ABSTRACT
The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.
ABSTRACT
Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.
Subject(s)
Aminoquinolines/chemistry , Drug Design , Proteins/metabolism , Administration, Oral , Aminoquinolines/metabolism , Aminoquinolines/pharmacokinetics , Aminoquinolines/therapeutic use , Animals , Benzoates/chemistry , Benzoates/metabolism , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Dogs , Half-Life , Humans , Male , Mice , Molecular Conformation , Molecular Dynamics Simulation , Neoplasms/drug therapy , Proteins/antagonists & inhibitors , Rats , Structure-Activity RelationshipABSTRACT
Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Boron Compounds/therapeutic use , Inflammation/drug therapy , Kallikreins/antagonists & inhibitors , Netherton Syndrome/drug therapy , Skin/pathology , Administration, Topical , Animals , Disease Models, Animal , Humans , Kallikreins/genetics , Mice , Mice, Transgenic , Signal Transduction , Skin/drug effects , Skin CreamABSTRACT
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Subject(s)
Benzamidines/chemistry , Kallikreins/antagonists & inhibitors , Netherton Syndrome/drug therapy , Serine Proteinase Inhibitors/chemistry , Amino Acid Sequence , Benzamidines/pharmacology , Binding Sites , Drug Evaluation, Preclinical , Humans , Isomerism , Models, Molecular , Molecular Structure , Mutation , Protein Binding , Serine Peptidase Inhibitor Kazal-Type 5/genetics , Serine Proteinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.
Subject(s)
Drug Design , Kallikreins/antagonists & inhibitors , Netherton Syndrome/drug therapy , HumansABSTRACT
The inclusion of different carrier materials in a dry powder inhaler (DPI) system can alter formulation performance, which might be attributable to variation in the adhesion between drug and carrier particles. The aim of this study was, therefore, to further examine the relationship between drug-carrier adhesion and performance, by comparing data relating to many different drug-carrier combinations. Four drugs and four carriers were employed, giving a total of 16 combinations. The relative magnitude of the drug-carrier adhesion for each combination was quantified using the cohesion-adhesion balance (CAB) approach to colloidal probe atomic force microscopy. The in vitro inhalation performance of the 16 formulations (1.5% w/w drug) was investigated and found to vary significantly. Plots of fine particle dose against drug-carrier CAB ratio revealed that performance was optimised when the drug-carrier CAB ratio was slightly cohesive. This trend was found to fit with those from similar previous studies, although due to the smaller number of formulations investigated previously, the full extent of this relationship had not been revealed. It was concluded, therefore, that when developing a carrier-based DPI, the selection of a drug-carrier combination with a slightly cohesive CAB ratio might result in optimal performance.
Subject(s)
Drug Carriers , Powders , Adhesiveness , Administration, Inhalation , Albuterol/administration & dosage , Albuterol/analogs & derivatives , Albuterol/chemistry , Androstadienes/administration & dosage , Androstadienes/chemistry , Anti-Allergic Agents/administration & dosage , Anti-Allergic Agents/chemistry , Capsules , Chemistry, Pharmaceutical , Crystallization , Excipients , Fluticasone , Lactose/chemistry , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Particle Size , Salmeterol Xinafoate , X-Ray DiffractionABSTRACT
PURPOSE: To investigate the dispersion mechanism(s) of ternary dry powder inhaler (DPI) formulations by comparison of the interparticulate adhesions and in vitro performance of a number of carrier-drug-fines combinations. MATERIALS AND METHODS: The relative levels of adhesion and cohesion between a lactose carrier and a number of drugs and fine excipients were quantified using the cohesion-adhesion balance (CAB) approach to atomic force microscopy. The in vitro performance of formulations produced using these materials was quantified and the particle size distribution of the aerosol clouds produced from these formulations determined by laser diffraction. RESULTS: Comparison between CAB ratios and formulation performance suggested that the improvement in performance brought about by the addition of fines to which the drug was more adhesive than cohesive might have been due to the formation of agglomerates of drug and fines particles. This was supported by aerosol cloud particle size data. The mechanism(s) underlying the improved performance of ternary formulations where the drug was more cohesive than adhesive to the fines was unclear. CONCLUSIONS: The performance of ternary DPI formulations might be increased by the preferential formation of drug-fines agglomerates, which might be subject to greater deagglomeration forces during aerosolisation than smaller agglomerates, thus producing better formulation performance.
Subject(s)
Nebulizers and Vaporizers , Powders/chemistry , Adhesiveness , Aerosols , Chemistry, Pharmaceutical , Microscopy, Atomic Force , Particle SizeABSTRACT
The physico-chemical behaviour of trehalose dihydrate during storage at low relative humidity and ambient temperature was investigated, using a combination of techniques commonly employed in pharmaceutical research. Weight loss, water content determinations, differential scanning calorimetry and X-ray powder diffraction showed that at low relative humidity (0.1% RH) and ambient temperature (25 degrees C) trehalose dihydrate dehydrates forming the alpha-polymorph. Physical examination of trehalose particles by scanning electron microscopy and of the dominant growth faces of trehalose crystals by environmentally controlled atomic force microscopy revealed significant changes in surface morphology upon partial dehydration, in particular the formation of cracks. These changes were not fully reversible upon complete rehydration at 50% RH. These findings should be considered when trehalose dihydrate is used as a pharmaceutical excipient in situations where surface properties are key to behaviour, for example as a carrier in a dry powder inhalation formulations, as morphological changes under common processing or storage conditions may lead to variations in formulation performance.
