Discovery of a CK2α'-Biased ATP-Competitive Inhibitor from a High-Throughput Screen of an Allosteric-Inhibitor-Like Compound Library.

Protein kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2ß) and two catalytic subunits (CK2α and CK2α'). CK2 controls several cellular processes, including proliferation, inflammation, and cell death. However, CK2α and CK2α' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α' has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α' due to their high structural homology, especially in the targeted ATP-binding site. Using computational analyses, we found a potential type IV ("D" pocket) allosteric site that contained different residues between CK2α and CK2α' and was distal from the ATP-binding pocket featured in both kinases. We decided to look for allosteric modulators that might interact in a biased fashion with the type IV pocket on both CK2α and CK2α'. We screened a commercial library containing ∼29,000 allosteric-kinase-inhibitor-like compounds using a CK2α' activity-dependent ADP-Glo Kinase assay. Obtained hits were counter-screened against CK2α using the ADP-Glo Kinase assay, revealing two CK2α'-biased compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

A patent review of SHP2 allosteric inhibitors (2018-present).

INTRODUCTION: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is a target of interest for cancer therapy due to its key role in the regulation of the RAS/MAPK signal transduction pathway downstream of Receptor Tyrosine Kinases (RTKs). Moreover, SHP2 can inhibit T cells via the PD-1/PD-L1 pathway. SHP2 plays a critical role in numerous physiological and pathological cellular processes, such as cell proliferation, survival, and migration. AREAS COVERED: This review examines SHP2 allosteric inhibitors reported in patents published in Espacenet and Scifinder databases from 2018 to present. An overview of claimed structures is conducted, focusing attention on structural modifications compared to SHP099, the first described allosteric inhibitor of SHP2. EXPERT OPINION: Multiple potent allosteric SHP2 inhibitors have been discovered, disclosed, and tested in a variety of preclinical cancer models with strong evidence of efficacy. Fifteen compounds are currently in clinical development, but none of them have been approved for marketing. Until now, long-term benefit of SHP2 inhibitors as monotherapy agents have not been demonstrated due to acquired mechanisms of resistance and/or lack of efficacy. However, combination therapies with a variety of agents, such as MEK, BRAF, EGFR, RAS-G12C and PDL-1 inhibitors, have high potential and are currently an extensive area of investigation.

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase.

The allosteric inhibition of insulin-like growth factor receptor 1 kinase (IGF1RK) is a potential strategy to overcome selectivity barriers for targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives that have been reported as allosteric inhibitors of IGF1RK. We further studied the dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation: forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen-bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the positioning of chemical substituents with different pharmacophore features at the R1 indole ring influences molecular interactions and binding conformations of indole-butyl-amine derivatives and, hence, dramatically affects their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

Computational insights into allosteric inhibition of focal adhesion kinase: A combined pharmacophore modeling and molecular dynamics approach.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that modulates integrin and growth factor signaling pathways and is implicated in cancer cell migration, proliferation, and survival. Over the past decade various, FAK kinase, FERM, and FAT domain inhibitors have been reported and a few kinase domain inhibitors are under clinical consideration. However, few of them were identified as multikinase inhibitors. In kinase drug design selectivity is always a point of concern, to improve selectivity allosteric inhibitor development is the best choice. The current research utilized a pharmacophore modeling (PM) approach to identify novel allosteric inhibitors of FAK. The all-available allosteric inhibitor bound 3D structures with PDB ids 4EBV, 4EBW, and 4I4F were utilized for the pharmacophore modeling. The validated PM models were utilized to map a database of 770,550 compounds prepared from ZINC, EXIMED, SPECS, ASINEX, and InterBioScreen, aiming to identify potential allosteric inhibitors. The obtained compounds from screening step were forwarded to molecular docking (MD) for the prediction of binding orientation inside the allosteric site and the results were evaluated with the known FAK allosteric inhibitor (REF). Finally, 14 FAK-inhibitor complexes were selected from the docking study and were studied under molecular dynamics simulations (MDS) for 500 ns. The complexes were ranked according to binding free energy (BFE) and those demonstrated higher affinity for allosteric site of FAK than REF inhibitors were selected. The selected complexes were further analyzed for intermolecular interactions and finally, three potential allosteric inhibitor candidates for the inhibition of FAK protein were identified. We believe that identified scaffolds may help in drug development against FAK as an anticancer agent.

