Identification of Allosteric PIF‐Pocket Ligands for PDK1 using NMR‐Based Fragment Screening and 1H‐15N TROSY Experiments

Aberrant activation of the phosphoinositide 3‐kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3‐phosphoinositide‐dependent kinase‐1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N‐terminal lobe of the 3‐phosphoinositide‐dependent kinase‐1 catalytic domain contains a docking site which recognizes the non‐catalytic C‐terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so‐called PDK1 Interacting Fragment pocket allows interaction with 3‐phosphoinositide‐dependent kinase‐1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3‐phosphoinositide‐dependent kinase‐1 domain construct against a library of chemically diverse fragments in order to identify small, ligand‐efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP‐site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand‐induced changes in ¹H‐¹⁵N TROSY spectra acquired using uniformly ¹⁵N‐enriched PDK1 provided evidence to distinguish ATP‐site from PDK1 Interacting Fragment‐site binding. Caliper assay data and ¹⁹F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.     

KRAS及其抑制剂的研究进展

RAS是人类癌症中最常发生突变的致癌基因,而KRAS则是RAS家族中最常发生突变的亚型,其中多数为12位密码子的突变。突变之后的KRAS使细胞的生长、增殖不受控制,进而导致癌症的发生与发展。尽管经过了30多年的努力,但直接靶向KRAS活性位点的药物开发均以失败告终。由于KRAS与GTP的亲和力极强,同时细胞中GTP浓度较高,以至于使KRAS成为"不可成药"靶点。近期,针对突变的KRAS G12C特异性共价抑制剂在临床试验中取得了突破性进展,为KRAS抑制剂的可成药性提供了临床证据。综述KRAS的结构功能及其小分子抑制剂的设计和临床研究概况,着重介绍具有代表意义的KRAS G12C抑制剂和其作为抗肿瘤药物的最新研发进展,为以KRAS为靶点的药物开发提供研究思路。     

Mapping of Allosteric Druggable Sites in Activation‐Associated Conformers of the M2 Muscarinic Receptor

G‐protein‐coupled receptors (GPCRs) are key cellular signaling proteins and have been targeted by approximately 30–40% of marketed drugs for treating many human diseases including cancer and heart failure. Recently, we directly observed activation of the M2 muscarinic receptor through long‐timescale accelerated molecular dynamics (aMD) simulation, which revealed distinct inactive, intermediate and active conformers of the receptor. Here, FTMAP is applied to search for ‘hot spots’ in these activation‐associated conformers using a library of 16 organic probe molecules that represent fragments of potential drugs. Seven allosteric (non‐orthosteric) binding sites are identified in the M2 receptor through the FTMAP analysis. These sites are distributed in the solvent‐exposed extracellular and intracellular mouth regions, as well as the lipid‐exposed pockets formed by the transmembrane α helices TM3‐TM4, TM5‐TM6 and TM7‐TM1/TM2. They serve as promising target sites for designing novel allosteric modulators as receptor‐selective drugs.     

Microsecond molecular dynamics simulations provide insight into the ATP‐competitive inhibitor‐induced allosteric protection of Akt kinase phosphorylation

Akt is a serine/threonine protein kinase, a critical mediator of growth factor‐induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long‐range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP‐competitive inhibitor, GDC‐0068‐bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC‐0068 binding to the ATP‐binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N‐ and C‐lobes, equilibrated between the open and closed conformations. GDC‐0068 occupancy of the ATP‐binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC‐0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC‐0068‐bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC‐0068 to the pT308 are identified. Our analysis of GDC‐0068‐induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity.     

Site‐directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non‐Nucleoside Inhibitors

Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non‐substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of non‐nucleoside non‐competitive inhibitors of human adenosine kinase (hAK), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies, and site‐directed mutagenesis to validate our hypothesis. Based on a three‐dimensional model of interaction between hAK and our molecules, we designed, cloned, and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A, and Q74A‐F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non‐nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild‐type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK and may pave the way to the development of novel selective and potent non‐nucleoside inhibitors of hAK endowed with therapeutic potential.     

