The pharmaceutical industry has recognized that many drug-like molecules can self-aggregate in aqueous media and have physicochemical properties that skew experimental results and decisions. Herein, we introduce the use of a simple NMR strategy for detecting the formation of aggregates using dilution experiments that can be performed on equipment prevalent in most synthetic chemistry departments. We show that (1)H NMR resonances are sensitive to large molecular-size entities and to smaller multimers and mixtures of species. Practical details are provided for sample preparation and for determining the concentrations of single molecule, aggregate entities, and precipitate. The critical concentrations above which aggregation begins can be found and were corroborated by comparisons with light scattering techniques. Disaggregation can also be monitored using detergents. This NMR assay should serve as a practical and readily available tool for medicinal chemists to better characterize how their compounds behave in aqueous media and influence drug design decisions.

The development of peptidomimetic inhibitors of the human cytomegalovirus (HCMV) protease showing sub-micromolar potency in an enzymatic assay is described. Selective substitution of the amino acid residues of these inhibitors led to the identification of tripeptide inhibitors showing improvements in inhibitor potency of 27-fold relative to inhibitor 39 based upon the natural tetrapeptide sequence. Small side chains at P1 were well tolerated by this enzyme, a fact consistent with previous observations. The S2 binding pocket of HCMV protease was very permissive, tolerating lipophilic and basic residues. The substitutions tried at P3 indicated that a small increase in inhibitor potency could be realized by the substitution of a tert-leucine residue for valine. Substitutions of the N-terminal capping group did not significantly affect inhibitor potency. Pentafluoroethyl ketones, alpha,alpha-difluoro-beta-keto amides, phosphonates and alpha-keto amides were all effective substitutions for the activated carbonyl component and gave inhibitors which were selective for HCMV protease. A slight increase in potency was observed by lengthening the P1' residue of the alpha-keto amide series of inhibitors. This position also tolerated a variety of groups making this a potential site for future modifications which could modulate the physicochemical properties of these molecules.

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIII␤). A good correlation between PI4KIII␤ activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIII␤ inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIII␤ inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIII␤ were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIII␤ is deleterious.

No abstract

The development of novel monobactam inhibitors of HCMV protease incorporating a carbon side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at the C-3 position of the beta-lactam ring gave an increase in enzymatic activity and in stability; however, a lack of selectivity against other serine proteases was noted. The use of both tri- and tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl substitution of the urea moiety was beneficial, especially when strong electron-withdrawing groups where attached at the para position. Modest antiviral activity was found in a plaque reduction assay.

A series of N-tert-butylacetyl-l-tert-butylglycyl-l-Ngamma, Ngamma-dimethylasparagyl-l-alanyl-derived inhibitors (trifluoromethyl ketone 1, pentafluoroethyl ketone, 2, methyl ketone 3, and alpha-ketoamide 4, with respective KI values of 1.1, 0.1, 2100, and 0.2 microM) of the human cytomegalovirus protease were used to study the effect of binding of peptidyl inhibitors on the intrinsic fluorescence and CD properties of the enzyme. In the presence of saturating concentrations of compounds 1, 2, and 4, an identical blue shift in the fluorescence maximum of the enzyme upon specific tryptophan excitation was observed relative to that of the free protease. In the case of the methyl ketone 3, whose inhibition of the enzyme does not involve formation of a covalent adduct as evidenced by 13C NMR studies of carbonyl-labeled inhibitors, the blue shift in the emission was also observed. For both compounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset of inhibition of substrate hydrolysis correlate well with the kobs values of the time-dependent change in the emission spectra. Studies employing a double mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 as the principal fluorescence reporter. Taken together with information provided by our recent elucidation of the crystallographic structure of the enzyme [Tong, L., Qian, C., Massariol, M.-J., Bonneau, P. R., Cordingley, M. G., & Lagacé, L. (1996) Nature 383, 272], these observations are consistent with the inhibition of HCMV protease by peptidyl ketones involving a conformational change of the protease. A mechanism involving a kon limited by dehydration of the hydrated species, followed by rapid ligand binding and a conformational change prior to covalent adduct formation, is proposed for activated inhibitors such as 1 and 2.

No abstract

There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain-penetrant ALK2 inhibitors based on the lead compound LDN-214117. Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009, M4K2117, and M4K2163, each with a superior potency, selectivity, and/or blood− brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced preclinical compounds suitable for further development and evaluation in orthotopic models of DIPG.