Career development in fragment-based drug discovery.

The pharmaceutical industry is highly reliant on researchers who not only possess the technical knowledge but also the professional skills to collaborate in drug development. To prepare future practitioners to thrive in this interdisciplinary environment, Innovative Training Networks (ITNs) have become increasingly important in doctoral training. In this piece, we explore the benefits of these ITNs in training future practitioners in drug discovery. Through a bibliometric review, we find that the top researchers in fragment-based drug discovery have a high degree of collaboration and mobility across institutes. We then investigate which aspects of the ITN training program enable PhD students to gain these skills. We find that secondments, the short-term stays that students have in partner research institutes, are useful in preparing students to have both broad knowledge of drug discovery and specialization in their field of interest. Aside from imparting technical skills, we find that the collaborative environment in ITNs enables students to communicate better and to work effectively in teams. Doctoral students benefit by being exposed to relevant experiences that they can later apply as they navigate through the complex web of relationships and competencies in the industry. We conclude by recommending best practices to further improve ITNs in the training of future practitioners.

FDA-approved drug screening in patient-derived organoids demonstrates potential of drug repurposing for rare cystic fibrosis genotypes.

BACKGROUND: Preclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF who carry the most prevalent F508del-CFTR mutation, but a large unmet need remains to identify new treatments for all pwCF. METHODS: We used 76 PDIOs not homozygous for F508del-CFTR to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays. The most promising hits were verified in a secondary FIS screen. Based on the results of this secondary screen, we further investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators. RESULTS: In the primary screen, 30 hits were characterized that elevated CFTR function. In the secondary validation screen, 19 hits were confirmed and categorized in three main drug families: CFTR modulators, PDE4 inhibitors and tyrosine kinase inhibitors. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs where residual CFTR function is either present, or created by additional compound exposure. Additionally, upon CFTR modulator treatment we show rescue of CF genotypes that are currently not eligible for this therapy. CONCLUSION: This study exemplifies the feasibility of high-throughput compound screening using PDIOs. We show the potential of repurposing drugs for pwCF carrying non-F508del genotypes that are currently not eligible for therapies. ONE-SENTENCE SUMMARY: We screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.

A novel class of adenosine A3 receptor ligands. 1. 3-(2-Pyridinyl)isoquinoline derivatives.

A series of 3-(2-pyridinyl)isoquinoline derivatives was synthesized as potential antagonists for the human adenosine A3 receptor by substitution of the 1-position. The compounds were obtained by various synthetic routes from 1-amino-3-(2-pyridinyl)isoquinoline. The affinity was determined in radioligand binding assays for rat brain A1 and A2A receptors and for the cloned human A3 receptor. A structure-activity relationship analysis indicated that a phenyl group when coupled by a spacer allowing conjugation on position 1 of the isoquinoline ring increased the adenosine A3 receptor affinity. In contrast, such a phenyl group directly bound to position 1 of the isoquinoline ring decreased affinity. Since the combination of a phenyl group together with a spacer raised adenosine A3 receptor affinity, various spacers were investigated. VUF8501 (N-[3-(2-pyridinyl)isoquinolin-1-yl]benzamidine (15) showed an affinity at the human adenosine A3 receptor of 740 nM. Substituent effects on the phenyl group were investigated by in vitro evaluation of a series of substituted benzamidines. Electron-donating groups at the para position of the benzamidine ring increased adenosine A3 receptor affinity. These investigations led to VUF8505 (4-methoxy-N-[3-(2-pyridinyl)isoquinolin-1-yl]benzamidine(22)), which is a moderately potent and selective ligand for the human adenosine A3 receptor with an affinity of 310 nM in our test system having negligible affinity for rat A1 and A2A receptors.

A novel class of adenosine A3 receptor ligands. 2. Structure affinity profile of a series of isoquinoline and quinazoline compounds.

