Application of a Fluorescence Recovery-Based Polo-Like Kinase 1 Binding Assay to Polo-Like Kinase 2 and Polo-Like Kinase 3.

Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC50 values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.

Enhancing precision in colorectal cancer surgery: development of an LGR5-targeting RSPO1 peptide mimetic as a contrast agent for intraoperative fluorescence molecular imaging.

Colorectal cancer (CRC) is the third most common cancer worldwide. In the United States alone, CRC was responsible for approximately 52,550 deaths in 2023, with an estimated 153,020 new cases. CRC presents with synchronous peritoneal spread in 5-10% of patients, and up to 20-50% of patients with recurrent disease will develop metachronous colorectal cancer peritoneal metastatic (CRC-PM) disease. Eradication of the tumor, tumor margins and microscopic residual disease is paramount, as microscopic residual disease is associated with local recurrences, with 5-year survival rates of less than 35%. The success of resection and reduction of residual disease depends on the accuracy with which cancer cells and normal tissue can be intra-operatively distinguished. Fluorescence Molecular Imaging (IFMI) and tumor-targeted contrast agents represent a promising approach for intraoperative detection and surgical intervention. Proper target selection, the development of scalable imaging agents and enhanced real-time tumor and tumor microenvironment imaging are critical to enabling enhanced surgical resection. LGR5 (leucine-rich repeat-containing G-protein-coupled receptor 5), a colonic crypt stem cell marker and the receptor for the R-spondins (RSPO) in the Wnt signaling pathway, is also expressed on colorectal cancer stem cells (CSC) and on CRC tumors and metastases, suggesting it could be a useful target for imaging of CRC. However, there are numerous diverging reports on the role of LGR5 in CRC therapy and outcomes. Herein, we report on the synthesis and validation of a 37 amino acid RSPO1-mimetic peptide, termed RC18, that was specifically designed to access the R-spondin binding site of LGR5 to potentially be used for interoperative imaging of CRC-PM. The receptor-binding capabilities of the RC18 indicate that direct interactions with LGR5 neither significantly increased LGR5 signaling nor blocked RSPO1 binding and signal transduction, suggesting that the RSPO1-mimetic is functionally inert, making it an attractive contrast agent for intraoperative CRC-PM imaging.

N-Substituted Bicyclic Carbamoyl Pyridones: Integrase Strand Transfer Inhibitors that Potently Inhibit Drug-Resistant HIV-1 Integrase Mutants.

HIV-1 integrase (IN) is an important molecular target for the development of anti-AIDS drugs. A recently FDA-approved second-generation integrase strand transfer inhibitor (INSTI) cabotegravir (CAB, 2021) is being marketed for use in long-duration antiviral formulations. However, missed doses during extended therapy can potentially result in persistent low levels of CAB that could select for resistant mutant forms of IN, leading to virological failure. We report a series of N-substituted bicyclic carbamoyl pyridones (BiCAPs) that are simplified analogs of CAB. Several of these potently inhibit wild-type HIV-1 in single-round infection assays in cultured cells and retain high inhibitory potencies against a panel of viral constructs carrying resistant mutant forms of IN. Our lead compound, 7c, proved to be more potent than CAB against the therapeutically important resistant double mutants E138K/Q148K (>12-fold relative to CAB) and G140S/Q148R (>36-fold relative to CAB). A significant number of the BiCAPs also potently inhibit the drug-resistant IN mutant R263K, which has proven to be problematic for the FDA-approved second-generation INSTIs.

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

Identification of multidentate tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors that simultaneously access the DNA, protein and catalytic-binding sites by oxime diversification.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family that can downregulate the anticancer effects of the type I topoisomerase (TOP1) inhibitors by hydrolyzing the 3'-phosphodiester bond between DNA and the TOP1 residue Y723 in the critical stalled intermediate that is the foundation of TOP1 inhibitor mechanism of action. Thus, TDP1 antagonists are attractive as potential enhancers of TOP1 inhibitors. However, the open and extended nature of the TOP1-DNA substrate-binding region has made the development of TDP1 inhibitors extremely challenging. In this study, starting from our recently identified small molecule microarray (SMM)-derived TDP1-inhibitory imidazopyridine motif, we employed a click-based oxime protocol to extend the parent platform into the DNA and TOP1 peptide substrate-binding channels. We applied one-pot Groebke-Blackburn-Bienayme multicomponent reactions (GBBRs) to prepare the needed aminooxy-containing substrates. By reacting these precursors with approximately 250 aldehydes in microtiter format, we screened a library of nearly 500 oximes for their TDP1 inhibitory potencies using an in vitro florescence-based catalytic assay. Select hits were structurally explored as their triazole- and ether-based isosteres. We obtained crystal structures of two of the resulting inhibitors bound to the TDP1 catalytic domain. The structures reveal that the inhibitors form hydrogen bonds with the catalytic His-Lys-Asn triads ("HKN" motifs: H263, K265, N283 and H493, K495, N516), while simultaneously extending into both the substrate DNA and TOP1 peptide-binding grooves. This work provides a structural model for developing multivalent TDP1 inhibitors capable of binding in a tridentate fashion with a central component situated within the catalytic pocket and extensions that project into both the DNA and TOP1 peptide substrate-binding regions.

