Inhibitor Report for: KT203

The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) associated Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to cellular angiotensin converting enzyme 2 (ACE2) receptor. The virus specific molecular interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure toward exponentially expanding COVID-19. Thereto, high throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the receptor binding domain of spike protein (S-RBD) and ACE2 host cell receptor. Primary screening identified a few promising molecules for both the targets, which were further analyzed in details by their binding energy, binding modes through molecular docking, dynamics and simulations. Evidently, GR 127935 hydrochloride hydrate, GNF-5, RS504393, TNP, and eptifibatide acetate were found binding to virus binding motifs of ACE2 receptor. Additionally, KT203, BMS195614, KT185, RS504393, and GSK1838705A were identified to bind at the receptor binding site on the viral S-protein. These identified molecules may effectively assist in controlling the rapid spread of SARS-CoV-2 by not only potentially inhibiting the virus at entry step but are also hypothesized to act as anti-inflammatory agents, which could impart relief in lung inflammation. Timely identification and determination of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-CoV-2 inhibition efficiency by these molecules could save lives is justified.

alpha/beta-Hydrolase domain-containing 6 (ABHD6) contributes to the hydrolysis of the major endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS) and in the periphery. ABHD6 blockade has been proposed as novel strategy to treat multiple sclerosis (MS), based on the observation that the inhibitor WWL70 exerts protective anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE). According to recent data, WWL70 exhibits off-target anti-inflammatory activity in microglial cells and the potential of ABHD6 as drug target in MS remains controversial. Here we further investigated the role of ABHD6 during autoimmune demyelination by comparing the efficacy of two novel inhibitors with different CNS permeability in vivo. Preventive treatment with the systemically active inhibitor KT182 ameliorated the neurological signs of EAE during the time-course of disease. By contrast, administration of the peripherally restricted compound KT203 was ineffective in attenuating EAE symptomatology. Both inhibitors failed to improve corticospinal tract conduction latency and to attenuate inflammation at EAE recovery phase, despite being equally active at targeting brain ABHD6. Chronic administration of KT182 was associated to a partial loss of brain CB(1) receptor coupling ability, suggesting the engagement of CB(1) receptor-mediated mechanisms during the EAE disease progression. In cultured neurons, KT182 attenuated NMDA-stimulated excitotoxicity and mitochondrial calcium overload. However, these protective effects were not attributable to ABHD6, as they were not mimicked by the alternative inhibitors KT203, KT195 and WWL70. These results indicate that ABHD6 blockade exerts only modest therapeutic effects against autoimmune demyelination and call into question its utility as novel drug target in MS.

Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) associated Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to cellular angiotensin converting enzyme 2 (ACE2) receptor. The virus specific molecular interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure toward exponentially expanding COVID-19. Thereto, high throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the receptor binding domain of spike protein (S-RBD) and ACE2 host cell receptor. Primary screening identified a few promising molecules for both the targets, which were further analyzed in details by their binding energy, binding modes through molecular docking, dynamics and simulations. Evidently, GR 127935 hydrochloride hydrate, GNF-5, RS504393, TNP, and eptifibatide acetate were found binding to virus binding motifs of ACE2 receptor. Additionally, KT203, BMS195614, KT185, RS504393, and GSK1838705A were identified to bind at the receptor binding site on the viral S-protein. These identified molecules may effectively assist in controlling the rapid spread of SARS-CoV-2 by not only potentially inhibiting the virus at entry step but are also hypothesized to act as anti-inflammatory agents, which could impart relief in lung inflammation. Timely identification and determination of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-CoV-2 inhibition efficiency by these molecules could save lives is justified.

alpha/beta-Hydrolase domain-containing 6 (ABHD6) contributes to the hydrolysis of the major endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS) and in the periphery. ABHD6 blockade has been proposed as novel strategy to treat multiple sclerosis (MS), based on the observation that the inhibitor WWL70 exerts protective anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE). According to recent data, WWL70 exhibits off-target anti-inflammatory activity in microglial cells and the potential of ABHD6 as drug target in MS remains controversial. Here we further investigated the role of ABHD6 during autoimmune demyelination by comparing the efficacy of two novel inhibitors with different CNS permeability in vivo. Preventive treatment with the systemically active inhibitor KT182 ameliorated the neurological signs of EAE during the time-course of disease. By contrast, administration of the peripherally restricted compound KT203 was ineffective in attenuating EAE symptomatology. Both inhibitors failed to improve corticospinal tract conduction latency and to attenuate inflammation at EAE recovery phase, despite being equally active at targeting brain ABHD6. Chronic administration of KT182 was associated to a partial loss of brain CB(1) receptor coupling ability, suggesting the engagement of CB(1) receptor-mediated mechanisms during the EAE disease progression. In cultured neurons, KT182 attenuated NMDA-stimulated excitotoxicity and mitochondrial calcium overload. However, these protective effects were not attributable to ABHD6, as they were not mimicked by the alternative inhibitors KT203, KT195 and WWL70. These results indicate that ABHD6 blockade exerts only modest therapeutic effects against autoimmune demyelination and call into question its utility as novel drug target in MS.

Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

The endocannabinoid system remains an attractive molecular target for pharmacological intervention due to its roles in the central nervous system in learning, thinking, emotional function, regulation of food intake or pain sensation, as well as in the peripheral nervous system, where it modulates the action of cardiovascular, immune, metabolic or reproductive function. alpha/beta hydrolase domain containing 6 (ABHD6)-an enzyme forming part of the endocannabinoid system-is a newly discovered post-genomic protein acting as a 2-AG (2-arachidonoylglycerol) serine hydrolase. We have recently reported a series of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors. Here, we present comparative molecular field analysis (CoMFA) and molecular dynamics studies of these compounds. First, we performed a homology modeling study of ABHD6 based on the assumption that the catalytic triad of ABHD6 comprises Ser148-His306-Asp 278 and the oxyanion hole is formed by Met149 and Phe80. A total of 42 compounds was docked to the homology model using the Glide module from the Schrodinger suite of software and the selected docking poses were used for CoMFA alignment. A model with the following statistics was obtained: R (2) = 0.98, Q (2) = 0.55. In order to study the molecular interactions of the inhibitors with ABHD6 in detail, molecular dynamics was performed with the Desmond program. It was found that, during the simulations, the hydrogen bond between the inhibitor carbonyl group and the main chain of Phe80 is weakened, whereas a new hydrogen bond with the side chain of Ser148 is formed, facilitating the possible formation of a covalent bond. Graphical Abstract Left-right: Docking pose of 1 in the binding pocket of alpha/beta hydrolase domain containing 6 (ABHD6) selected for molecular alignment; CoMFA steric and electrostatic contour fields; changes in potential energy of the complex during simulations for the complex of 6 and ABHD6.