Publications by authors named "Tad A Holak"

112 Publications

Ultrasensitive electrochemical determination of the cancer biomarker protein sPD-L1 based on a BMS-8-modified gold electrode.

Bioelectrochemistry 2021 Jun 18;139:107742. Epub 2021 Jan 18.

Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.

This work describes the modification of a gold electrode with the BMS-8 compound that interacts with the Programmed Death-Ligand 1 (PD-L1), an immune checkpoint protein. The results show that we can confirm the presence of the sPD-L1 in the concentration range of 10 to 10 M using electrochemical impedance spectroscopy (EIS) with a limit of detection (LOD) of 1.87 × 10 M for PD-L1 (S/N = 3.3) and at a concentration of 10 M via cyclic voltammetry (CV). Additionally, high-resolution X-ray photoelectron spectroscopy (XPS), contact angle, and surface free energy measurements were applied to confirm the functionalization of the electrode. We investigated the selectivity of the electrode for other proteins: Programmed Death-1 (PD-1), cluster of differentiation 160 (CD160), and B- and T-lymphocyte attenuator (BTLA) at concentrations of 10 M. Differentiation between PD-L1 and PD-1 was achieved based on the analysis of the capacitance effect frequency dispersion at the surface of the modified Au electrode with BMS-8 after incubation at various concentrations of PD-L1 and PD-1 proteins in the range of 10 to 10 M. Significant differences were observed in the heterogeneity of PD-L1 and PD-1. The results of the quasi-capacitance studies demonstrate that BMS-8 strongly and specifically interacts with the PD-L1 protein.
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http://dx.doi.org/10.1016/j.bioelechem.2021.107742DOI Listing
June 2021

Human and mouse PD-L1: similar molecular structure, but different druggability profiles.

iScience 2021 Jan 24;24(1):101960. Epub 2020 Dec 24.

Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.

In the development of PD-L1-blocking therapeutics, it is essential to transfer initial findings into proper animal models. Classical immunocompetent mice are attractive due to high accessibility and low experimental costs. However, it is unknown whether inter-species differences in PD-L1 sequence and structure would allow for human-mouse cross applications. Here, we disclose the first structure of the mouse () PD-L1 and analyze its similarity to the human () PD-L1. We show that PD-L1 interacts with PD-1 and provides a negative signal toward activated Jurkat T cells. We also show major differences in druggability between the PD-L1 and PD-L1 using therapeutic antibodies, a macrocyclic peptide, and small molecules. Our study indicates that while the amino acid sequence is well conserved between the PD-L1 and PD-L1 and overall structures are almost identical, crucial differences determine the interaction with anti-PD-L1 agents, that cannot be easily predicted .
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http://dx.doi.org/10.1016/j.isci.2020.101960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788105PMC
January 2021

Systematic 'foldamerization' of peptide inhibiting p53-MDM2/X interactions by the incorporation of trans- or cis-2-aminocyclopentanecarboxylic acid residues.

Eur J Med Chem 2020 Dec 11;208:112814. Epub 2020 Sep 11.

Department of Bioorganic Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland. Electronic address:

A 'foldamerization' strategy for the discovery of biologically active peptide is evaluated using as an example the peptides that inhibit the p53-MDM2/X interactions. Application of a peptide scan with two constrained β-residue of trans and cis stereochemistry indicated a substitution pattern that leads to active molecules with enhanced conformational stability and high resistance to proteolysis. This procedure led to the discovery of a peptide that showed subnanomolar inhibition of the p53-MDM2 interaction (K = 0.4 nM) with resistance to proteolysis enhanced by ca. two orders of magnitude. Crystallographic analysis and molecular modelling allowed for understanding of these peptide-protein interactions at the molecular level.
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http://dx.doi.org/10.1016/j.ejmech.2020.112814DOI Listing
December 2020

Di-bromo-Based Small-Molecule Inhibitors of the PD-1/PD-L1 Immune Checkpoint.

J Med Chem 2020 10 23;63(19):11271-11285. Epub 2020 Sep 23.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, Krakow 30-387, Poland.