Analysis of Enoyl‐Acyl Carrier Protein Reductase Structure and Interactions Yields an Efficient Virtual Screening Approach and Suggests a Potential Allosteric Site

Enoyl‐acyl carrier protein reductases have an important role in fatty acid biosynthesis and are considered essential for bacterial and protozoal survival. Here, we perform a computational assessment of enoyl‐acyl carrier protein reductase structures, providing insights for inhibitor design that we incorporate into a virtual screening approach. Firstly, we analyse 80 crystal structures of 16 different enoyl‐acyl carrier protein reductases for their active site characteristics and druggability, finding these sites contain a readily druggable pocket, of varying size and shape. Interestingly, a high affinity, potentially allosteric site was identified for pfFabI. Analysis of the ligand–protein interactions of four enoyl‐acyl carrier protein reductases from different micro‐organisms (InhA, pfFabI, saFabI and ecFabI), involving 59 available crystal structures, found three commonly shared interactions; constraining these interactions in docking improved enrichment of enoyl‐acyl carrier protein reductase virtual screens, by up to 60% in the top 3% of the ranked library. This docking protocol also improved pose prediction, decreasing the root‐mean‐square deviation to crystallographic pose by up to 75% on average. The binding site analysis and knowledge‐based docking protocol presented here can potentially assist in the structure‐based design of new enoyl‐acyl carrier protein reductase inhibitors.     

Virtual screening,SAR, and discovery of 5‐(indole‐3‐yl)‐2‐[(2‐nitrophenyl)amino] [1,3,4]‐oxadiazole as a novel Bcl‐2 inhibitor

A new series of oxadiazoles were designed to act as inhibitors of the anti‐apoptotic Bcl‐2 protein. Virtual screening led to the discovery of new hits that interact with Bcl‐2 at the BH3 binding pocket. Further study of the structure–activity relationship of the most active compound of the first series, compound 1 , led to the discovery of a novel oxadiazole analogue, compound 16j , that was a more potent small‐molecule inhibitor of Bcl‐2. 16j had good in vitro inhibitory activity with submicromolar IC₅₀ values in a metastatic human breast cancer cell line (MDA‐MB‐231) and a human cervical cancer cell line (HeLa). The antitumour effect of 16j is concomitant with its ability to bind to Bcl‐2 protein as shown by an enzyme‐linked immunosorbent assay (IC₅₀ = 4.27 μm ). Compound 16j has a great potential to develop into highly active anticancer agent.     

Structure‐based pharmacophore models to probe anticancer activity of inhibitors of protein kinase B‐beta (PKB β)

Protein kinase B ‐ beta (PKBβ/Akt2) is a non‐receptor kinase that has attracted a great deal of attention as a promising cancer therapy drug target. In mammalian cells, hyperactivation of Akt2 exclusively facilitates the survival of solid tumors by interfering with cell cycle progression. This definite function of Akt2 in tumor survival/maintenance provides the basis for the development of its antagonists with the aim of desensitizing cell proliferation. In order to find novel and potent Akt2 inhibitors, structure ‐ based pharmacophore models have been developed and validated by the test set prediction. The final pharmacophore model was used for hits identification using public chemical databases. The hits were further prioritized using drug ‐ like filters which revealed 14 potential hit compounds having novel chemical scaffolds. Our results elucidate the importance of three hydrogen bond acceptors (A), one hydrogen bond donor (D), one hydrophobic group (H), and one positive ionic charge (P) toward inhibition of the Ak2. One of our selected hits showed 68% cell apoptosis at 8 μg/ml concentration. We proposed various chemical scaffolds including benzamide, carboxamide, and methyl benzimidazole targeting Akt2 and thus may act as potential leads for the further development of new anticancer agents.     

Exploration of the structure–activity relationship and druggability of novel oxazolidinone‐based compounds as Gram‐negative antibacterial agents

To gain further knowledge of the structure–activity relationship and druggability of novel oxazolidinone‐based UDP‐3‐O‐acyl‐N‐acetylglucosamine deacetylase (LpxC) inhibitors as Gram‐negative antibacterial agents, compounds containing the hydrophobic tails with different lengths and terminal substitutions were synthesized and their antibacterial activities against standard and clinically isolated Gram‐negative strains were evaluated. We summarized their structure–activity relationships and found that oxazolidinone‐based compounds exhibited a narrower antibacterial spectrum compared with threonine‐based compounds. Furthermore, we parallelly compared the metabolic stabilities of the compounds with the classic threonine scaffold and the novel oxazolidinone scaffold in liver microsomes. The results indicated that the druggability of the oxazolidinone scaffold may be inferior to the classic threonine scaffold in the design of LpxC inhibitors.     

