Camptothecin structure simplification elaborated new imidazo[2,1-b]quinazoline derivative as a human topoisomerase I inhibitor with efficacy against bone cancer cells and colon adenocarcinoma.

Camptothecin is a pentacyclic natural alkaloid that inhibits the hTop1 enzyme involved in DNA transcription and cancer cell growth. Camptothecin structure pitfalls prompted us to design new congeners using a structure simplification strategy to reduce the ring extension number from pentacyclic to tetracyclic while maintaining potential stacking of the new compounds with the DNA base pairs at the Top1-mediated cleavage complex and aqueous solubility, as well as minimizing compound-liver toxicity. The principal axis of this study was the verification of hTop1 inhibiting activity as a possible mechanism of action and the elaboration of new simplified inhibitors with improved pharmacodynamic and pharmacokinetic profiling using three structure panels (A-C) of (isoquinolinoimidazoquinazoline), (imidazoquinazoline), and (imidazoisoquinoline), respectively. DNA relaxation assay identified five compounds as hTop1 inhibitors belonging to the imidazoisoquinolines 3a,b, the imidazoquinazolines 12, and the isoquinolinoimidazoquinazolines 7a,b. In an MTT cytotoxicity assay against different cancer cell lines, compound 12 was the most potent against HOS bone cancer cells (IC50 = 1.47 µM). At the same time, the other inhibitors had no detectable activity against any cancer cell type. Compound (12) demonstrated great penetrating power in the HOS cancer cells' 3D-multicellular tumor spheroid model. Bioinformatics research of the hTop1 gene revealed that the TP53 cell proliferative gene is in the network of hTop1. The finding is confirmed empirically using the gene expression assay that proved the increase in p53 expression. The impact of structure simplification on compound 12 profile, characterized by the absence of acute oral liver toxicity when compared to Doxorubicin as a standard inhibitor, the lethal dose measured on Swiss Albino female mice and reported at LD50 = 250 mg/kg, and therapeutic significance in reducing colon adenocarcinoma tumor volume by 75.36 % after five weeks of treatment with compound 12. The molecular docking solutions of the active CPT-based derivative 12 and the inactive congener 14 into the active site of hTop1 and the activity cliffing of such MMP directed us to recommend the addition of HBD and HBA variables to compound 12 imidazoquinazoline core scaffold to enhance the potency via hydrogen bond formation with the major groove amino acids (Asp533, Lys532) as well as maintaining the hydrogen bond with the minor groove amino acid Arg364.

Lead generation of UPPS inhibitors targeting MRSA: Using 3D-QSAR pharmacophore modeling, virtual screening, molecular docking, and molecular dynamic simulations.

Undecaprenyl Pyrophosphate Synthase (UPPS) is a vital target enzyme in the early stages of bacterial cell wall biosynthesis. UPPS inhibitors have antibacterial activity against resistant strains such as MRSA and VRE. In this study, we used several consecutive computer-based protocols to identify novel UPPS inhibitors. The 3D QSAR pharmacophore model generation (HypoGen algorithm) protocol was used to generate a valid predictive pharmacophore model using a set of UPPS inhibitors with known reported activity. The developed model consists of four pharmacophoric features: one hydrogen bond acceptor, two hydrophobic, and one aromatic ring. It had a correlation coefficient of 0.86 and a null cost difference of 191.39, reflecting its high predictive power. Hypo1 was proven to be statistically significant using Fischer's randomization at a 95% confidence level. The validated pharmacophore model was used for the virtual screening of several databases. The resulting hits were filtered using SMART and Lipinski filters. The hits were docked into the binding site of the UPPS protein, affording 70 hits with higher docking affinities than the reference compound (6TC, - 21.17 kcal/mol). The top five hits were selected through extensive docking analysis and visual inspection based on docking affinities, fit values, and key residue interactions with the UPPS receptor. Moreover, molecular dynamic simulations of the top hits were performed to confirm the stability of the protein-ligand complexes, yielding five promising novel UPPS inhibitors.

Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging. Each sulfatase may differ in their degree of post-translational modification by FGE, which may influence the phenotypic spectrum of MSD. Here, we evaluate if residual sulfatase activity and accumulating GAG patterns distinguish cases from controls and stratify clinical severity groups in MSD. We quantify sulfatase activities and GAG accumulation using three complementary methods in MSD participants. Sulfatases differed greatly in their tolerance of reduction in FGE-mediated activation. Enzymes that degrade heparan sulfate (HS) demonstrated lower residual activities than those that act on other GAGs. Similarly, HS-derived urinary GAG subspecies preferentially accumulated, distinguished cases from controls, and correlated with disease severity. Accumulation patterns of specific sulfatase substrates in MSD provide fundamental insights into sulfatase regulation and will serve as much-needed biomakers for upcoming clinical trials. This work highlights that biomarker investigation of an ultra-rare disease can simultaneously inform our understanding of fundamental biology and advance clinical trial readiness efforts.

