and glucose deprivation induces calcium overload, extensive oxidative stress, neuroinflammation, and, finally, massive neuronal loss. In the search of a neuroprotective compound to mitigate this neuronal loss, we have designed and synthesized a new multitarget hybrid (ITH14001) directed at the reduction of calcium overload by acting on two regulators of calcium homeostasis; the mitochondrial Na+/Ca2+ exchanger (mNCX) and L-type voltage dependent calcium channels (VDCCs). This compound is a hybrid of CGP37157 (mNCX inhibitor) and nimodipine (L-type VDCCs blocker), and its pharmacological evaluation revealed a moderate ability to selectively inhibit both targets. These activities conferred concentration-dependent neuroprotection in two models of Ca2+ overload, such as toxicity induced by high K+ in the SH-SY5Y cell line (60% protection at 30 μM) and veratridine in hippocampal slices (26% protection at 10 μM). It also showed neuroprotective effect against oxidative stress, an activity related to its nitrogen radical scavenger effect and moderate induction of the Nrf2-ARE pathway. Its Nrf2 induction capability was confirmed by the increase of the expression of the antioxidant and anti-inflammatory enzyme heme-oxygenase I (3-fold increase). In addition, the multitarget profile of ITH14001 led to anti-inflammatory properties, shown by the reduction of nitrites production induced by lipopolysaccharide in glial cultures. Finally, it showed protective effect in two acute models of cerebral ischemia in hippocampal slices, excitotoxicity induced by glutamate (31% protection at 10 μM) and oxygen and glucose deprivation (76% protection at 10 μM), reducing oxidative stress and iNOS deleterious induction. In conclusion, our hybrid derivative showed improved neuroprotective properties when compared to its parent compounds CGP37157 and nimodipine.

Automated Protocol for Large-Scale Modeling of Gene Expression Data.

With the continued rise of phenotypic- and genotypic-based screening projects, computational methods to analyze, process, and ultimately make predictions in this field take on growing importance. Here we show how automated machine learning workflows can produce models that are predictive of differential gene expression as a function of a compound structure using data from A673 cells as a proof of principle. In particular, we present predictive models with an average accuracy of greater than 70% across a highly diverse ∼1000 gene expression profile. In contrast to the usual in silico design paradigm, where one interrogates a particular target-based response, this work opens the opportunity for virtual screening and lead optimization for desired multitarget gene expression profiles.

Novel 8-Hydroxyquinoline Derivatives as Multitarget Compounds for the Treatment of Alzheimer's Disease.

We discovered a small series of hit compounds that show multitargeting activities against key targets in Alzheimer's disease (AD). The compounds were designed by combining the structural features of the anti-AD drug donepezil with clioquinol, which is able to chelate redox-active metals, thus decreasing metal-driven oxidative phenomena and β-amyloid (Aβ)-mediated neurotoxicity. The majority of the new hybrid compounds selectively target human butyrylcholinesterase at micromolar concentrations and effectively inhibit Aβ self-aggregation. In addition, compounds 5-chloro-7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (1 b), 7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (2 b), and 7-(((1-benzylpiperidin-4-yl)amino)methyl)-5-chloro-8-hydroxyquinoline (3 a) are able to chelate copper(II) and zinc(II) and exert antioxidant activity in vitro. Importantly, in the case of 2 b, the multitarget profile is accompanied by high predicted blood-brain barrier permeability, low cytotoxicity in T67 cells, and acceptable toxicity in HUVEC primary cells.

Quinoline based mono- and bis-(thio)carbohydrazones: synthesis, anticancer activity in 2D and 3D cancer and cancer stem cell models

Study of antitumor activity of mono- and bis-quinoline based (thio)carbohydrazones on THP-1 and AsPC-1 cancer stem cells, revealed that thiocarbohydrazones had superior pro-apoptotic activity than carbohydrazones with multi-target profile activities.

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound.

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer’s disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

Abstract 5387: Dual kinase/bromodomain inhibitors for rationally designed polypharmacology

Abstract Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multi-target profile has, however, necessitated the application of combination therapies, which can pose significant clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Here we have used BROMOscan™ bromodomain ligand binding assays to identify several clinical kinase inhibitors that also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase/bromodomain inhibitors. Nanomolar activity on BRD4 by clinical PLK1 and JAK2/FLT3 kinase inhibitors is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a novel strategy for rational single agent polypharmacological targeting. Importantly, cell-based data show that these dual inhibitors suppress c-Myc expression (a hallmark of BRD4 inhibition) and induce complex polypharmacological phenotypes reflecting dual kinase/bromodomain inhibition across a panel of primary human cell assay systems that model complex tissue and disease state environments (BioMap™). Furthermore, rich structure-activity relationships for related inhibitors and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase/bromodomain inhibitors. Citation Format: Elizabeth R. Quinn, Pietro Ciceri, Susanne Müller-Knapp, Alison O'Mahony, Oleg Fedorov, Panagis Filippakopoulos, Jeremy P. Hunt, Elisabeth A. Lasater, Gabriel Pallares, Sarah Picaud, Christopher Wells, Lisa M. Wodicka, Neil P. Shah, Stefan Knapp, Daniel K. Treiber. Dual kinase/bromodomain inhibitors for rationally designed polypharmacology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5387. doi:10.1158/1538-7445.AM2014-5387

Syntheses of coumarin–tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aβ aggregation, and β-secretase

Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer’s disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine–coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (Ki=16.7nM) against human AChE and about 2-fold lower potency (Ki=16.1nM) against BChE than tacrine (Ki=35.7nM for AChE, Ki=8.7nM for BChE), respectively. In addition, some of the tacrine–coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine–coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.

Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting β-amyloid, tau, and cholinesterase pathologies

Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine–6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5b–d have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5a–d has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC50 values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5a–d, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities.

Dual kinase-bromodomain inhibitors for rationally designed polypharmacology.

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multi-target profile has, however, necessitated the application of combination therapies, which can pose significant clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase/bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, clinical PLK1 and JAK2/FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a novel strategy for rational single agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase/bromodomain inhibitors.

Novel insights on the structural determinants of clozapine and olanzapine multi-target binding profiles

The clinical efficacy of antipsychotic drugs has been associated with a certain binding profile for a set of G protein-coupled receptors (GPCR)s. In this work, we use the structurally-related clozapine–olanzapine pair to progress in the understanding of the structural properties that determine their divergent binding profiles and, thereby, their differing therapeutic efficacy. First, we present novel site-directed mutagenesis results that confirm our previous hypothesis on the importance of ligand interaction with positions 5.42 and 5.46 in transmembrane helix 5. Then, we use refined models of ligand-receptor complexes, built from recently published GPCR crystal structures, to gain further insight into the molecular mechanisms responsible for the observed experimental outcomes. In particular, we observe that preventing or potentiating hydrogen bonding with position 5.46, could allow obtaining ligands with, respectively, clozapine or olanzapine-like affinities. Results presented in this study could guide the design of antipsychotic candidates with tailored binding profiles.

Cannabinoid agonists showing BuChE inhibition as potential therapeutic agents for Alzheimer's disease

Designing drugs with a specific multi-target profile is a promising approach against multifactorial illnesses as Alzheimer's disease. In this work, new indazole ethers that possess dual activity as both cannabinoid agonists CB2 and inhibitors of BuChE have been designed by computational methods. On the basis of this knowledge, the synthesis, pharmacological evaluation and docking studies of a new class of indazoles has been performed. Pharmacological evaluation includes radioligand binding assays with [3H]-CP55940 for CB1R and CB2R and functional activity for cannabinoid receptors on isolated tissue. Additionally, in vitro inhibitory assays of AChE/BuChE and the corresponding competition studies have been carried out. The results of pharmacological tests have revealed that three of these derivatives behave as CB2 cannabinoid agonists and simultaneously show BuChE inhibition. In particular, compounds 3 and 24 have emerged as promising candidates as novel cannabinoids that inhibit BuChE by a non-competitive or mixed mechanism, respectively. On the other hand, both molecules show antioxidant properties.

Prediction of the P. falciparum Target Space Relevant to Malaria Drug Discovery

Malaria is still one of the most devastating infectious diseases, affecting hundreds of millions of patients worldwide. Even though there are several established drugs in clinical use for malaria treatment, there is an urgent need for new drugs acting through novel mechanisms of action due to the rapid development of resistance. Resistance emerges when the parasite manages to mutate the sequence of the drug targets to the extent that the protein can still perform its function in the parasite but can no longer be inhibited by the drug, which then becomes almost ineffective. The design of a new generation of malaria drugs targeting multiple essential proteins would make it more difficult for the parasite to develop full resistance without lethally disrupting some of its vital functions. The challenge is then to identify which set of Plasmodium falciparum proteins, among the millions of possible combinations, can be targeted at the same time by a given chemotype. To do that, we predicted first the targets of the close to 20,000 antimalarial hits identified recently in three independent phenotypic screening campaigns. All targets predicted were then projected onto the genome of P. falciparum using orthologous relationships. A total of 226 P. falciparum proteins were predicted to be hit by at least one compound, of which 39 were found to be significantly enriched by the presence and degree of affinity of phenotypically active compounds. The analysis of the chemically compatible target combinations containing at least one of those 39 targets led to the identification of a priority set of 64 multi-target profiles that can set the ground for a new generation of more robust malaria drugs.