Immune checkpoint blockade is one of the most promising strategies of cancer immunotherapy. However, unlike classical targeted therapies, it is currently solely based on expensive monoclonal antibodies, which often inflict immune-related adverse events. Herein, we propose a novel small-molecule inhibitor targeted at the most clinically relevant immune checkpoint, PD-1/PD-L1. The compound is capable of disrupting the PD-1/PD-L1 complex by antagonizing PD-L1 and, therefore, restores activation of T cells similarly to the antibodies, while being cheap in production and possibly nonimmunogenic. The final compound is significantly smaller than others reported in the literature while being nontoxic to cells even at high concentrations. The scaffold was designed using a structure-activity relationship screening cascade based on a new antagonist-induced dissociation NMR assay, called the weak-AIDA-NMR. Weak-AIDA-NMR finds true inhibitors, as opposed to only binders to the target protein, in early steps of lead compound development, and this process makes it less time and cost consuming.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584369PMC
October 2020

Anti-CD44 DNA Aptamers Selectively Target Cancer Cells.

Nucleic Acid Ther 2020 10 6;30(5):289-298. Epub 2020 May 6.

Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.

CD44 is a type I transmembrane glycoprotein interacting with a number of extracellular components, including hyaluronic acid (HA). CD44-HA axis is involved in a variety of processes, including adhesion, migration, differentiation, trafficking, and others. CD44 is overexpressed in several cancers where binding of HA induces signal transduction leading to activation of antiapoptotic proteins and factors linked to drug resistance. As such, CD44 has been implicated in cancer growth, progression, and metastasis. It has been convincingly demonstrated that blocking CD44-HA interaction decreases cancer cell survival and metastasis. In this study, using selection, we have developed DNA aptamers recognizing a HA-binding domain of CD44 with high affinity and specificity. The aptamers bind to CD44 with nanomolar affinities and efficiently inhibit the growth of leukemic cancer cells characterized by high expression of CD44. The selectivity is demonstrated by an irrelevant effect on cells characterized by low CD44 levels. The obtained aptamers broaden the existing landscape of potential approaches to the development of antitumor strategies based on inhibition of the CD44 axis.
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http://dx.doi.org/10.1089/nat.2019.0833DOI Listing
October 2020

Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein-Protein Interactions.

Angew Chem Int Ed Engl 2020 03 6;59(13):5235-5241. Epub 2020 Feb 6.

Department of Pharmacy, Drug Design group, University of, Groningen, The Netherlands.

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein-protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.
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http://dx.doi.org/10.1002/anie.201916257DOI Listing
March 2020

Optimized Inhibitors of MDM2 via an Attempted Protein-Templated Reductive Amination.

ChemMedChem 2020 02 12;15(4):370-375. Epub 2019 Dec 12.

Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.

Innovative and efficient hit-identification techniques are required to accelerate drug discovery. Protein-templated fragment ligations represent a promising strategy in early drug discovery, enabling the target to assemble and select its binders from a pool of building blocks. Development of new protein-templated reactions to access a larger structural diversity and expansion of the variety of targets to demonstrate the scope of the technique are of prime interest for medicinal chemists. Herein, we present our attempts to use a protein-templated reductive amination to target protein-protein interactions (PPIs), a challenging class of drug targets. We address a flexible pocket, which is difficult to achieve by structure-based drug design. After careful analysis we did not find one of the possible products in the kinetic target-guided synthesis (KTGS) approach, however subsequent synthesis and biochemical evaluation of each library member demonstrated that all the obtained molecules inhibit MDM2. The most potent library member (K =0.095 μm) identified is almost as active as Nutlin-3, a potent inhibitor of the p53-MDM2 PPI.
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http://dx.doi.org/10.1002/cmdc.201900574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064911PMC
February 2020

Hitting on the move: Targeting intrinsically disordered protein states of the MDM2-p53 interaction.

Eur J Med Chem 2019 Nov 6;182:111588. Epub 2019 Aug 6.

Department of Pharmacy, Drug Design Group, University of Groningen, Antonius Deusinglaan 1, 9700 AD, Groningen, the Netherlands. Electronic address:

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal ''lid'' segment of MDM2 from the proximity of the p53-complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds.
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http://dx.doi.org/10.1016/j.ejmech.2019.111588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008132PMC
November 2019

CA-170 - A Potent Small-Molecule PD-L1 Inhibitor or Not?