1,3‐Bis(aryloxy)propan‐2‐ols as potential antileishmanial agents

We describe herein the synthesis and antileishmanial activity of 1,3‐bis(aryloxy)propan‐2‐ols. Five compounds ( 2 , 3 , 13 , 17 , and 18 ) exhibited an effective antileishmanial activity against stationary promastigote forms of Leishmania amazonensis (IC₅₀ < 15.0 μm ), and an influence of compound lipophilicity on activity was suggested. Most of the compounds were poorly selective, as they showed toxicity toward murine macrophages, except 17 and 18 , which presented good selective indexes (SI ≥ 10.0). The five more active compounds ( 2 , 3 , 13 , 17 , and 18 ) were selected for the treatment of infected macrophages, and all of them were able to reduce the number of internalized parasites by more than 80%, as well as the number of infected macrophages by more than 70% in at least one of the tested concentrations. Altogether, these results demonstrate the potential of these compounds as new hits of antileishmanial agents and open future possibilities for them to be tested in in vivo studies.     

N‐hydroxy‐substituted 2‐aryl acetamide analogs: A novel class of HIV‐1 integrase inhibitors

An in silico method has been used to discover N‐hydroxy‐substituted 2‐aryl acetamide analogs as a new class of HIV‐1 integrase inhibitors. Based on the molecular requirements of the binding pocket of catalytic active site, two molecules (compounds 2 and 4b ) were designed as fragments. These were further synthesized and biologically evaluated. In vitro potency along with docking studies highlighted compound 4b as an active fragment which was further used to synthesize new leads as HIV‐1 integrase inhibitors. Finally, six promising compounds (compounds 5b , 5c , 5e, 6–2c, 6–3b, and 6–5b ) were identified by integrase inhibition assay (>50% inhibition). Based on in vitro anti‐HIV‐1 activity in a reporter gene‐based cell assay system, compounds 5d , 6s , and 6k were found as novel HIV‐1 integrase inhibitors due to its better selectivity index. Additionally, docking study revealed the importance of H‐bond as well as hydrophobic interactions with Asn155, Lys156, and Lys159 which were required for their anti‐HIV‐1 activity.     

In silico approaches and chemical space of anti‐P‐type ATPase compounds for discovering new antituberculous drugs

Tuberculosis (TB) is one of the most important public health problems around the world. The emergence of multi‐drug‐resistant (MDR) and extensively drug‐resistant (XDR) Mycobacterium tuberculosis strains has driven the finding of alternative anti‐TB targets. In this context, P‐type ATPases are interesting therapeutic targets due to their key role in ion homeostasis across the plasma membrane and the mycobacterial survival inside macrophages. In this review, in silico and experimental strategies used for the rational design of new anti‐TB drugs are presented; in addition, the chemical space distribution based on the structure and molecular properties of compounds with anti‐TB and anti‐P‐type ATPase activity is discussed. The chemical space distribution compared to public compound libraries demonstrates that natural product libraries are a source of novel chemical scaffolds with potential anti‐P‐type ATPase activity. Furthermore, compounds that experimentally display anti‐P‐type ATPase activity belong to a chemical space of molecular properties comparable to that occupied by those approved for oral use, suggesting that these kinds of molecules have a good pharmacokinetic profile (drug‐like) for evaluation as potential anti‐TB drugs.     

Proposing novel MDM2 inhibitors: Combined physics‐driven high‐throughput virtual screening and in vitro studies

The mouse double minute 2 (MDM2) protein acts as a negative regulator of the p53 tumor suppressor. It directly binds to the N terminus of p53 and promotes p53 ubiquitination and degradation. Since the most common p53‐suppressing mechanisms involve the MDM2, proposing novel inhibitors has been the focus of many in silico and also experimental studies. Thus, here we screened around 500,000 small organic molecules from Enamine database at the binding pocket of this oncogenic target. The screening was achieved systematically with starting from the high‐throughput virtual screening method followed by more sophisticated docking approaches. The initial high number of screened molecules was reduced to 100 hits which then were studied extensively for their therapeutic activity and pharmacokinetic properties using binary QSAR models. The structural and dynamical profiles of the selected molecules at the binding pocket of the target were studied thoroughly by all‐atom molecular dynamics simulations. The free energy of the binding of the hit molecules was estimated by the MM/GBSA method. Based on docking simulations, binary QSAR model results, and free energy calculations, 11 compounds ( E1 – E11 ) were selected for in vitro studies. HUVEC vascular endothelium, colon cancer, and breast cancer cell lines were used for testing the binding affinities of the identified hits and for further cellular effects on human cancer cell. Based on in vitro studies, six compounds ( E1 , E2 , E5 , E6 , E9 , and E11 ) in breast cancer cell lines and six compounds ( E1 , E2 , E5 , E6 , E8 , and E10 ) in colon cancer cell lines were found as active. Our results showed that these compounds inhibit proliferation and lead to apoptosis.     

