Highlights d Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed d Conformational changes in nsp8 and its interaction with the exiting RNA are observed d Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated d Transition model from primase complex to polymerase complex is proposed

The antineoplastic drug carmofur is shown to inhibit the SARS-CoV-2 main protease (M pro ). Here, the X-ray crystal structure of M pro in complex with carmofur reveals that the carbonyl reactive group of carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC 50 = 24.30 μM) and is a promising lead compound to develop new antiviral treatment for COVID-19.

Graphical Abstract Highlights d The crystal structure of Mycobacterium smegmatis MmpL3 has been determined d Two Asp-Tyr pairs in the TM region of MmpL3 facilitate proton-translocation d SQ109, an anti-TB drug, binds inside the protontranslocation channel of MmpL3 d Rimonabant, an antagonist for the cannabinoid receptor CB 1 , also inhibits MmpL3

Anaplastic lymphoma kinase (ALK) activation has been associated with many types of human cancer. Significant efforts have been devoted to the development of ALK inhibitors to antagonize the kinase activity of ALK. Four ALK inhibitors have been approved by the FDA to date for treating patients with ALK-positive non-small cell lung cancers (NSCLC). However, drug resistance has been observed in the majority of patients treated with these inhibitors. New therapeutic strategies (e.g., compounds with novel mechanisms of action) are needed to overcome the drug resistance issue. The emerging PROTAC (Proteolysis Targeting Chimera) technology has been successfully applied to selective degradation of multiple protein targets, but not ALK. Since ALK protein levels are not important for viability in mammals, ALK PROTACs could lead to novel therapeutics with minimal toxicity. Here we report the design, synthesis and biological evaluation of novel PROTACs (degraders) of ALK. MS4077 (5) and MS4078 (6) potently decreased cellular levels of oncogenic active ALK fusion proteins in a concentration- and time-dependent manner in SU-DHL-1 lymphoma and NCI-H2228 lung cancer cells. The ALK protein degradation induced by compounds 5 and 6 was cereblon and proteasome dependent. In addition, compounds 5 and 6 potently inhibited proliferation of SU-DHL-1 cells. Furthermore, compound 6 displayed good plasma exposure in a mouse pharmacokinetic study, thus is suitable for in vivo efficacy studies. We also developed MS4748 (7) and MS4740 (8), very close analogs of 5 and 6 respectively, which are incapable to degrade the ALK fusion proteins, as negative controls. Compounds 5-8 are valuable chemical tools for investigating effects of ALK pharmacological degradation. Our study paved the way for developing the next generation of ALK PROTACs.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed β-arrestin2 (βarr2)-biased D2R partial agonists to simultaneously target hyperand hypodopaminergia. Using neuron-specific βarr2-KO mice, we show that the antipsychotic-like effects of a βarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-βarr2 signaling. Furthermore, βarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of βarr2 and G proteincoupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that βarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.arrestin | antipsychotics | biased signaling | dopamine D2R | fast-spiking interneurons G protein-coupled receptors (GPCRs) represent the largest family of receptors in the human genome and are one of the most common targets of pharmaceutical drugs (1, 2). Upon ligand binding, GPCRs activate downstream G protein-dependent signaling pathways followed by phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs) (3). Phosphorylation enhances association of the GPCR with β-arrestins (βarrs), and this combined process mediates desensitization of G-protein signaling (4) and internalization of GPCRs (5-7). Two isoforms of βarrs, βarr1 and βarr2, are widely coexpressed in most tissues in mammals and are 80% identical, but they can have either overlapping or distinct functions (8, 9). It is now firmly established that GPCRs activate downstream signaling pathways through not only canonical G-protein pathways but also, the ability of βarrs to scaffold distinct intracellular signaling complexes (10-12). Elucidation of these distinct G-protein and βarr signaling pathways has provided support for the concept of functional selectivity or biased signaling, wherein each signaling pathway has the ability to mediate distinct physiological responses (13). There are now several physiologically relevant examples of selective engagement of signaling pathways or selective GPCR ligands that target these different signaling pathways (13-15). Therefore, leveraging the concept of GPCR functional selectivity holds promise for the development of more selective therapeutic approaches. Dopamine (DA) is a catecholamine neurotransmitter that has been implicated in movement, reward, and cognition (16-19) as well as CNS disorders, such as schizophrenia, attention deficit hyperactivity disorder, Parkinson's disease, and obsessive-compulsive disorder (20-23). DA mediates its effects via GPCRs belonging to two major ...