Molecules 2019 Aug 1;24(15). Epub 2019 Aug 1.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.

is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between and PD-L1. To strengthen our reasoning, we performed control experiments on - a 29-mer peptide, which is a precursor of . Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: and .
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http://dx.doi.org/10.3390/molecules24152804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695792PMC
August 2019

Helping the Released Guardian: Drug Combinations for Supporting the Anticancer Activity of HDM2 (MDM2) Antagonists.

Cancers (Basel) 2019 Jul 19;11(7). Epub 2019 Jul 19.

Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, ul. Gronostajowa 2, 30-387 Krakow, Poland.

The protein p53, known as the "Guardian of the Genome", plays an important role in maintaining DNA integrity, providing protection against cancer-promoting mutations. Dysfunction of p53 is observed in almost every cancer, with 50% of cases bearing loss-of-function mutations/deletions in the gene. In the remaining 50% of cases the overexpression of HDM2 (mouse double minute 2, human homolog) protein, which is a natural inhibitor of p53, is the most common way of keeping p53 inactive. Disruption of HDM2-p53 interaction with the use of HDM2 antagonists leads to the release of p53 and expression of its target genes, engaged in the induction of cell cycle arrest, DNA repair, senescence, and apoptosis. The induction of apoptosis, however, is restricted to only a handful of p53 cells, and, generally, cancer cells treated with HDM2 antagonists are not efficiently eliminated. For this reason, HDM2 antagonists were tested in combinations with multiple other therapeutics in a search for synergy that would enhance the cancer eradication. This manuscript aims at reviewing the recent progress in developing strategies of combined cancer treatment with the use of HDM2 antagonists.
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http://dx.doi.org/10.3390/cancers11071014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678622PMC
July 2019

Design, Synthesis, Evaluation, and Structural Studies of -Symmetric Small Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein-Protein Interaction.

J Med Chem 2019 08 25;62(15):7250-7263. Epub 2019 Jul 25.

The Cancer Institute of New Jersey , New Brunswick , New Jersey 08901 , United States.

A series of -symmetric inhibitors was designed and evaluated for inhibitory activity against the programmed cell death-1/programmed death-ligand 1(PD-1/PD-L1) protein-protein interaction (PPI) in a homogenous time-resolved fluorescence (HTRF) assay and PD-1 signaling in cell-based coculture assays. -symmetric inhibitors (LH1306) and (LH1307) exhibited IC values of 25 and 3.0 nM, respectively, in the HTRF assay. While was ∼3.8-fold more potent than previously reported inhibitor , could not be differentiated from due to their high potency and the limit of our HTRF assay conditions. In one cell-based coculture PD-1 signaling assay, and were 8.2- and 2.8-fold more potent in inhibiting PD-1 signaling than and , respectively. NMR and X-ray cocrystal structural studies provided more structural insights into the interaction between and PD-L1; binds to PD-L1 at the PD-1 binding site and induces the formation of a more symmetrically arranged PD-L1 homodimer than that previously reported for other inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00795DOI Listing
August 2019

Development of the Inhibitors that Target the PD-1/PD-L1 Interaction-A Brief Look at Progress on Small Molecules, Peptides and Macrocycles.

Molecules 2019 May 30;24(11). Epub 2019 May 30.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.

Cancer immunotherapy based on antibodies targeting the immune checkpoint PD-1/PD-L1 pathway has seen unprecedented clinical responses and constitutes the new paradigm in cancer therapy. The antibody-based immunotherapies have several limitations such as high production cost of the antibodies or their long half-life. Small-molecule inhibitors of the PD-1/PD-L1 interaction have been highly anticipated as a promising alternative or complementary therapeutic to the monoclonal antibodies (mAbs). Currently, the field of developing anti-PD-1/PD-L1 small-molecule inhibitors is intensively explored. In this paper, we review anti-PD-1/PD-L1 small-molecule and peptide-based inhibitors and discuss recent structural and preclinical/clinical aspects of their development. Discovery of the therapeutics based on small-molecule inhibitors of the PD-1/PD-L1 interaction represents a promising but challenging perspective in cancer treatment.
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http://dx.doi.org/10.3390/molecules24112071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600339PMC
May 2019

Design of indole- and MCR-based macrocycles as p53-MDM2 antagonists.

Beilstein J Org Chem 2019 20;15:513-520. Epub 2019 Feb 20.

