1,2,6-thiadiazinones as novel narrow spectrum calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) inhibitors

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors

The C-terminal domains SnRK2 box and ABA box have a role in sugarcane SnRK2S auto-activation and activity

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s-known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain alpha C helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP465651/2014-3; 141368/2018-733003017024P2; 88887.158494/2017-002013/50724-5; 2013/155765; 2014/50897This work was supported by the Brazilian agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (2013/50724-5, 2013/155765 and 2014/50897) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (465651/2014-3). The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and Wellcome [106169/ZZ14/Z]. Germanna Righetto received fellowships from CAPES (Coordenação de Aperfeiçomento de Pessoal de Nível Superior) (33003017024P2) and CNPq (141368/2018-7). CC received a CAPES INCT fellowship (88887.158494/2017-00

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S.pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S.pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S.pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization

1,2,6-thiadiazinones as novel narrow spectrum calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) inhibitors

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors

Binding and structural analyses of potent inhibitors of the human Ca2+/calmodulin dependent protein kinase kinase 2 (CAMKK2) identified from a collection of commercially-available kinase inhibitors

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP465651/2014-388887.136342/2017-00; 88887.136432/2017-00; 88887.136437/2017-00; 88887.146077/2017-002019/14275-8; 2016/09041-0; 2017/05697-0Brazilian agency FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/50724-5, 2014/50897-0]; Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)National Council for Scientific and Technological Development (CNPq) [465651/2014-3]; AbbVie; Bayer Pharma AG; Boehringer IngelheimBoehringer Ingelheim; Canada Foundation for InnovationCanada Foundation for Innovation; Eshelman Institute for Innovation; Genome CanadaGenome Canada; Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD] [115766]; JanssenJohnson & Johnson USAJanssen Biotech Inc; Merck KGaA Darmstadt Germany; MSD; Novartis Pharma AG; Ontario Ministry of Economic Development and Innovation; PfizerPfizer; TakedaTakeda Pharmaceutical Company Ltd; Wellcome [106169/ZZ14/Z]; National Cancer Institute of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [R01CA218442]; CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)CAPES [88887.136342/2017-00, 88887.136432/2017-00, 88887.136437/2017-00, 88887.146077/2017-00]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2019/14275-8, 2016/09041-0, 2017/05697-0]; National Institute of General Medical Sciences from the National Institutes of Health [GU51510, GU56413, P41 GM103403]; NIH-ORIP HEI grant [S10 RR029205]; DOE Office of Science by Argonne National LaboratoryUnited States Department of Energy (DOE) [DE-AC02-06CH11357

Development of pyridine-based inhibitors for the human vaccinia-related kinases 1 and 2

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no potent and specific inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compound for VRK1 (26) displayed an IC50 value of 150 nM and was fairly selective in a panel of 48 human kinases (selectivity score S(50%) of 0.04). Differences in compound binding mode and substituent preferences between the two VRKs were identified by the structure-activity relationship combined with the crystallographic analysis of key compounds. We expect our results to serve as a starting point for the design of more specific and potent inhibitors against each of the two VRKs10912661271CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP465651/2014-3; 400906/2014-788887.146077/2017-00; 88887.136342/2017-002013/50724-5; 2014/5087-0; 2016/25320-6; 2018/09475-5; 2018/03359-3This work was supported by the Brazilian agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (2013/50724-5 and 2014/5087-0), Embrapii (Empresa Brasileira de Pesquisa e Inovação Industrial), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (465651/2014-3 and 400906/2014-7). The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and Wellcome [106169/ZZ14/Z]. R.A.M.S., S.N.S.V. and F.D.P. received FAPESP fellowships (2016/25320-6, 2018/09475-5 and 2018/03359-3 respectively). C.V.R. and A.S.S. received CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) postdoctoral fellowships (88887.146077/2017-00 and 88887.136342/2017-00, respectively

A Chemical Probe Targeting AAK1 and BMP2K

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.</p

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934 (CAMKK2 IC50 = 3 nM), a total of 32 compounds, composed of single ring, 5,6-, and 6,6-fused heteroaromatic cores were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. These compounds were evaluated in vitro in biochemical and cellular assays for CAMKK2 inhibition. Compared to GSK650394 and STO-609, thirteen of our compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had greatly improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs aimed at the identification of CAMKK2 chemical probes and clinical candidates</div