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Over the past decade, two independent technologies have emerged and been widely adopted by the neuroscience community for remotely controlling neuronal activity: optogenetics which utilize engineered channelrhodopsin and other opsins, and chemogenetics which utilize engineered G protein-coupled receptors (Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)) and other orthologous ligand–receptor pairs. Using directed molecular evolution, two types of DREADDs derived from human muscarinic acetylcholine receptors have been developed: hM3Dq which activates neuronal firing, and hM4Di which inhibits neuronal firing. Importantly, these DREADDs were not activated by the native ligand acetylcholine (ACh), but selectively activated by clozapine N-oxide (CNO), a pharmacologically inert ligand. CNO has been used extensively in rodent models to activate DREADDs, and although CNO is not subject to significant metabolic transformation in mice, a small fraction of CNO is apparently metabolized to clozapine in humans and guinea pigs, lessening the translational potential of DREADDs. To effectively translate the DREADD technology, the next generation of DREADD agonists are needed and a thorough understanding of structure–activity relationships (SARs) of DREADDs is required for developing such ligands. We therefore conducted the first SAR studies of hM3Dq. We explored multiple regions of the scaffold represented by CNO, identified interesting SAR trends, and discovered several compounds that are very potent hM3Dq agonists but do not activate the native human M3 receptor (hM3). We also discovered that the approved drug perlapine is a novel hM3Dq agonist with >10 000-fold selectivity for hM3Dq over hM3.

25COVID-19 virus is the cause of a debilitating and life-threatening infectious 26 pulmonary disease that is now responsible for a global pandemic. Currently, there are 27 no specific drugs or vaccines to contain this virus. The main protease (M pro ) of COVID-28 19 virus is a key enzyme, which plays an essential role in viral replication and 29 transcription, making it an ideal drug target. An FDA-approved antineoplastic drug, 30 carmofur, has been identified as an inhibitor that targets COVID-19 virus M pro . 31However, its inhibitory mechanism is unknown. Here, we report the 1.6-Å crystal 32 structure of COVID-19 virus M pro in complex with carmofur. The crystal structure 33 shows that carmofur contains an electrophilic carbonyl reactive group, which 34 covalently binds to C145, a member of the catalytic dyad. As a result, its fatty acid tail 35 occupies the hydrophobic S2 subsite of M pro whilst its 5-fluorouracil head is cleaved as 36 product of the new covalent bond that has formed. Carmofur is active in a cell based 37 antiviral assay with an EC50 of 24.87 μM. It is therefore a promising lead compound 38 for the development of new antivirals to target COVID-19. : bioRxiv preprint 41 Starting in December 2019, a highly infectious viral disease has now spread and 42 reached over 200 countries leading to a global public health emergency and pandemic. 43The etiological agent of the disease is a coronavirus (identified as . 44According to the WHO COVID-2019 Situation Report-77, there were 1,210,956 45 confirmed cases and 67,594 deaths, with a mortality rate at 5.58%. The number of 46 confirmed cases worldwide continues to grow at a rapid rate and is far from peaking. 47However, there are no specific drugs or vaccines available to control symptoms or the 48 spread of this disease. 49The COVID-19 virus has a ~30,000 nt RNA genome encoded with two translation 50 products, polyproteins 1a and 1ab (pp1a and pp1ab) which are crucial for replication 51 and transcription 1,2 . These polyproteins become matured non-structural and structural 52 proteins through auto-cleavage by the main protease (M pro ) and by a papain-like 53 protease 3 . Because of this, M pro is an excellent target for anti-coronavirus (CoV) drug 54 development 4-6 . In order to rapidly discover new drug leads that target COVID-19 virus 55 M pro , our group screened over 10,000 compounds from a library that consisted of 56 approved drugs, drug candidates in clinical trials, and other pharmacologically active 57 compounds. Amongst these we identified carmofur as compound that can inhibit M pro 58 with an IC50 of 1.82 μM 7 . 59Carmofur is an FDA-approved antineoplastic drug, and a derivative of 5-60 fluorouracil (5-FU) a widely drug used against solid cancers. 5-FU is especially 61 efficient for controlling head, neck, and gastrointestinal tumors 8 . Carmofur (Figure 1A) 62 is a derivative of this compound and has been used in colon cancer therapy since 1981 9 . 63Clinical research has also shown that carmofur has a curative effect on breast, gastri...