Department of Drug Design, University of Groningen, Antonius Deusinglaan 1, 9700 AD Groningen, The Netherlands.

Macrocycles were designed to antagonize the protein-protein interaction p53-MDM2 based on the three-finger pharmacophore FWL. The synthesis was accomplished by a rapid, one-pot synthesis of indole-based macrocycles based on Ugi macrocyclization. The reaction of 12 different α,ω-amino acids and different indole-3-carboxaldehyde derivatives afforded a unique library of macrocycles otherwise difficult to access. Screening of the library for p53-MDM2 inhibition by fluorescence polarization and H,N HSQC NMR measurements confirm MDM2 binding.
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http://dx.doi.org/10.3762/bjoc.15.45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404402PMC
February 2019

A therapeutic patent overview of MDM2/X-targeted therapies (2014-2018).

Expert Opin Ther Pat 2019 03 1;29(3):151-170. Epub 2019 Mar 1.

a Department of Organic Chemistry, Faculty of Chemistry , Jagiellonian University , Krakow , Poland.

Introduction: MDM2 and MDMX proteins provide the inhibition of p53 tumor suppressor, thus allowing for accelerated mutation-driven cancer microevolution. A pharmacological blockade of MDM2/X-p53 interaction results in p53 reactivation in p53 cells, leading to cancer growth inhibition. Throughout the past 20 years, multiple chemical entities have been proposed to reactivate p53 by antagonizing MDM2/X proteins.

Areas Covered: This manuscript reviews 2014-2018 therapeutic patents in the field of MDM2/X antagonists and is a continuation of previous reviews on similar matter. The patents covering the use of MDM2/X antagonists in drug combinations are also presented in this review, as they constitute an important trend in the field of cancer treatment with MDM2/X antagonists.

Expert Opinion: In the years 2014-2018, several previously-known chemical scaffolds have been further developed and disclosed. Importantly, in the same time period, many lead compounds have entered clinical trials for the treatment of cancer patients. Meanwhile, several important reports have pointed to serious limitations of anticancer properties of MDM2 antagonists. As a result, many efforts have been made to seek for positive, synergistic therapeutic effects of combined anti-cancer treatment strategies. One recent example is a dual targeting of MDM2 and additional protein targets by utilizing the PROTAC technology.
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http://dx.doi.org/10.1080/13543776.2019.1582645DOI Listing
March 2019

A fluorinated indole-based MDM2 antagonist selectively inhibits the growth of p53 osteosarcoma cells.

FEBS J 2019 04 16;286(7):1360-1374. Epub 2019 Feb 16.

Faculty of Chemistry, Jagiellonian University, Krakow, Poland.

The p53 protein is engaged in the repair of DNA mutations and elimination of heavily damaged cells, providing anticancer protection. Dysregulation of p53 activity is a crucial step in carcinogenesis. This dysregulation is often caused by the overexpression of negative regulators of p53, among which MDM2 is the most prominent one. Antagonizing MDM2 with small molecules restores the activity of p53 in p53 wild-type (p53 ) cells and thus provides positive outcomes in the treatment of p53 cancers. Previously, we have reported the discovery of a panel of fluoro-substituted indole-based antagonists of MDM2. Here, we demonstrate the biological activity and stereoselectivity of the most active compound from this series. Both enantiomers of the esterified form of the compound, as well as its corresponding carboxylic acids, were found active in fluorescence polarization (FP) assay, nuclear magnetic resonance (NMR) and microscale thermophoresis (MST) assay, with K and K values around 1 μm. From these four compounds, the esterified enantiomer (R)-5a was active in cells, which was evidenced by the increase of p53 levels, the induced expression of p53-target genes (CDKN1A and MDM2), the selective induction of cell cycle arrest, and selective growth inhibition of p53 U-2 OS and SJSA-1 compared to p53 SAOS-2 cells. The analysis of the crystal structure of human MDM2 in complex with the compound (R)-6a (carboxylic acid of the active (R)-5a compound) revealed the classical three-finger binding mode. Altogether, our data demonstrate the activity of the compound and provide the structural basis for further structure optimization.
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http://dx.doi.org/10.1111/febs.14774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634946PMC
April 2019

NMR fragment-based screening for development of the CD44-binding small molecules.