1-Substituted 3-(2-pyridinyl)isoquinolines have been shown to form a novel class of adenosine A3 receptor ligands. In the present study further investigations of this new lead and the structure affinity relationships of this class of compounds are described. First, the influence of an amide group at position 1 of the isoquinoline ring on the adenosine A3 receptor affinity was determined. A carboxamide proved to be a useful spacer between the isoquinoline and a phenyl ring. N-[2-(2-pyridinyl)isoquinolin-4-yl]benzamide (VUF8507, compound 6) had an affinity of 200 nM at the adenosine A3 receptor. Second, we investigated the effects of substitution of the benzamide ring of 6 with a series of mono- and disubstituted N-[3-(2-pyridinyl)isoquinoline]benzamides. The ratio of the tautomers of the benzamides was determined in the solid state and in solution by spectroscopic techniques (IR and NMR). Affinities were determined in radioligand binding assays at rat brain A1 and A2A receptors and at cloned human A3 receptor. The benzamides showed higher adenosine A3 receptor affinity than aliphatic amides. We propose that the adenosine A3 receptor affinity of the different benzamides is related to their presence in either the iminol or amide form. Ligands present in the iminol form showed relatively high adenosine A3 receptor affinity. Finally, we explored the influence of replacement of C4 of the isoquinoline ring by a nitrogen atom. Comparison of isoquinolines with the corresponding quinazolines revealed that both compounds showed similar adenosine A3 receptor affinity. These investigations led to potent and selective human adenosine A3 receptor ligands with affinities in the nanomolar range. The subtype-selective compound 4-methoxy-N-[2-(2-pyridinyl)quinazolin-4-yl]benzamide (VUF8504, 13) with an affinity of 17.0 nM at the human adenosine A3 receptor might become a useful tool in the pharmacological characterization or the investigation of the physiological function of this receptor.

Isoquinoline and quinazoline urea analogues as antagonists for the human adenosine A(3) receptor.

Isoquinoline and quinazoline urea derivatives were found to bind to human adenosine A(3) receptors. Series of N-phenyl-N'-quinazolin-4-ylurea derivatives and N-phenyl-N'-isoquinolin-1-ylurea derivatives were synthesized and tested in radioligand binding assays on their adenosine receptor affinities. A structure-affinity analysis indicated that on the 2-position of the quinazoline ring or the equivalent 3-position of the isoquinoline ring a phenyl or heteroaryl substituent increased the adenosine A(3) receptor affinity in comparison to unsubstituted or aliphatic derivatives. Furthermore, the structure-affinity relationship of substituted phenylurea analogues was investigated. Substituents such as electron-withdrawing or electron-donating groups were introduced at different positions of the benzene ring to probe electronic and positional effects of substitution. Substitution on the 3- or 4-position of the phenyl ring decreased the adenosine A(3) receptor affinity. Substitution at position 2 with an electron-donating substituent, such as methyl or methoxy, increased human adenosine A(3) receptor affinity, whereas substitution on the 2-position with an electron-withdrawing substituent did not influence affinity. Combination of the optimal substituents in the two series had an additive effect, which led to the potent human adenosine A(3) receptor antagonist N-(2-methoxyphenyl)-N'-(2-(3-pyridyl)quinazolin-4-yl)urea (VUF5574, 10a) showing a K(i) value of 4 nM and being at least 2500-fold selective vs A(1) and A(2A) receptors. Compound 10a competitively antagonized the effect of an agonist in a functional A(3) receptor assay, i.e., inhibition of cAMP production in cells expressing the human adenosine A(3) receptor; a pA(2) value of 8.1 was derived from a Schild plot. In conclusion, compound 10a is a potent and selective human adenosine A(3) receptor antagonist and might be a useful tool in further characterization of the human A(3) receptor.

Thiazole and thiadiazole analogues as a novel class of adenosine receptor antagonists.