Computational‐based discovery of FAK FERM domain chemical probes that inhibit HER2‐FAK cancer signaling

The N‐terminal FERM domain of focal adhesion kinase (FAK) contributes to FAK scaffolding and interacts with HER2, an oncogene and receptor tyrosine kinase. The interaction between HER2 and FAK drives resistance to FAK‐kinase domain inhibitors through FAK Y397 transphosphorylation and FAK re‐activation upon inhibition. As such, FAK FERM remains an attractive drug discovery target. In this report, we detail an alternative approach to targeting FAK through virtual screening‐based discovery of chemical probes that target FAK FERM. We validated the binding interface between HER2 and FAK using site‐directed mutagenesis and GST pull‐down experiments. We assessed the ligandability of key‐binding residues of HER2 and FAK utilizing computational tools. We developed a virtual screening method to screen ~200,000 compounds against the FAK FERM domain, identifying 20 virtual chemical probes. We performed GST pull‐down screening on these compounds, discovering two hits, VS4 and VS14, with nanomolar IC₅₀s in disrupting HER2‐FAK. We performed further testing, including molecular docking, immunofluorescence, phosphorylation, and cellular invasion assays to evaluate the compounds’ biological effects. One probe, VS14, was identified with the ability to block both auto‐ and transphosphorylation of Y397. In all, these studies identify two new probes that target FAK FERM, enabling future investigation of this domain.     

Structure elucidation of the designer drug N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide and the relevance of predicted 13C NMR shifts – a case study

The detailed structure elucidation process of the new cannabimimetic designer drug, N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5‐fluoropentyl group; a 4‐fluorophenyl substituent; and a carbamoyl group, which is N‐substituted with a methyl residue carrying a tert.‐butyl and a carbamoyl substituent. The 5‐fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the ¹H,¹³C HMBC were performed, but due to the small number of signals based on long‐range couplings, the comparison of predicted and observed ¹³C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of ¹³C shift values which allowed an efficient and unambiguous structure determination. For the identified N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, the best agreement between predicted ¹³C shifts and the observed chemical shifts and long‐range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ‐037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd.     

Design,synthesis and biological evaluation of novel quinoline‐based carboxylic hydrazides as anti‐tubercular agents

In this study, seventeen novel quinoline‐based carboxylic hydrazides were designed as potential anti‐tubercular agents using molecular hybridization approach and evaluated in‐silico for drug‐likeness behavior. The compounds were synthesized, purified, and characterized using spectral techniques (like FTIR, ¹H NMR, and Mass). The in‐vitro anti‐tubercular activity (against Mycobacterium tuberculosisH37Ra) and cytotoxicity against human lung fibroblast cells were studied. Among the tested hydrazides, four compounds ( 6h , 6j , 6l, and 6m ) exhibited significant anti‐tubercular activity with MIC values below 20 μg/mL. The two most potent compounds of the series, 6j and 6m exhibited MIC values 7.70 and 7.13 μg/mL, respectively, against M. tuberculosis with selectivity index >26. Structure–activity relationship studies were performed for the tested compounds in order to explore the effect of substitution pattern on the anti‐tubercular activity of the synthesized compounds.     

Design,Synthesis, Structural Characterization by IR, 1H, 13C, 15N, 2D‐NMR,X‐Ray Diffraction and Evaluation of a New Class of Phenylaminoacetic Acid Benzylidene Hydrazines as pfENR Inhibitors