Bioorg Chem 2019 02 25;82:284-289. Epub 2018 Oct 25.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland. Electronic address:

The cell-surface protein CD44, a primary receptor for hyaluronic acid (HA), is one of the most promising targets for cancer therapies. It is prominently involved in the process of tumor growth and metastasis. The possibility of modulating the CD44-HA interaction with a pharmacological inhibitor is therefore of great importance, yet until now there are only few small molecules reported to bind to CD44. Here, we describe the results of the NMR fragment-based screening conducted against CD44 by which we found eight new hit compounds that bind to the receptor with the affinity in milimolar range. The NMR-based characterization revealed that there are two possible binding modes for these compounds, and for some of them the binding is no longer possible in the presence of hyaluronic acid. This could provide an interesting starting point for the development of new high-affinity ligands targeting the CD44-HA axis.
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http://dx.doi.org/10.1016/j.bioorg.2018.10.043DOI Listing
February 2019

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells.

Cancers (Basel) 2018 Oct 24;10(11). Epub 2018 Oct 24.

Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53 cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53 cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53 cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.
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http://dx.doi.org/10.3390/cancers10110396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266412PMC
October 2018

Specific Antibody Fragment Ligand Traps Blocking FGF1 Activity.

Int J Mol Sci 2018 Aug 21;19(9). Epub 2018 Aug 21.

Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383 Wroclaw, Poland.

Fibroblast growth factor 1 (FGF1) and its receptors (FGFRs) regulate crucial biological processes such as cell proliferation and differentiation. Aberrant activation of FGFRs by their ligands can promote tumor growth and angiogenesis in many tumor types, including lung or breast cancer. The development of FGF1-targeting molecules with potential implications for the therapy of FGF1-driven tumors is recently being considered a promising approach in the treatment of cancer. In this study we have used phage display selection to find scFv antibody fragments selectively binding FGF1 and preventing it from binding to its receptor. Three identified scFv clones were expressed and characterized with regard to their binding to FGF1 and ability to interfere with FGF1-induced signaling cascades activation. In the next step the scFvs were cloned to scFv-Fc format, as dimeric Fc fusions prove beneficial in prospective therapeutic application. As expected, scFvs-Fc exhibited significantly increased affinity towards FGF1. We observed strong antiproliferative activity of the scFvs and scFvs-Fc in the in vitro cell models. Presented antibody fragments serve as novel FGF1 inhibitors and can be further utilized as powerful tools to use in the studies on the selective cancer therapy.
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http://dx.doi.org/10.3390/ijms19092470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163658PMC
August 2018

A patent review on PD-1/PD-L1 antagonists: small molecules, peptides, and macrocycles (2015-2018).

Expert Opin Ther Pat 2018 09 10;28(9):665-678. Epub 2018 Sep 10.

a Department of Drug Design , University of Groningen , Groningen , The Netherlands.

Introduction: The protein-protein interaction PD1/PD-L1 is an important immune checkpoint and several recently approved monoclonal antibodies show promising anti cancer activities in the clinical practice. However, only a small percentage of cancer patients benefit from PD1/PD-L1 directed mAbs. Moreover, some patients experience immune related side effects upon treatment with these mAbs. Recently, several atomic-resolution structures of human PD1/PD-L1, and small molecules, peptides and mAbs with PD-L1 and PD1 open the field for structure based drug design. Small molecules and peptides targeting PD1/PD-L1 promise to enhance tumor activity while showing less immune related side effects.

Areas Covered: We reviewed the small molecules classes and peptides targeting PD1/PD-L1.

Expert Opinion: Currently approved PD1/PD-L1 directed therapeutics show room for improvement. Three classes of non mAb small molecule classes have been discovered so far: (cyclic) peptides as direct competitive PD1/PD-L1 antagonists; small molecules disrupting PD1/PD-L1 and inducing a PD-L1 dimerization; and a small molecule class of unknown mode-of-action. An example of the later group CA-170 is currently investigated in a Phase 1 trial in patients with advanced solid tumors and lymphomas. Potential advantages of small molecules over mAbs include high distribution and better tumor penetration, improved PK/PD, less side effects and oral bioavailability.
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http://dx.doi.org/10.1080/13543776.2018.1512706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323140PMC
September 2018

Crystal structure of the FAS1 domain of the hyaluronic acid receptor stabilin-2.