Disease correction in mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.

Lead generation of cysteine based mesenchymal epithelial transition (c-Met) kinase inhibitors: Using structure-based scaffold hopping, 3D-QSAR pharmacophore modeling, virtual screening, molecular docking, and molecular dynamics simulation.

Cysteine-based mesenchymal-epithelial transition (c-Met) is a receptor tyrosine kinase that plays a definitive role during cancer progression and was identified as a possible target for anti-angiogenesis drugs. In the present study, different protocols of computer-based drug design were performed. Construction of predictive pharmacophore model using HypoGen algorithm resulted in a validated model of four features of positive ionizable, hydrogen bond acceptor, hydrophobic, and ring aromatic features with a correlation coefficient of 0.87, a configuration cost of 14.95, and a cost difference of 357.92. The model revealed a promising predictive power and had >90% probability of representing true correlation with the activity data. The model was established using Fisher's validation test at the 95% confidence level and test set prediction (r = 0.96), furthermore, the model was validated by mapping of set of compounds undergoing clinical trials as class Ⅱ c-met inhibitors. The generated valid pharmacophore model was then anticipated for virtual screening of three data bases. Moreover, scaffold hopping using replace fragments protocol was implemented. Hits generated were filtered according to Lipinski's rule; 510 selected hits were anatomized and subjected to molecular docking studies into the crystal structure of c-Met kinase. The good correlation between docking scores and ligand pharmacophore mapping fit values provided a reliable foundation for designing new potentially active candidates that may target c-Met kinase. Eventually, eight hits were selected as potential leads. Subsequently, seven (Hits) have displayed a higher dock score and demonstrated key residue interactions with stable molecular dynamics simulation. Therefore, these c-Met kinase inhibitors may further serve as new chemical spaces in designing new compounds.

Structural Basis of Selective Human Indoleamine-2,3-dioxygenase 1 (hIDO1) Inhibition.

hIDO1 is a heme-dioxygenase overexpressed in the tumor microenvironment and is implicated in the survival of cancer cells. Metabolism of tryptophan to N-formyl-kynurenine by hIDO1 leads to immune suppression to result in cancer cell immune escape. In this article, we discuss the discovery of selective hIDO1 inhibitors for therapeutic intervention that have been promoted to clinical trials and for which crystallographic structural information is available for the respective inhibitor-enzyme complex. The structural insights are based on the complex crystal structures and the relative biological data profiles. The structural basis of selective hIDO1 inhibition, as discussed herein, opens new avenues to the discovery of novel inhibitors with improved activity profiles, selectivity, and distinct structure frameworks.

Molecular design, synthesis and in vitro biological evaluation of thienopyrimidine-hydroxamic acids as chimeric kinase HDAC inhibitors: a challenging approach to combat cancer.

A series of thieno[2,3-d]pyrimidine-based hydroxamic acid hybrids was designed and synthesised as multitarget anti-cancer agents, through incorporating the pharmacophore of EGFR, VEGFR2 into the inhibitory functionality of HDAC6. Three compounds (12c, 15b and 20b) were promising hits, whereas (12c) exhibited potent VEGFR2 inhibition (IC50=185 nM), potent EGFR inhibition (IC50=1.14 µM), and mild HDAC6 inhibition (23% inhibition). Moreover, compound (15c) was the most potent dual inhibitor among all the synthesised compounds, as it exhibited potent EGFR and VEGFR2 inhibition (IC50=19 nM) and (IC50=5.58 µM), respectively. While compounds (20d) and (7c) displayed nanomolar selective kinase inhibition with EGFR IC50= 68 nM and VEGFR2 IC50= 191 nM, respectively. All of the synthesised compounds were screened in vitro for their cytotoxic effect on 60 human NCI tumour cell lines. Additionally, molecular docking studies and ADMET studies were carried out to gain further insight into their binding mode and predict the pharmacokinetic properties of all the synthesised inhibitors.

Structure-based design generated novel hydroxamic acid based preferential HDAC6 lead inhibitor with on-target cytotoxic activity against primary choroid plexus carcinoma.

Histone deacetylase 6 (HDAC6) is an attractive target for cancer therapeutic intervention. Selective HDAC6 inhibitors is important to minimise the side effects of pan inhibition. Thus, new class of hydroxamic acid-based derivatives were designed on structural basis to perform preferential activity against HDAC6 targeting solid tumours. Interestingly, 1-benzylbenzimidazole-2-thio-N-hydroxybutanamide 10a showed impressive preference with submicromolar potency against HDAC6 (IC50 = 510 nM). 10a showed cytotoxic activity with interesting profile against CCHE-45 at (IC50 = 112.76 µM) when compared to standard inhibitor Tubacin (IC50 = 20 µM). Western blot analysis of acetylated-α-tubulin verified the HDAC6 inhibiting activity of 10a. Moreover, the insignificant difference in acetylated-α-tubulin induced by 10a and Tubacin implied the on-target cytotoxic activity of 10a. Docking of 10a in the binding site of HDAC6 attributed the activity of 10a to π-π stacking with the amino acids of the hydrophobic channel of HDAC6 and capture of zinc metal in bidentate fashion. The therapeutic usefulness besides the on-target activity may define 10a as an interesting safe-lead inhibitor for future development.