Novel classes of heterocyclic compounds as adenosine antagonists were developed based on a template approach. Structure-affinity relationships revealed insights for extended knowledge of the receptor-ligand interaction. We replaced the bicyclic heterocyclic ring system of earlier described isoquinoline and quinazoline adenosine A(3) receptor ligands by several monocyclic rings and investigated the influence thereof on adenosine receptor affinity. The thiazole or thiadiazole derivatives seemed most promising, so we continued our investigations with these two classes of compounds. The large difference between a pyridine and isoquinoline ring in binding adenosine A(1) and A(3) receptors showed the importance of the second ring of the isoquinoline ligands. We prepared several N-[4-(2-pyridyl)thiazol-2-yl]benzamides, and these compounds showed adenosine affinities in the micromolar range. Most surprising in the series of the N-[4-(2-pyridyl)thiazol-2-yl]amides were the retained adenosine affinities by introduction of a cylopentanamide instead of the benzamide. A second series of compounds, the thiadiazolobenzamide series of compounds, revealed potent and selective adenosine receptor antagonists, especially N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-hydroxybenzamide (LUF5437, 8h) showing a K(i) value of 7 nM at the adenosine A(1) receptor and N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-methoxybenzamide (LUF5417, 8e) with a K(i) value of 82 nM at the adenosine A(3) receptor. 4-Hydroxybenzamide 8h is the most potent adenosine A(1) receptor antagonist of this new class of compounds. Structure--affinity relationships showed the existence of a steric restriction at the para-position of the benzamide ring for binding adenosine A(1) and A(3) receptors. The electronic nature of the 4-substituents played an important role in binding the adenosine A(3) receptor. Cis- and trans-4-substituted cyclohexyl derivatives were made next to the 4-substituted benzamide analogues. We used them to study the proposed specific interaction between the adenosine A(1) receptor and the 4-hydroxy group of this class of thiadiazolo compounds, as well as a suggested special role for the 4-methoxy group in binding the A(3) receptor. Both the adenosine A(1) and A(3) receptor slightly preferred the trans-analogues over the cis-analogues, while all compounds showed low affinities at the adenosine A(2A) receptor. Our investigations provided the potent and highly selective adenosine A(1) antagonist N-(3-phenyl-1,2,4-thiadiazol-5-yl)-trans-4-hydroxycyclohexanamide (VUF5472, 8m) showing a K(i) value of 20 nM. A third series of compounds was formed by urea analogues, N-substituted with thiazolo and thiadiazolo heterocycles. The SAR of this class of compounds was not commensurate with the SAR of the previously described quinazoline urea. On the basis of these findings we suggest the existence of a special interaction between adenosine receptors and a region of high electron density positioned between the thia(dia)zole ring and phenyl(pyridyl) ring. Molecular electrostatic potential contour plots showed that for this reason the ligands need either a thiadiazole ring instead of a thiazole or a 2-pyridyl group instead of a phenyl. The derived novel classes of antagonists will be useful for a better understanding of the molecular recognition at the adenosine receptors.

Allosteric modulation of A(3) adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives.

Allosteric modulators of A(1) and A(2A) adenosine receptors have been described; however, for the A(3) adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A(3) adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [(125)I]N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [(125)I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [(3)H]PSB-11 from the A(3) adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A(1) receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [(125)I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [(3)H]N(6)-[(R)-phenylisopropyl]adenosine from the A(1) adenosine receptor or [(3)H]CGS21680 from the A(2A) adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.

Synthesis and use of FSCPX, an irreversible adenosine A1 antagonist, as a 'receptor knock-down' tool.

A new preparative synthetic route for the irreversible adenosine A1 antagonist 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1-N-propyl-xanthine (FSCPX, 1) is described. The availability of ample amounts of the irreversible antagonist FSCPX allowed us to use FSCPX as a research tool for adenosine A1 receptors in in vivo experiments. After verification of the irreversible antagonistic function of FSCPX in in vitro experiments, FSCPX was used successfully as a 'receptor knock-down' tool in in vivo experiments on conscious rats.