Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

Low-latency gravitational wave alert products and their performance at the time of the fourth LIGO-Virgo-KAGRA observing run.

Multimessenger searches for binary neutron star (BNS) and neutron star-black hole (NSBH) mergers are currently one of the most exciting areas of astronomy. The search for joint electromagnetic and neutrino counterparts to gravitational wave (GW)s has resumed with ALIGO's, AdVirgo's and KAGRA's fourth observing run (O4). To support this effort, public semiautomated data products are sent in near real-time and include localization and source properties to guide complementary observations. In preparation for O4, we have conducted a study using a simulated population of compact binaries and a mock data challenge (MDC) in the form of a real-time replay to optimize and profile the software infrastructure and scientific deliverables. End-toend performance was tested, including data ingestion, running online search pipelines, performing annotations, and issuing alerts to the astrophysics community. We present an overview of the low-latency infrastructure and the performance of the data products that are now being released during O4 based on the MDC. We report the expected median latency for the preliminary alert of full bandwidth searches (29.5 s) and show consistency and accuracy of released data products using the MDC. We report the expected median latency for triggers from early warning searches (-3.1 s), which are new in O4 and target neutron star mergers during inspiral phase. This paper provides a performance overview for LIGO-Virgo-KAGRA (LVK) low-latency alert infrastructure and data products using theMDCand serves as a useful reference for the interpretation of O4 detections.

A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains.

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Comprehensive characterization and preclinical assessment of an imidazopyridine-based anticancer lead molecule.

Imidazopyridine scaffold holds significant pharmacological importance in the treatment of cancer. An in-house synthesized imidazopyridine-based molecule was found to have promising anticancer activity against breast cancer, lung cancer, and colon cancer. The molecule is an inhibitor of pyruvate kinase M2, the enzyme that elevates tumor growth, metastasis and chemoresistance by directly controlling tumor cell metabolism. Screening of the physicochemical properties of any lead molecules is essential to avoid failure in late-stage drug development. In this research, the physicochemical properties of the molecule including log P, log D, pKa, and plasma protein binding were assessed to check its drug-likeness. Plasma and metabolic stability of the molecule were also evaluated. Moreover, pharmacokinetic profiles of the lead molecule in Sprague-Dawley rats and in vitro metabolite identification studies were also performed. Finally, an in silico software, Pro-Tox-II, was used to predict toxicity of the molecule and its metabolites. Log P, Log D (pH 7.4), pKa, and plasma protein binding of the molecule were found to be 2.03%, 2.42%, 10.4%, and 98%, respectively. The molecule was stable in plasma and metabolic conditions. A total of nine new metabolites were identified and characterized. Cmax and t½ of this molecule were found to be 4016 ± 313.95 ng/mL and 9.57 ± 3.05 h, respectively. Based on the previously reported study and this finding, the molecule can be considered as a promising anticancer lead with potential drug-likeness properties. Further preclinical and clinical drug discovery studies may be initiated in continuation of this study in search of a potential anticancer lead.

PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain.

Leucine-rich-repeat-kinase 1 (LRRK1) and its homolog LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson's disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ∼3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the αC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.

Conformational plasticity of the ULK3 kinase domain.

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Lessons from LIMK1 enzymology and their impact on inhibitor design.

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique 'rock-and-poke' mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding.

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina.

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.

Influence of Arrestin on the Photodecay of Bovine Rhodopsin.

Continued activation of the photocycle of the dim-light receptor rhodopsin leads to the accumulation of all-trans-retinal in the rod outer segments (ROS). This accumulation can damage the photoreceptor cell. For retinal homeostasis, deactivation processes are initiated in which the release of retinal is delayed. One of these processes involves the binding of arrestin to rhodopsin. Here, the interaction of pre-activated truncated bovine visual arrestin (Arr(Tr)) with rhodopsin in 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) micelles is investigated by solution NMR techniques and flash photolysis spectroscopy. Our results show that formation of the rhodopsin-arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin, which involves the simultaneous formation of a meta II and a meta III state from the meta I state. Binding of Arr(Tr) leads to an increase in the population of the meta III state and consequently to an approximately twofold slower release of all-trans-retinal from rhodopsin.