Design, synthesis and biological evaluation of Thiazolo[3, 2-a]Pyrimidine derivatives as novel RNase H inhibitors.

Targeting Ribonuclease H (RNase H) has been considered a viable strategy for HIV therapy. In this study, a series of novel thiazolo[3, 2-a]pyrimidine derivatives were firstly designed and synthesized as potential inhibitors of HIV-1 RNase H. Among these compounds, A28 exhibited the most potent inhibition against HIV-1 RNase H with an IC50 value of 4.14 µM, which was about 5-fold increase in potency than the hit compound A1 (IC50 = 21.49 µM). To gain deeper insights into the structure-activity relationship (SAR), a CoMFA model was constructed to yield reasonable statistical results (q2 = 0.658 and R2 = 0.969). Results from magnesium ion chelation experiments and molecular docking studies revealed that these thiazolopyrimidine inhibitors may exert their inhibitory activity by binding to an allosteric site on RNase H at the interface between subunits p51 and p66. Furthermore, this analog demonstrated favorable physicochemical properties. Our findings provide valuable groundwork for further development of allosteric inhibitors targeting HIV-1 RNase H.

Exploring Binding Pockets in the Conformational States of the SARS-CoV-2 Spike Trimers for the Screening of Allosteric Inhibitors Using Molecular Simulations and Ensemble-Based Ligand Docking.

Understanding mechanisms of allosteric regulation remains elusive for the SARS-CoV-2 spike protein, despite the increasing interest and effort in discovering allosteric inhibitors of the viral activity and interactions with the host receptor ACE2. The challenges of discovering allosteric modulators of the SARS-CoV-2 spike proteins are associated with the diversity of cryptic allosteric sites and complex molecular mechanisms that can be employed by allosteric ligands, including the alteration of the conformational equilibrium of spike protein and preferential stabilization of specific functional states. In the current study, we combine conformational dynamics analysis of distinct forms of the full-length spike protein trimers and machine-learning-based binding pocket detection with the ensemble-based ligand docking and binding free energy analysis to characterize the potential allosteric binding sites and determine structural and energetic determinants of allosteric inhibition for a series of experimentally validated allosteric molecules. The results demonstrate a good agreement between computational and experimental binding affinities, providing support to the predicted binding modes and suggesting key interactions formed by the allosteric ligands to elicit the experimentally observed inhibition. We establish structural and energetic determinants of allosteric binding for the experimentally known allosteric molecules, indicating a potential mechanism of allosteric modulation by targeting the hinges of the inter-protomer movements and blocking conformational changes between the closed and open spike trimer forms. The results of this study demonstrate that combining ensemble-based ligand docking with conformational states of spike protein and rigorous binding energy analysis enables robust characterization of the ligand binding modes, the identification of allosteric binding hotspots, and the prediction of binding affinities for validated allosteric modulators, which is consistent with the experimental data. This study suggested that the conformational adaptability of the protein allosteric sites and the diversity of ligand bound conformations are both in play to enable efficient targeting of allosteric binding sites and interfere with the conformational changes.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 µM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase.

The allosteric inhibition of Insulin-like Growth Factor Receptor 1 Kinase (IGF1RK) is a potential strategy to overcome selectivity barriers in targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives which have been reported as allosteric inhibitors of IGF1RK. We further studied dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation, forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the position of chemical substituents at the R1 indole ring with different pharmacophore features influences molecular interactions and binding conformations of the indole-butyl-amine derivatives, hence dramatically affecting their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection.

In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.

Antiviral actions of natural compounds against dengue virus RNA dependent RNA polymerase: insights from molecular dynamics and Gibbs free energy landscape.