Acta Crystallogr D Struct Biol 2018 07 27;74(Pt 7):695-701. Epub 2018 Jun 27.

Faculty of Chemistry, Jagiellonian University, 30-387 Cracow, Poland.

Recent research has identified a potential role of the hyaluronic acid receptor stabilin-2 (Stab2) in cancer metastasis. Stab2 belongs to a group of scavenger receptors and is responsible for the clearance of more than ten ligands, including hyaluronic acid (HA). In vivo experiments on mice have shown that the absence of Stab2, or its blocking by an antibody, effectively opposes cancer metastasis, which is accompanied by an increase in the level of circulating HA. Knowledge of ligand recognition and signal transduction by Stab2 is limited and no three-dimensional structures of any protein fragments of this receptor have been solved to date. Here, a high-resolution X-ray structure of the seventh FAS1 domain of Stab2 is reported. This structure provides the first insight into the Stab2 structure.
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http://dx.doi.org/10.1107/S2059798318007271DOI Listing
July 2018

Identification of small-molecule inhibitors of USP2a.

Eur J Med Chem 2018 Apr 5;150:261-267. Epub 2018 Mar 5.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Krakow, Poland. Electronic address:

USP2a is a deubiquitinating protease that rescues its target proteins from destruction by the proteasome by reversing the process of protein ubiquitination. USP2a shows oncogenic properties in vivo and has been found to be a specific activator of cyclin D1. Many types of cancers are addicted to cyclin D1 expression. Targeting USP2a is a promising strategy for cancer therapy but little progress has been made in the field of inhibition of USP2a. Using NMR-based fragment screening and biophysical binding assays, we have discovered small molecules that bind to USP2a. Iterations of fragment combination and structure-driven design identified two 5-(2-thienyl)-3-isoxazoles as the inhibitors of the USP2a-ubiquitin protein-protein interaction. The affinity of these molecules for the catalytic domain of USP2a parallels their ability to interfere with USP2a binding to ubiquitin in vitro. Altogether, our results establish the 5-(2-thienyl)-3-isoxazole pharmacophore as an attractive starting point for lead optimization.
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http://dx.doi.org/10.1016/j.ejmech.2018.03.009DOI Listing
April 2018

Immune Checkpoint PD-1/PD-L1: Is There Life Beyond Antibodies?

Angew Chem Int Ed Engl 2018 04 23;57(18):4840-4848. Epub 2018 Feb 23.

Department of Drug Design, University of Groningen, A. Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

The PD-1/PD-L1 interaction has emerged as a significant target in cancer immunotherapy. Current medications include monoclonal antibodies, which have shown impressive clinical results in the treatment of several types of tumors. The cocrystal structure of human PD-1 and PD-L1 is expected to be a valuable starting point for the design of novel inhibitors, along with the recent crystal structures with monoclonal antibodies, small molecules, and macrocycles.
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http://dx.doi.org/10.1002/anie.201710407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280658PMC
April 2018

Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells.

Oncotarget 2017 Sep 7;8(42):72167-72181. Epub 2017 Aug 7.

Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, 30-060 Krakow, Poland.

Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.
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http://dx.doi.org/10.18632/oncotarget.20050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641120PMC
September 2017

Artificial Macrocycles as Potent p53-MDM2 Inhibitors.

ACS Med Chem Lett 2017 Oct 20;8(10):1025-1030. Epub 2017 Sep 20.

Department of Drug Design, University of Groningen, A. Deusinglaan 1, Groningen 9700AV, The Netherlands.

Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53-MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73, Val93, and Lys94 yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence polarization (FP) assay and H-N two-dimensional HSQC nuclear magnetic resonance experiments.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641952PMC
October 2017

Bioactive Macrocyclic Inhibitors of the PD-1/PD-L1 Immune Checkpoint.

Angew Chem Int Ed Engl 2017 10 26;56(44):13732-13735. Epub 2017 Sep 26.

Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Krakow, Poland.

