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Small molecule macrocyclic kinase inhibitors have attracted significant attention in drug discovery over the past years with drugs approval such as lorlatinib demonstrating the clinical relevance of this approach. We developed our expertise to further optimize those cyclic molecules. Having low molecular weight, they favorably alter the biological and physiochemical properties as well as selectivity, as compared to their linear parent, yielding high-quality drug candidates. While focused on kinase inhibitors, macrocyclic derivatives could be potentially turned into bifunctional protein degrader molecules useful for selective cellular knockdown of targeted proteins and investigation of the pharmacological effects. With macrocyclic “probes” from our proprietary library in nanomolar range IC50, we turned our attention to CDK9 inhibitors with good selectivity profile against CDK1/2/5/7. Previous CDK9 degraders based on acyclic inhibitors have used thalidomide as recruiter of the CRL4CRBN, resulting in successful ubiquitination and proteasomal degradation of the protein of interest. Applying a three-step approach, we first defined the vector of substitution on our macrocyclic “probes” by homology modeling with known acyclic ligands/CDK9 co-crystal structures. Having defined 2 substitution patterns, we synthesized and tested in a biochemical assay the novels “probes” vectorized with the pro-linker moieties, without loss of activity. ODS’7152 was further derived into fully bifunctional molecules with variation on the linker’s nature and length, keeping thalidomide as the E3 ligase recruiter. Biophysical and biological properties were further evaluated in an MTS assay against several cell lines (HCT116, Jurkat E6.2, MOLT-4 and MV4-11) to assess cell proliferation and viability. The E3 Ligase engagement along with cell penetration was measured in a NanoBRET™ assay and compared to MDR_MDCK permeability. Finally, a selection of 4 PROTACs were evaluated in a dose response assay on MV4-11 for protein degradation phosphorylation of CTD-pol II and selectivity among several CDKs. Our findings support the rapid derivatization of macrocyclic “probes” inhibitors into macrocyclic-PROTAC as a strategy to generate early chemical biology tools with maintained potency and selectivity between homologous targets. Optimization of the physico-chemical and ADME properties of molecules can lead to drug candidates with distinct pharmacological effects as compared to the parent linear kinase inhibitors. Citation Format: Nerina Dodic, Nicolas Ancellin, Morgane Bordessoules, Anne Bouillot, Nathan Butin, Cedric Charrier, Marie-Helene Fouchet, Alain Laroze, Anne-Pascale Luzy, Alexandre Moquette, Julia Pilot, Guillaume Serin, Jean-François Mirjolet, Fabrice Viviani, Christophe Parsy. Structure-based design of small macrocyclic CDK9 degraders as chemical biology tools and beyond [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1398.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α

Severe α1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z α1-antitrypsin. The lead compound blocks Z α1-antitrypsin polymerisationin vitro, reduces intracellular polymerisation and increases the secretion of Z α1-antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1-antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that ‘mutation ameliorating’ small molecules are a viable approach to treat protein conformational diseases.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.

To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.

We describe the discovery of novel potent inhibitors of 2,3-oxidosqualene:lanosterol cyclase inhibitors (OSCi) from a focused pharmacophore-based screen. Optimization of the most tractable hits gave a series of compounds showing inhibition of cholesterol biosynthesis at 2 mg/kg in the rat with distinct pharmacokinetic profiles. Two compounds were selected for toxicological study in the rat for 21 days in order to test the hypothesis that low systemic exposure could be used as a strategy to avoid the ocular side effects previously described with OSCi. We demonstrate that for this series of inhibitors, a reduction of systemic exposure is not sufficient to circumvent cataract liabilities.

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.

A series of 6-phenylpyrazolo[3,4-d]pyrimidones is described which are specific inhibitors of cGMP specific (type V) phosphodiesterase. Enzymatic and cellular activity as well as in vivo oral antihypertensive activity are evaluated. A n-propoxy group at the 2-position of the phenyl ring is necessary for activity. A series of products substituted at the 5-position in addition to the 2-n-propoxy was prepared and evaluated. This position can accommodate many unrelated groups. Amino derivatives were very potent but lacked metabolic stability. Substitution by carbon-linked small heterocycles provided both high levels of activity and stability. Cellular activity very often correlated with in vivo activity. Among the compounds, 1,3-dimethyl-6(2-propoxy-5-methanesulfonamidophenyl)-1,5-dihydr opyrazolo[3,4-d]pyrimidin-4-one (38) and 1-ethyl-3-methyl-6-(2-propoxy-5-(4-methylthiazol-2-yl)phenyl -1,5-dihy dropyrazolo[3,4-d]pyrimidin-4-one (59) displayed outstanding in vivo activities at 5 mg/kg/os and good metabolic stabilities.

Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.

A series of 6-phenylpyrazolo[3,4-d]pyrimidones is described which are specific inhibitors of cGMP specific (type V) phosphodiesterase. Enzymatic and cellular activity as well as in vivo oral antihypertensive activity are evaluated. A n-propoxy group at the 2-position of the phenyl ring is necessary for activity. A series of products substituted at the 5-position in addition to the 2-n-propoxy was prepared and evaluated. This position can accommodate many unrelated groups. Amino derivatives were very potent but lacked metabolic stability. Substitution by carbon-linked small heterocycles provided both high levels of activity and stability. Cellular activity very often correlated with in vivo activity. Among the compounds, 1,3-dimethyl-6-(2-propoxy-5-methanesulfonamidophenyl)-1,5-dihydropyrazolo[3,4-d]pyrimidin-4-one (38) and 1-ethyl-3-methyl-6-(2-propoxy-5-(4-methylthiazol-2-yl)phenyl)-1,5-dihydropyrazolo[3,4-d]pyrimidin-4-one (59) displayed outstanding in vivo activities at 5 mg/kg/os and good metabolic st...

Starting from quinazoline 3a, we designed potent and selective ALK5 inhibitors over p38MAP kinase from a rational drug design approach based on co-crystal structures in the human ALK5 kinase domain. The quinazoline 3d exhibited also in vivo activity in an acute rat model of DMN-induced liver fibrosis when administered orally at 5 mg/kg (bid).

A number of tricyclic carboxamides have been synthesized and tested to evaluate their ability to reverse multidrug resistance in the CHRC/5 cell line. Among them the acridone derivatives were the most potent. A key feature is the presence of a dimethoxybenzyl or phenethylamine cationic site, separated from the tricyclic lipophilic part by a carbamoylphenyl chain. Optimization led to compounds 2 orders of magnitude more active than the prototype inhibitors verapamil and amiodarone. On the basis of in vitro and in vivo activities, 9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7- dimethoxyisoquinol-2-yl)ethyl]phenyl]-4-acridinecarboxamide (84) has been selected for further development.