Dengue fever, a major global health challenge, affects nearly half the world's population and lacks effective treatments or vaccines. Addressing this, our study focused on natural compounds that potentially inhibit the dengue virus's RNA-dependent RNA polymerase (RdRp), a crucial target in the viral replication cycle. Utilizing the MTiOpenScreen webserver, we screened 1226 natural compounds from the NP-lib database. This screening identified four promising compounds ZINC000059779788, ZINC0000044404209, ZINC0000253504517 and ZINC0000253499146), each demonstrating high negative binding energies between -10.4 and -9.9 kcal/mol, indicative of strong potential as RdRp inhibitors. These compounds underwent rigorous validation through re-docking and a detailed 100 ns molecular dynamics (MD) simulation. This analysis affirmed the dynamic stability of the protein-ligand complexes, a critical factor in the effectiveness of potential drug candidates. Additionally, we conducted essential dynamics and free energy landscape calculations to understand the structural transitions in the RdRp protein upon ligand binding, providing valuable insights into the mechanism of inhibition. Our findings present these natural molecules as promising therapeutic agents against the dengue virus. By targeting the allosteric site of RdRp, these compounds offer a novel approach to hinder the viral replication process. This research significantly contributes to the search for effective anti-dengue treatments, positioning natural compounds as potential key players in dengue virus control strategies.Communicated by Ramaswamy H. Sarma.

Casitas b cell lymphoma­B (Cbl-b): A new therapeutic avenue for small-molecule immunotherapy.

Immunotherapy has revolutionized the area of cancer treatment. Although most immunotherapies now are antibodies targeting membrane checkpoint molecules, there is an increasing demand for small-molecule drugs that address intracellular pathways. The E3 ubiquitin ligase Casitas B cell lymphoma­b (Cbl-b) has been regarded as a promising intracellular immunotherapy target. Cbl-b regulates the downstream proteins of multiple membrane receptors and co-receptors, restricting the activation of the innate and adaptive immune system. Recently, Cbl-b inhibitors have been reported with promising effects on immune surveillance activation and anti-tumor efficacy. Several molecules have entered phase Ⅰ clinical trials. In this review, the biological rationale of Cbl-b as a promising target for cancer immunotherapy and the latest research progress of Cbl-b are summarized, with special emphasis on the allosteric small-molecule inhibitors of Cbl-b.

Identification of CK2α' selective inhibitors by the screening of an allosteric-kinase-inhibitor-like compound library.

Protein Kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2ß) and two catalytic subunits (CK2α and CK2α'). CK2 controls several cellular processes including proliferation, inflammation, and cell death. However, CK2α and CK2α' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α' has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α' due to their high structural homology, especially in the targeted ATP binding site. Using computational analyses, we found a potential Type IV ("D" pocket) allosteric site on CK2α' that contained different residues than CK2α and was distal from the ATP binding pocket featured in both kinases. With this potential allosteric site in mind, we screened a commercial library containing ~29,000 allosteric-kinase-inhibitor-like compounds using a CK2α' activity-dependent ADP-Glo™ Kinase assay. Obtained hits were counter-screened against CK2α revealing two CK2α' selective compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

Direct-to-biology platform: From synthesis to biological evaluation of SHP2 allosteric inhibitors.

Tyrosine phosphatase SHP2 is a proto-oncogenic protein involved in cell growth and differentiation via diverse intracellular signaling pathways. With the scope of identifying new SHP2 allosteric inhibitors, we report here the development and optimization of a high-throughput "Direct-to-Biology" (D2B) workflow including the synthesis and the biological evaluation of the reaction crude, thus eliminating the need for purification. During this labor-saving procedure, the structural diversity was introduced through a SNAr reaction. A wide array of analogues with good chemical purity was generated, allowing the obtention of reliable biological data which validated this efficient technique. This approach enabled the fast evaluation of a variety of structurally diverse fragments leading to nanomolar SHP2 allosteric inhibitors and a new series bearing a novel bicyclo[3.1.0]hexane moiety.

Identification of putative allosteric inhibitors of BCKDK via virtual screening and biological evaluation.