Synthetic Approaches to a Key Pyridone-carboxylic Acid Precursor Common to the HIV-1 Integrase Strand Transfer Inhibitors Dolutegravir, Bictegravir, and Cabotegravir.

Dolutegravir (DTG), Bictegravir (BIC), and Cabotegravir (CAB) are the second-generation integrase strand transfer inhibitors (INSTIs) that have been FDA-approved for the treatment of HIV-1 infection. Preparation of these INSTIs utilizes the common intermediate 1-(2,2-dimethoxyethyl)-5-methoxy-6-(methoxycarbonyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid (6). Presented herein is a literature and patent review of synthetic routes used to access the pharmaceutically important intermediate 6. The review highlights the ways in which small fine-tuned synthetic modifications have been used to achieve good yields and regioselectivity of ester hydrolysis.

Examination of aminophenol-containing compounds designed as antiproliferative agents and potential atypical retinoids.

Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure-activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.

A Practical Approach to Bicyclic Carbamoyl Pyridones with Application to the Synthesis of HIV-1 Integrase Strand Transfer Inhibitors.

An efficient one-pot synthetic method has been developed for the preparation of bicyclic carbamoyl pyridones from the known common intermediate methyl 5-((2,4-difluorobenzyl)carbamoyl)-1-(2,2-dimethoxyethyl)-3-methoxy-4-oxo-1,4-dihydropyridine-2-carboxylate (8). The scalable protocol is facile and employs readily available reagents, needing only a single purification as the final step. The utility of the approach was demonstrated by preparing a library of HIV-1 integrase strand transfer inhibitors (INSTIs) that differ by the presence or absence of a double bond in the B-ring of the bicyclic carbamoyl pyridines 6 and 7. Several of the analogs show good antiviral potencies in single-round HIV-1 replication antiviral assays and show no cytotoxicity in cell culture assays. In general, the compounds with a B-ring double bond have higher antiviral potencies than their saturated congeners. Our methodology should be applicable to the synthesis of a range of new metal-chelating analogs.

Phosphonic acid-containing inhibitors of tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs stalled type I topoisomerase (TOP1)-DNA complexes by hydrolyzing the phosphodiester bond between the TOP1 Y723 residue and the 3'-phosphate of its DNA substrate. Although TDP1 antagonists could potentially reduce the dose of TOP1 inhibitors needed to achieve effective anticancer effects, the development of validated TDP1 inhibitors has proven to be challenging. This may, in part, be due to the open and extended nature of the TOP1 substrate binding region. We have previously reported imidazopyrazines and imidazopyridines that can inhibit TDP1 catalytic function in vitro. We solved the TDP1 crystal structures with bound inhibitors of this class and found that the dicarboxylic acid functionality within the N-(3,4-dicarboxyphenyl)-2-diphenylimidazo [1,2-a]pyridin-3-amine platform overlaps with aspects of phosphoryl substrate recognition. Yet phosphonic acids could potentially better-replicate cognate TOP1-DNA substrate binding interactions than carboxylic acids. As reported herein, we designed phosphonic acid-containing variants of our previously reported carboxylic acid-containing imidazopyrazine and imidazopyridine inhibitors and effected their synthesis using one-pot Groebke-Blackburn-Bienayme multicomponent reactions. We obtained crystal structures of TDP1 complexed with a subset of inhibitors. We discuss binding interactions of these inhibitors within the context of phosphate-containing substrate and carboxylic acid-based inhibitors. These compounds represent a new structural class of small molecule ligands that mimic aspects of the 3'-processed substrate that results from TDP1 catalysis.

Development of ultra-high affinity bivalent ligands targeting the polo-like kinase 1.