3D-QSAR pharmacophore modelling, virtual screening and docking studies for lead discovery of a novel scaffold for VEGFR 2 inhibitors: Design, synthesis and biological evaluation.

A series of novel 6,7-dihydro-5H-cyclopenta[d]pyrimidine derivatives was successfully designed, synthesized and evaluated as a new chemical scaffold with vascular endothelial growth factor receptor (VEGFR 2) inhibitory activity. Compounds 6c and 6b showed enzyme inhibition of 97% and 87% at 10 µM, respectively, and exhibited potent dose-related VEGFR 2 inhibition with IC50 values of 0.85 µM and 2.26 µM, respectively. The design of the 6,7-dihydro-5H-cyclopenta[d]pyrimidine scaffold was implemented via consecutive molecular modelling protocols prior to the synthesis and biological evaluation of the derivatives. First, sorafenib was docked in the binding site of VEGFR 2 to study its binding orientation and affinity, followed by the generation of a valid 3D QSAR pharmacophore model for use in the virtual screening of different 3D databases. Structures with promising pharmacophore-based virtual screening results were refined using molecular docking studies in the binding site of VEGFR 2. A novel scaffold was designed by incorporating the results of the pharmacophore model generation and molecular docking studies. The new scaffold showed hydrophobic interactions with the kinase front pocket that may be attributed to increasing residence time in VEGFR 2, which is a key success factor for ligand optimization in drug discovery. Different derivatives of the novel scaffold were validated using docking studies and pharmacophore mapping, where they exhibited promising results as VEGFR 2 inhibitors to be synthesized and biologically evaluated. 6,7-dihydro-5H-cyclopenta[d]pyrimidine is a new scaffold that can be further optimized for the synthesis of promising VEGFR 2 inhibitors.

Studies of TAK1-centered polypharmacology with novel covalent TAK1 inhibitors.

Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5, 27, was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases.

Structure-guided development of covalent TAK1 inhibitors.

TAK1 (transforming growth factor-ß-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted pyrimidine covalent TAK1 inhibitors that target Cys174, a residue immediately adjacent to the 'DFG-motif' of the kinase activation loop. Co-crystal structures of TAK1 with candidate compounds enabled iterative rounds of structure-based design and biological testing to arrive at optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which possess an equivalently positioned cysteine residue. These compounds are smaller, more easily synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol.

Toward discovery of mutant EGFR inhibitors; Design, synthesis and in vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives.

A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type (WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type (EGFR WT) with IC50 values in the low nanomolar range (<0.495-9.05nM) and displayed more potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed. Compounds 4d, 6f, 7e showed significant inhibition (IC50=1.76-2.38µM) in these mutant lines and significant Her2 enzyme inhibition (IC50=19.2-40.6nM) compared to lapatinib (60.1nM). The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested, compounds 6f and 7e showed about eight and three folds respectively greater potency than gefitinib. Our structure-activity relationships (SAR) studies suggested that presence of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for developing inhibitors against wild type and mutant EGFR.

Design, synthesis and in vitro anti-proliferative activity of 4,6-quinazolinediamines as potent EGFR-TK inhibitors.

4-Anilino-6-substituted-quinazolines were designed, synthesized and evaluated for EGFR-TK and tumor growth inhibitory activities. The target compounds were designed with enamine ester or urea moieties appended at the C-6 of quinazoline as additional hydrogen bond acceptor functions. Most of the synthesized compounds displayed potent EGFR-TK inhibitory activity at 10 µM and the 6-ureido-anilinoquinazoline derivative 7a showed IC50 value of 0.061 µM. Moreover, six compounds were tested by National Cancer Institute (NCI), USA for their anti-proliferative activity at 10 µM in full NCI 60 cell panel. Compound 7a was further assayed for five dose molar ranges in full NCI 60 cell panel and exhibited remarkable growth inhibitory activity pattern against Non-Small Cell Lung Cancer EKVX (GI50 = 0.37 µM), NCI-H322M (GI50 = 0.36 µM), Renal Cancer A498 (GI50 = 0.46 µM), TK-10 (GI50 = 0.99 µM) and Breast Cancer MDA-MB-468 (GI50 = 1.096 µM) which are of high EGFR expression. Docking study was performed for the active compounds into ATP binding site of EGFR-TK which showed similar binding mode to gefitinib and additional binding with Cys-773 at the gatekeeper of EGFR-TK enzyme.