Design of photocaged puromycin for nascent polypeptide release and spatiotemporal monitoring of translation.

The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC-puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome-bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC-puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2% and the NVOC-puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two-photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons.

Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy.

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype G protein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε-(15)N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The meta II and meta III states emerge in parallel with a relative ratio of about 3:1. Transient formation of the meta III state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2 -loop in the formation of the meta III state.

Mechanistic Investigation of Thiazole-Based Pyruvate Kinase M2 Inhibitor Causing Tumor Regression in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a deadly breast cancer with a poor prognosis. Pyruvate kinase M2 (PKM2), a key rate-limiting enzyme in glycolysis, is abnormally highly expressed in TNBC. Overexpressed PKM2 amplifies glucose uptake, enhances lactate production, and suppresses autophagy, thereby expediting the progression of oncogenic processes. A high mortality rate demands novel chemotherapeutic regimens at once. Herein, we report the rational development of an imidazopyridine-based thiazole derivative 7d as an anticancer agent inhibiting PKM2. Nanomolar range PKM2 inhibitors with favorable drug-like properties emerged through enzyme assays. Experiments on two-dimensional (2D)/three-dimensional (3D) cell cultures, lactate release assay, surface plasmon resonance (SPR), and quantitative real-time polymerase chain reaction (qRT-PCR) validated 7d preclinically. In vivo, 7d outperformed lapatinib in tumor regression. This investigation introduces a lead-based approach characterized by its clear-cut chemistry and robust efficacy in designing an exceptionally potent inhibitor targeting PKM2, with a focus on combating TNBC.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

Roadmap to Pyruvate Kinase M2 Modulation - A Computational Chronicle.

Pyruvate kinase M2 (PKM2) has surfaced as a potential target for anti-cancer therapy. PKM2 is known to be overexpressed in the tumor cells and is a critical metabolic conduit in supplying the augmented bioenergetic demands of the recalcitrant cancer cells. The presence of PKM2 in structurally diverse tetrameric as well as dimeric forms has opened new avenues to design novel modulators. It is also a truism to state that drug discovery has advanced significantly from various computational techniques like molecular docking, virtual screening, molecular dynamics, and pharmacophore mapping. The present review focuses on the role of computational tools in exploring novel modulators of PKM2. The structural features of various isoforms of PKM2 have been discussed along with reported modulators. An extensive analysis of the structure-based and ligand- based in silico methods aimed at PKM2 modulation has been conducted with an in-depth review of the literature. The role of advanced tools like QSAR and quantum mechanics has been established with a brief discussion of future perspectives.

PROTACting the kinome with covalent warheads.

The dawn of targeted degradation using proteolysis-targeting chimeras (PROTACs) against recalcitrant proteins has prompted numerous efforts to develop complementary drugs. Although many of these are specifically directed against undruggable proteins, there is increasing interest in small molecule-based PROTACs that target intracellular pathways, and some have recently entered clinical trials. Concurrently, small molecule-based PROTACs that target protumorigenic pathways in cancer cells, the tumor microenvironment (TME), and angiogenesis have been found to have potent effects that synergize with the action of antibodies. This has led to the augmentation of PROTACs with variable substitution patterns. Several combinations with small molecules targeting undruggable proteins are now under clinical investigation. In this review, we discuss the recent milestones achieved as well as challenges encountered in this area of drug development, as well as our opinion on the best path forward.

Inhibition of Parkinson's disease-related LRRK2 by type I and type II kinase inhibitors: Activity and structures.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD) and a risk factor for the sporadic form. Increased kinase activity was shown in patients with both familial and sporadic PD, making LRRK2 kinase inhibitors a major focus of drug development efforts. Although much progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-electron microscopy structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type I inhibitor MLi-2 and the broad-spectrum type II inhibitor GZD-824. Our structures revealed an active-like LRRK2 kinase in the type I inhibitor complex, and an inactive DYG-out in the type II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structures provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.

Structure of LRRK1 and mechanisms of autoinhibition and activation.

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinson's disease while LRRK1 is linked to bone diseases. Furthermore, Parkinson's disease-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. Here, to understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.