Abnormal accumulation of branched-chain amino acids (BCAAs) can lead to metabolic diseases and cancers. Branched-chain α-keto acid dehydrogenase kinase (BCKDK) is a key negative regulator of BCAA catabolism, and targeting BCKDK provides a promising therapeutic approach for diseases caused by BCAA accumulation. Here, we screened PPHN and POAB as novel putative allosteric inhibitors by integrating allosteric binding site prediction, large-scale ligand database virtual screening, and bioactivity evaluation assays. Both of them showed a high binding affinity to BCKDK, with Kd values of 3.9 µM and 1.86 µM, respectively. In vivo experiments, the inhibitors demonstrated superior kinase inhibitory activity and notable antiproliferative and proapoptotic effects on diverse cancer cells. Finally, bulk RNA-seq analysis revealed that PPHN and POAB suppressed cell growth through a range of signalling pathways. Taken together, our findings highlight two novel BCKDK inhibitors as potent therapeutic candidates for metabolic diseases and cancers associated with BCAA dysfunctional metabolism.

Role of GSK-3ß Inhibitors: New Promises and Opportunities for Alzheimer's Disease.

Glycogen synthase kinase-3 (GSK-3) was discovered to be a multifunctional enzyme involved in a wide variety of biological processes, including early embryo formation, oncogenesis, as well cell death in neurodegenerative diseases. Several critical cellular processes in the brain are regulated by the GSK-3ß, serving as a central switch in the signaling pathways. Dysregulation of GSK-3ß kinase has been reported in diabetes, cancer, Alzheimer's disease, schizophrenia, bipolar disorder, inflammation, and Huntington's disease. Thus, GSK-3ß is widely regarded as a promising target for therapeutic use. The current review article focuses mainly on Alzheimer's disease, an age-related neurodegenerative brain disorder. GSK-3ß activation increases amyloid-beta (Aß) and the development of neurofibrillary tangles that are involved in the disruption of material transport between axons and dendrites. The drug-binding cavities of GSK-3ß are explored, and different existing classes of GSK-3ß inhibitors are explained in this review. Non-ATP competitive inhibitors, such as allosteric inhibitors, can reduce the side effects compared to ATP-competitive inhibitors. Whereas ATP-competitive inhibitors produce disarrangement of the cytoskeleton, neurofibrillary tangles formation, and lead to the death of neurons, etc. This could be because they are binding to a site separate from ATP. Owing to their interaction in particular and special binding sites, allosteric ligands interact with substrates more selectively, which will be beneficial in resolving drug-induced resistance and also helpful in reducing side effects. Hence, in this review, we focussed on the allosteric GSK-3ß inhibitors and discussed their futuristic opportunities as anti-Alzheimer's compounds.

Design, synthesis and biological evaluation of EGFR kinase inhibitors that spans the orthosteric and allosteric sites.

Acquired drug resistance occurred in the treatment of non-small-cell lung cancer is a persistent challenge, especially in EGFR mutant type. In this study, we present design, synthesis and biological evaluation of novel quinazoline and pyrrolopyrimidine derivatives that simultaneously occupy the orthosteric and allosteric sites of EGFR. Among them, compound A-7 was confirmed as a potential EGFRL858R/T790M/C797S and EGFRDel19/T790M/C797S inhibitor. Docking study indicated that compound A-7 could simultaneously occupy two binding sites of EGFR and form three key H-bonds with the residues Met793, Lys745 and Met766 in two regions.

JAK2 inhibitors for the treatment of Philadelphia-negative myeloproliferative neoplasms: current status and future directions.

The overactivation of Janus kinases 2 (JAK2) by gain-of-function mutations in the JAK2, Myeloproliferative leukemia virus oncogene, or Calreticulin genes are the most important factor in the development of Philadelphia-negative myeloproliferative neoplasms (MPNs). The discovery of the JAK2V617F mutation is a significant breakthrough in understanding the pathogenesis of MPNs, and inhibition of JAK2 abnormal activation has become one of the most effective strategies against MPNs. Currently, three JAK2 inhibitors for treating MPNs have been approved, and several are being evaluated in clinical trials. However, persistent challenges in terms of drug resistance and off-target effects remain unresolved. In this review, we introduce and classify the available JAK2 inhibitors in terms of their mechanisms and clinical considerations. Additionally, through an analysis of target points, binding modes, and structure-activity inhibitor relationships, we propose strategies such as combination therapy and allosteric inhibitors to overcome specific challenges. This review offers valuable insights into current trends and future directions for optimal management of MPNs using JAK2 inhibitors.