The polo-like kinase 1 (Plk1) is an important mediator of cell cycle regulation and a recognized anti-cancer molecular target. In addition to its catalytic kinase domain (KD), Plk1 contains a polo-box domain (PBD), which engages in protein-protein interactions (PPIs) essential to proper Plk1 function. We have developed a number of extremely high-affinity PBD-binding peptide inhibitors. However, we have reached an apparent limit to increasing the affinities of these monovalent ligands. Accordingly, we undertook an extensive investigation of bivalent ligands, designed to engage both KD and PBD regions of Plk1. This has resulted in bivalent constructs exhibiting more than 100-fold Plk1 affinity enhancement relative to the best monovalent PBD-binding ligands. Startlingly, and in contradiction to widely accepted notions of KD-PBD interactions, we have found that full affinities can be retained even with minimal linkers between KD and PBD-binding components. In addition to significantly advancing the development of PBD-binding ligands, our findings may cause a rethinking of the structure - function of Plk1.

INSTIs and NNRTIs Potently Inhibit HIV-1 Polypurine Tract Mutants in a Single Round Infection Assay.

Integrase strand transfer inhibitors (INSTIs) are a class of antiretroviral compounds that prevent the insertion of a DNA copy of the viral genome into the host genome by targeting the viral enzyme integrase (IN). Dolutegravir (DTG) is a leading INSTI that is given, usually in combination with nucleoside reverse transcriptase inhibitors (NRTIs), to treat HIV-1 infections. The emergence of resistance to DTG and other leading INSTIs is rare. However, there are recent reports suggesting that drug resistance mutations can occur at positions outside the integrase gene either in the HIV-1 polypurine tract (PPT) or in the envelope gene (env). Here, we used single round infectivity assays to measure the antiviral potencies of several FDA-approved INSTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) against a panel of HIV-1 PPT mutants. We also tested several of our promising INSTIs and NNRTIs in these assays. No measurable loss in potency was observed for either INSTIs or NNRTIs against the HIV-1 PPT mutants. This suggests that HIV-1 PPT mutants are not able, by themselves, to confer resistance to INSTIs or NNRTIs.

Design and synthesis of a new orthogonally protected glutamic acid analog and its use in the preparation of high affinity polo-like kinase 1 polo-box domain - binding peptide macrocycles.

Targeting protein - protein interactions (PPIs) has emerged as an important area of discovery for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the polo-like kinase 1 (Plk1) polo-box domain (PBD), a phosphorylated protein residue can provide high-affinity recognition and binding to target protein hot spots. Developing antagonists of the Plk1 PBD can be particularly challenging if one relies solely on interactions within and proximal to the phospho-binding pocket. Fortunately, the affinity of phospho-dependent PPI antagonists can be significantly enhanced by taking advantage of interactions in both the phospho-binding site and hidden "cryptic" pockets that may be revealed on ligand binding. In our current paper, we describe the design and synthesis of macrocyclic peptide mimetics directed against the Plk1 PBD, which are characterized by a new glutamic acid analog that simultaneously serves as a ring-closing junction that provides accesses to a cryptic binding pocket, while at the same time achieving proper orientation of a phosphothreonine (pT) residue for optimal interaction in the signature phospho-binding pocket. Macrocycles prepared with this new amino acid analog introduce additional hydrogen-bonding interactions not found in the open-chain linear parent peptide. It is noteworthy that this new glutamic acid-based amino acid analog represents the first example of extremely high affinity ligands where access to the cryptic pocket from the pT-2 position is made possible with a residue that is not based on histidine. The concepts employed in the design and synthesis of these new macrocyclic peptide mimetics should be useful for further studies directed against the Plk1 PBD and potentially for ligands directed against other PPI targets.

Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures.

Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg2+ ion pair involved in the interaction with the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.

Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand-protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.

HIV-1 Integrase Inhibitors with Modifications That Affect Their Potencies against Drug Resistant Integrase Mutants.

Integrase strand transfer inhibitors (INSTIs) block the integration step of the retroviral lifecycle and are first-line drugs used for the treatment of HIV-1/AIDS. INSTIs have a polycyclic core with heteroatom triads, chelate the metal ions at the active site, and have a halobenzyl group that interacts with viral DNA attached to the core by a flexible linker. The most broadly effective INSTIs inhibit both wild-type (WT) integrase (IN) and a variety of well-known mutants. However, because there are mutations that reduce the potency of all of the available INSTIs, new and better compounds are needed. Models based on recent structures of HIV-1 and red-capped mangabey SIV INs suggest modifications in the INSTI structures that could enhance interactions with the 3'-terminal adenosine of the viral DNA, which could improve performance against INSTI resistant mutants. We designed and tested a series of INSTIs having modifications to their naphthyridine scaffold. One of the new compounds retained good potency against an expanded panel of HIV-1 IN mutants that we tested. Our results suggest the possibility of designing inhibitors that combine the best features of the existing compounds, which could provide additional efficacy against known HIV-1 IN mutants.