Recent studies have revealed that plasmodial enoyl‐ACP reductase (pfENR, FabI), one of the crucial enzymes in the plasmodial type II fatty acid synthesis II (FAS II) pathway, is a promising target for liver stage malaria infections. Hence, pfENR inhibitors have the potential to be used as causal malarial prophylactic agents. In this study, we report the design, synthesis, structural characterization and evaluation of a new class of pfENR inhibitors. The search for inhibitors began with a virtual screen of the iResearch database by molecular docking. Hits obtained from the virtual screen were ranked according to their Glide score. One hit was selected as a lead and modified to improve its binding to pfENR; from this, a series of phenylamino acetic acid benzylidene hydrazides were designed and synthesized. These molecules were thoroughly characterized by IR, ¹H, ¹³C, ¹⁵N, 2D‐NMR (COSY, NOESY, ¹H‐¹³C, ¹H‐¹⁵N HSQC and HMBC), and X‐ray diffraction. NMR studies revealed the existence of conformational/configurational isomers around the amide and imine functionalities. The major species in DMSO solution is the E, E form, which is in dynamic equilibrium with the Z, E isomer. In the solid state, the molecule has a completely extended conformation and forms helical structures that are stabilized by strong hydrogen bond interactions, forming a helical structure stabilized by N‐H…O interactions, a feature unique to this class of compounds. Furthermore, detailed investigation of the NMR spectra indicated the presence of a minor impurity in most compounds. The structure of this impurity was deduced as an imidazoline‐4‐one derivative based on ¹H‐¹³C and ¹H‐¹⁵H HMBC spectra and was confirmed from the NOESY spectra. The molecules were screened for in vitro activity against recombinant pfENR enzyme by a spectrophotometric assay. Four molecules, viz. 17, 7, 10, and 12 were found to be active at 7, 8, 10, and 12 μm concentration, respectively, showing promising pfENR inhibitory potential. A classification model was derived based on a binary QSAR approach termed recursive partitioning (RP) to highlight structural characteristics that could be tuned to improve activity.     

Allosteric modulators of cannabinoid receptor 1: developing compounds for improved specificity

Abstract

The cannabinoid receptor 1 (CB₁) is a G protein-coupled receptor (GPCR) that is located primarily in the central nervous system. CB₁ is a therapeutic target which may impact pathways to mediate pain, neurodegenerative disorders, hunger, and drug-seeking behavior. Despite these benefits, development of orthosteric therapeutic compounds, which target the endogenous ligand-binding site of CB₁, has been challenging due to detrimental side effects including psychoactivity, depression, and suicidal thoughts. However, CB₁ also has an allosteric binding site(s), which is topographically distinct from the orthosteric site. Allosteric modulation of CB₁ has a number of potential advantages including providing a mechanism for more precise control of downstream pathways and circumventing these side effects. In this review, we summarize the concept of allosteric modulation and focus on the structure–activity relationship studies of the well-characterized allosteric modulators, ORG27569 and PSNCBAM-1 and their derivatives, and a few other recent modulators. We review studies on the properties of these modulators on CB₁ signaling in cells and their effects in vivo. While many current allosteric modulators also produce complex outcomes, they provide new advances for the design of CB₁ centered therapeutics.     

Identification of inhibitors of Tartrate‐resistant acid phosphatase (TRAP/ACP5) activity by small‐molecule screening

Tartrate‐resistant acid phosphatase (TRAP/ACP5) occurs as two isoforms—TRAP 5a with low enzymatic activity due to a loop interacting with the active site and the more active TRAP isoform 5b generated upon proteolytic cleavage of this loop. TRAP has been implicated in several diseases, including cancer. Thus, this study set out to identify small‐molecule inhibitors of TRAP activity. A microplate‐based enzymatic assay for TRAP 5b was applied in a screen of 30,315 compounds, resulting in the identification of 90 primary hits. After removal of promiscuous compounds, unwanted groups, and false positives by orthogonal assays and three‐concentration validation, the properties of 52 compounds were further investigated to better understand their mechanism of action. Full‐concentration–response curves for these compounds were established under different enzyme concentrations and (pre)incubation times to remove compounds with inconsistent results and low potencies. Full‐concentration–response curves were also performed for both isoforms, to examine isoform prevalence. Filtering led to six prioritized compounds, representing different clusters. One of these, CBK289001 or (6S)‐6‐[3‐(2H‐1,3‐benzodioxol‐5‐yl)‐1,2,4‐oxadiazol‐5‐yl]‐N‐(propan‐2‐yl)‐1H,4H,5H,6H,7H‐imidazo[4,5‐c]pyridine‐5‐carboxamide, demonstrated efficacy in a migration assay and IC₅₀ values from 4 to 125 μm . Molecular docking studies and analog testing were performed around CBK289001 to provide openings for further improvement toward more potent blockers of TRAP activity.