Recent developments in catalysis and inhibition of the Jumonji histone demethylases.

Histone methylation, one of the most common histone modifications, has fundamental roles in regulating chromatin-based processes. Jumonji histone lysine demethylases (JMJC KDMs) influence regulation of gene transcription through both their demethylation and chromatin scaffolding functions. It has recently been demonstrated that dysregulation of JMJC KDMs contributes to pathogenesis and progression of several diseases, including cancer. These observations have led to an increased interest in modulation of enzymes that regulate lysine methylation. Here, we highlight recent progress in understanding catalysis of JMJC KDMs. Specifically, we focus on recent research advances on elucidation of JMJC KDM substrate recognition and interactomes. We also highlight recently reported JMJC KDM inhibitors and describe their therapeutic potentials and challenges. Finally, we discuss alternative strategies to target these enzymes, which rely on targeting JMJC KDMs accessory domains as well as utilization of the targeted protein degradation strategy.

Dihydroxyphenyl- and Heteroaromatic-Based Thienopyrimidinones to Tackle HIV-1 LEDGF/p75-Dependent IN Activity.

The spread of Human Immunodeficiency Virus (HIV) still represents a global public health issue of major concern, and would benefit from unveiling unique viral features as targets for drug design. In this respect, HIV-1 integrase (IN), due to the absence of homologs in human cells, is a popular target for the synthesis of novel selective compounds. Moreover, as drug-resistant viral strains are rapidly evolving, the development of novel allosteric inhibitors is acutely required. Recently, we have observed that Kuwanon-L, quinazolinones and thienopyrimidinones containing at least one polyphenol unit, effectively inhibited HIV-1 IN activity. Thus, in the present research, novel dihydroxyphenyl-based thienopyrimidinone derivatives were investigated for their LEDGF/p75-dependent IN inhibitory activity. Our findings indicated a close correlation between the position of the OH group on the phenyl moiety and IN inhibitory activity of these compounds. As catechol may be involved in cytotoxicity, its replacement by other aromatic scaffolds was also exploited. As a result, compounds 21-23, 25 and 26 with enhanced IN inhibitory activity provided good lead candidates, with 25 being the most selective for IN. Lastly, UV spectrometric experiments suggested a plausible allosteric mode of action, as none of the thienopirimidinones showed Mg2+ chelation properties otherwise typical of IN strand transfer inhibitors (INSTIs).

Hypusination-induced DHPS/eIF5A pathway as a new therapeutic strategy for human diseases: A mechanistic review and structural classification of DHPS inhibitors.

The discovery of new therapeutic strategies for diseases is essential for drug research. Deoxyhypusine synthase (DHPS) is a critical enzyme that modifies the conversion of the eukaryotic translation initiation factor 5A (eIF5A) precursor into physiologically active eIF5A (eIF5A-Hyp). Recent studies have revealed that the hypusine modifying of DHPS on eIF5A has an essential regulatory role in human diseases. The hypusination-induced DHPS/eIF5A pathway has been shown to play an essential role in various cancers, and it could regulate immune-related diseases, glucose metabolism-related diseases, neurological-related diseases, and aging. In addition, DHPS has a more defined substrate and a well-defined structure within the active pocket than eIF5A. More and more researchers are focusing on the prospect of advanced development of DHPS inhibitors. This review summarizes the regulatory mechanisms of the hypusination-induced DHPS/eIF5A pathway in a variety of diseases in addition to the inhibitors related to this pathway; it highlights and analyzes the structural features and mechanisms of action of DHPS inhibitors and expands the prospects of future drug development using DHPS as an anticancer target.