Blockade of the immunoinhibitory PD-1/PD-L1 pathway using monoclonal antibodies has shown impressive results with durable clinical antitumor responses. Anti-PD-1 and anti-PD-L1 antibodies have now been approved for the treatment of a number of tumor types, whereas the development of small molecules targeting immune checkpoints lags far behind. We characterized two classes of macrocyclic-peptide inhibitors directed at the PD-1/PD-L1 pathway. We show that these macrocyclic compounds act by directly binding to PD-L1 and that they are capable of antagonizing PD-L1 signaling and, similarly to antibodies, can restore the function of T-cells. We also provide the crystal structures of two of these small-molecule inhibitors bound to PD-L1. The structures provide a rationale for the checkpoint inhibition by these small molecules, and a description of their small molecule/PD-L1 interfaces provides a blueprint for the design of small-molecule inhibitors of the PD-1/PD-L1 pathway.
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http://dx.doi.org/10.1002/anie.201707707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400216PMC
October 2017

Structural Biology of the Immune Checkpoint Receptor PD-1 and Its Ligands PD-L1/PD-L2.

Structure 2017 08;25(8):1163-1174

Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland; Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland; Max Planck Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. Electronic address:

Cancer cells can avoid and suppress immune responses through activation of inhibitory immune checkpoint proteins, such as PD-1, PD-L1, and CTLA-4. Blocking the activities of these proteins with monoclonal antibodies, and thus restoring T cell function, has delivered breakthrough therapies against cancer. In this review, we describe the latest work on structural characterization of the checkpoint proteins, their interactions with cognate ligands and with therapeutic antibodies. Structures of the extracellular portions of these proteins reveal that they all have a similar modular structure, composed of small domains similar in topology to the domains found in antibodies. Structural basis for blocking the PD-1/PD-L1 interaction by small molecules is illustrated with the compound BMS-202 that binds to and induces dimerization of PD-L1.
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http://dx.doi.org/10.1016/j.str.2017.06.011DOI Listing
August 2017

Two-Step Synthesis of Complex Artificial Macrocyclic Compounds.

Angew Chem Int Ed Engl 2017 08 1;56(36):10725-10729. Epub 2017 Aug 1.

Drug Design, University of Groningen, Address Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

The design and synthesis of head-to-tail linked artificial macrocycles using the Ugi-reaction has been developed. This synthetic approach of just two steps is unprecedented, short, efficient and works over a wide range of medium (8-11) and macrocyclic (≥12) loop sizes. The substrate scope and functional group tolerance is exceptional. Using this approach, we have synthesized 39 novel macrocycles by two or even one single synthetic operation. The properties of our macrocycles are discussed with respect to their potential to bind to biological targets that are not druggable by conventional, drug-like compounds. As an application of these artificial macrocycles we highlight potent p53-MDM2 antagonism.
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http://dx.doi.org/10.1002/anie.201704426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660312PMC
August 2017

Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) Interaction via Transiently Induced Protein States and Dimerization of PD-L1.

J Med Chem 2017 07 23;60(13):5857-5867. Epub 2017 Jun 23.

Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University , Ingardena 3, 30-060 Krakow, Poland.

Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00293DOI Listing
July 2017

1,4,5-Trisubstituted Imidazole-Based p53-MDM2/MDMX Antagonists with Aliphatic Linkers for Conjugation with Biological Carriers.

J Med Chem 2017 05 16;60(10):4234-4244. Epub 2017 May 16.

Faculty of Chemistry, Jagiellonian University , Ingardena 3, 30-060 Cracow, Poland.

The tumor suppressor protein p53, the "guardian of the genome", is inactivated in nearly all cancer types by mutations in the TP53 gene or by overexpression of its negative regulators, oncoproteins MDM2/MDMX. Recovery of p53 function by disrupting the p53-MDM2/MDMX interaction using small-molecule antagonists could provide an efficient nongenotoxic anticancer therapy. Here we present the syntheses, activities, and crystal structures of the p53-MDM2/MDMX inhibitors based on the 1,4,5-trisubstituted imidazole scaffold which are appended with aliphatic linkers that enable coupling to bioactive carriers. The compounds have favorable properties at both biochemical and cellular levels. The most effective compound 19 is a tight binder of MDM2 and activates p53 in cancer cells that express the wild-type p53, leading to cell cycle arrest and growth inhibition. Crystal structures reveal that compound 19 induces MDM2 dimerization via the aliphatic linker. This unique dimerization-binding mode opens new prospects for the optimization of the p53-MDM2/MDMX inhibitors and conjugation with bioactive carriers.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00104DOI Listing
May 2017