Imbricaric Acid and Perlatolic Acid: Multi-Targeting Anti-Inflammatory Depsides from Cetrelia monachorum

In vitro screening of 17 Alpine lichen species for their inhibitory activity against 5-lipoxygenase, microsomal prostaglandin E2 synthase-1 and nuclear factor kappa B revealed Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb. As conceivable source for novel anti-inflammatory compounds. Phytochemical investigation of the ethanolic crude extract resulted in the isolation and identification of 11 constituents, belonging to depsides and derivatives of orsellinic acid, olivetolic acid and olivetol. The two depsides imbricaric acid (4) and perlatolic acid (5) approved dual inhibitory activities on microsomal prostaglandin E2 synthase-1 (IC50 = 1.9 and 0.4 µM, resp.) and on 5-lipoxygenase tested in a cell-based assay (IC50 = 5.3 and 1.8 µM, resp.) and on purified enzyme (IC50 = 3.5 and 0.4 µM, resp.). Additionally, these two main constituents quantified in the extract with 15.22% (4) and 9.10% (5) showed significant inhibition of tumor necrosis factor alpha-induced nuclear factor kappa B activation in luciferase reporter cells with IC50 values of 2.0 and 7.0 µM, respectively. In a murine in vivo model of inflammation, 5 impaired the inflammatory, thioglycollate-induced recruitment of leukocytes to the peritoneum. The potent inhibitory effects on the three identified targets attest 4 and 5 a pronounced multi-target anti-inflammatory profile which warrants further investigation on their pharmacokinetics and in vivo efficacy.

The Bivalent Ligand Approach as a Tool for Improving the in vitro Anti‐Alzheimer Multitarget Profile of Dimebon

The Bivalent Ligand Approach as a Tool for Improving the in vitro Anti‐Alzheimer Multitarget Profile of Dimebon

The multitarget opioid ligand LP1's effects in persistent pain and in primary cell neuronal cultures

Persistent pain states, such as those caused by nerve injury or inflammation, are associated with altered sensations, allodynia and hyperalgesia, that are resistant to traditional analgesics. A contribution to development and maintenance in altered pain perception comes from nociceptive processing and descending modulation from supraspinal sites. A multitarget ligand seems to be useful for pain relief with a decreased risk of adverse events and a considerable analgesic efficacy. The multitarget MOR agonist–DOR antagonist LP1, (3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benazocin-3(2H)-yl]-N-phenylpropanamide, is a central acting antinociceptive agent with low potential to induce tolerance. LP1 was tested in models of neuropathic pain – induced by chronic constriction injury (CCI) of the left sciatic nerve – and inflammatory pain – produced by intraplantar injection of carrageenan. In CCI rats, subcutaneous (s.c.) LP1 (3 mg/kg) showed a significant antiallodynic effect, measured with von Frey filaments, and antihyperalgesic effect, evoked in response to a radiant heat stimulus with plantar test. Analogously, LP1 significantly reduced allodynic and hyperalgesic thresholds in a model of inflammatory pain induced by carrageenan. To evaluate the contribution of opioid receptor subtypes in LP1 antinociceptive effects, the multitarget LP1 profile was assessed using selective opioid antagonists. Moreover, functional electrophysiological in vitro assays, using primary cortical and spinal cord networks, allowed to define the “pharmacological fingerprint” of LP1. The EC50 values in this functional screening seem to confirm LP1 as a potent opioid ligand (EC50 = 0.35 fM and EC50 = 44 pM in spinal cord and frontal cortex, respectively). Using a NeuroProof data-base of well characterised reference compounds, a similarity profile of LP1 to opioid and non-opioid drugs involved in pain modulation was detected. Our studies seem to support that multitarget ligand approach should be useful for persistent pain conditions in which mechanical allodynia and thermal hyperalgesia are significant components of the nociceptive response.

Pharmacological Characterization of Memoquin, a Multi-Target Compound for the Treatment of Alzheimer's Disease

Alzheimer's disease (AD) is characterized by progressive loss of cognitive function, dementia and altered behavior. Over 30 million people worldwide suffer from AD and available therapies are still palliative rather than curative. Recently, Memoquin (MQ), a quinone-bearing polyamine compound, has emerged as a promising anti-AD lead candidate, mainly thanks to its multi-target profile. MQ acts as an acetylcholinesterase and β-secretase-1 inhibitor, and also possesses anti-amyloid and anti-oxidant properties. Despite this potential interest, in vivo behavioral studies with MQ have been limited. Here, we report on in vivo studies with MQ (acute and sub-chronic treatments; 7–15 mg/kg per os) carried out using two different mouse models: i) scopolamine- and ii) beta-amyloid peptide- (Aβ-) induced amnesia. Several aspects related to memory were examined using the T-maze, the Morris water maze, the novel object recognition, and the passive avoidance tasks. At the dose of 15 mg/kg, MQ was able to rescue all tested aspects of cognitive impairment including spatial, episodic, aversive, short and long-term memory in both scopolamine- and Aβ-induced amnesia models. Furthermore, when tested in primary cortical neurons, MQ was able to fully prevent the Aβ-induced neurotoxicity mediated by oxidative stress. The results support the effectiveness of MQ as a cognitive enhancer, and highlight the value of a multi-target strategy to address the complex nature of cognitive dysfunction in AD.

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

Background and MethodologyRecently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach.Principal FindingsA functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity.Conclusions and SignificanceOverall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Automated design of ligands to polypharmacological profiles

The clinical efficacy and safety of a drug is determined by its activity profile across multiple proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to rationally design drugs a priori against profiles of multiple proteins would have immense value in drug discovery. We describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads where multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.