Integrase Strand Transfer Inhibitors Are Effective Anti-HIV Drugs.

Integrase strand transfer inhibitors (INSTIs) are currently recommended for the first line treatment of human immunodeficiency virus type one (HIV-1) infection. The first-generation INSTIs are effective but can select for resistant viruses. Recent advances have led to several potent second-generation INSTIs that are effective against both wild-type (WT) HIV-1 integrase and many of the first-generation INSTI-resistant mutants. The emergence of resistance to these new second-generation INSTIs has been minimal, which has resulted in alternative treatment strategies for HIV-1 patients. Moreover, because of their high antiviral potencies and, in some cases, their bioavailability profiles, INSTIs will probably have prominent roles in pre-exposure prophylaxis (PrEP). Herein, we review the current state of the clinically relevant INSTIs and discuss the future outlook for this class of antiretrovirals.

Uncoupling the Folding-Function Paradigm of Lytic Peptides to Deliver Impermeable Inhibitors of Intracellular Protein-Protein Interactions.

Here, we describe the use of peptide backbone N-methylation as a new strategy to transform membrane-lytic peptides (MLPs) into cytocompatible intracellular delivery vehicles. The ability of lytic peptides to engage with cell membranes has been exploited for drug delivery to carry impermeable cargo into cells, but their inherent toxicity results in narrow therapeutic windows that limit their clinical translation. For most linear MLPs, a prerequisite for membrane activity is their folding at cell surfaces. Modification of their backbone with N-methyl amides inhibits folding, which directly correlates to a reduction in lytic potential but only minimally affects cell entry. We synthesized a library of N-methylated peptides derived from MLPs and conducted structure-activity studies that demonstrated the broad utility of this approach across different secondary structures, including both ß-sheet and helix-forming peptides. Our strategy is highlighted by the delivery of a notoriously difficult class of protein-protein interaction inhibitors that displayed on-target activity within cells.

A new genre of fluorescence recovery assay to evaluate polo-like kinase 1 ATP-competitive inhibitors.

Using a probe consisting of a fluorescein-labeled variant of the potent polo-like kinase 1 (Plk1) inhibitor BI2536 [FITC-PEG-Lys(BI2536) 4], we were able to determine half maximal inhibitory concentration (IC50) of ATP-competitive Type 1 inhibitors of Plk1 by means of a fluorescence recovery assay. This methodology represents a cost-effective and simple alternative to traditional kinase assays for initial screening of potential Plk1 inhibitors.

HIV-1 Integrase Inhibitors That Are Active against Drug-Resistant Integrase Mutants.

The currently recommended first-line therapy for HIV-1-infected patients is an integrase (IN) strand transfer inhibitor (INSTI), either dolutegravir (DTG) or bictegravir (BIC), in combination with two nucleoside reverse transcriptase inhibitors (NRTIs). Both DTG and BIC potently inhibit most INSTI-resistant IN mutants selected by the INSTIs raltegravir (RAL) and elvitegravir (EVG). BIC has not been reported to select for resistance in treatment-naive patients, and DTG has selected for a small number of resistant viruses in treatment-naive patients. However, some patients who had viruses with substitutions selected by RAL and EVG responded poorly when switched to DTG-based therapies, and there are mutants that cause a considerable decrease in the potencies of DTG and BIC in in vitro assays. The new INSTI cabotegravir (CAB), which is in late-stage clinical trials, has been shown to select for novel resistant mutants in vitro Thus, it is important to develop new and improved INSTIs that are effective against all the known resistant mutants. This led us to test our best inhibitors, in parallel with DTG, BIC, and CAB, in a single-round infection assay against a panel of the new CAB-resistant mutants. Of the INSTIs we tested, BIC and our compound 4d had the broadest efficacy. Both were superior to DTG, as evidenced by the data obtained with the IN mutant T66I/L74M/E138K/S147G/Q148R/S230N, which was selected by CAB using an EVG-resistant lab strain. These results support the preclinical development of compound 4d and provide information that can be used in the design of additional INSTIs that will be effective against a broad spectrum of resistant mutants.