molecules Review Targeting Toxins toward Tumors Henrik Franzyk and Søren Brøgger Christensen * Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark; [email protected] * Correspondence: [email protected] Abstract: Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme- activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned. Citation: Franzyk, H.; Christensen, Keywords: chemotherapy; prodrug; drug targeting; overexpressed enzymes; ADC; ADEPT; GDEPT; S.B. Targeting Toxins toward Tumors. LEAPT; PROTAC Molecules 2021, 26, 1292. https:// doi.org/10.3390/molecules26051292 Academic Editors: 1. Introduction Marialuigia Fantacuzzi and According to the International Union of Pure and Applied Chemistry (IUPAC), pro- Alessandra Ammazzalorso drugs are defined as chemically modified drugs that undergo biological and/or chemical transformation(s) before eliciting pharmacological responses [1]. Drugs may be converted Received: 9 January 2021 into prodrugs in order to: (i) increase their bioavailability, (ii) target the drugs toward Accepted: 22 February 2021 tissues such as tumors, (iii) decrease toxicity, (iv) increase chemical stability, (v) increase Published: 27 February 2021 solubility, or (vi) mask unpleasant taste [2,3]. Prodrugs are formed by covalent attachment of the drug to a carrier, also often termed as the promoiety, which is subjected to cleavage Publisher’s Note: MDPI stays neutral within the body to release the active drugs. Promoieties and their degradation products with regard to jurisdictional claims in published maps and institutional affil- should be nontoxic and nonimmunogenic [2]. Pharmacologically inactive compounds, iations. which in the organism are modified into active drugs, are known as bio-precursor prodrugs. Examples of such prodrugs are proguanil that in the liver is converted into the antimalarial drug cycloguanil [4], salicin that is converted into salicylic acid [5], and acetanilide that is converted into acetaminophen [3]. Both salicylic acid and acetaminophen are antipyretics and analgesics. Another class of prodrugs is the co-drugs that consist of two drugs cova- Copyright: © 2021 by the authors. lently attached to each other—either directly or via a linker in a way so that they act as Licensee MDPI, Basel, Switzerland. promoieties for each other [2,6]. Examples of co-drugs comprise sulfasalazine, which in the This article is an open access article distributed under the terms and body is degraded to 5-aminosalicylic acid and sulfapyridine [6,7], and benorylate, which is conditions of the Creative Commons an ester of acetylsalicylic acid with paracetamol [6]. Attribution (CC BY) license (https:// A review focused on the prodrug approach revealed the state of the art in 2017 [8]. creativecommons.org/licenses/by/ In the present review new developments in the fields of boronic acids as prodrugs, of 4.0/). anthracyclines, and of antibody–drug conjugates, targeting of paclitaxel and refined use of Molecules 2021, 26, 1292. https://doi.org/10.3390/molecules26051292 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 25 Molecules 2021, 26, 1292 2 of 23 anthracyclines, and of antibody–drug conjugates, targeting of paclitaxel and refined use of prostate-specific membrane antigen (PSMA) for delivery of payloads are described. The prostate-specificproblem of targeting membrane oncogenes antigen for (PSMA)neoplastic for tissue delivery in gene-directed of payloads are enzyme described. prodrug The problemtherapy is of discussed targeting (which oncogenes often for has neoplastic been omitted tissue in in previous gene-directed reviews). enzyme Finally, prodrug a new therapygroup of is prodrugs, discussed which (which link often two has pharmacophores, been omitted in i.e., previous PROtease reviews). Targeting Finally, Chimeras a new group(PROTACs; of prodrugs, [9,10]) and which lectin-directed link two pharmacophores, enxym activated i.e., prodrugs, PROtease are Targeting discussed. Chimeras One of (PROTACs;these pharmacophores [9,10]) and has lectin-directed a high affinity enxym for an activated E3 ubiquitin prodrugs, ligase (an are enzyme discussed. that, Onewith ofassistance these pharmacophores of an E2 ubiquitin-conjugating has a high affinity enzyme, for an transfers E3 ubiquitin ubiquitin ligase to (an its enzyme protein that,sub- withstrate), assistance while the of other an E2 has ubiquitin-conjugating an affinity for the targeted enzyme, protein transfers e.g., ubiquitin a receptor to or its ion protein chan- substrate),nel. After binding while the of the other protein has anand affinity the ubiqui for thetin ligase, targeted the protein biomolecule e.g., a will receptor be modified or ion channel.with ubiquitin After bindingand subsequently of the protein cleaved and by the a ubiquitin26S proteasome, ligase, thewhich biomolecule degrades willubiqui- be modifiedtinated proteins with ubiquitin [9,10]. and subsequently cleaved by a 26S proteasome, which degrades ubiquitinatedThus, the proteins present [review9,10]. comprises the following: (i) prodrugs cleaved in the acidic microenvironmentThus, the present of cancer review cells comprises (Section the 2.1. following:1), (ii) prodrugs (i) prodrugs cleaved cleaved by reactive in the oxygen acidic microenvironmentspecies (ROS) in cancer of cancer cells (Section cells (Section 2.1.2), 2.1.1(iii) prodrugs), (ii) prodrugs cleaved cleaved by glutathione by reactive (Section oxy- gen2.1.3), species (iv) prodrugs (ROS) in cleaved cancer by cells enzymes (Section overex 2.1.2pressed), (iii) prodrugs in cancer cleaved cells ( Section by glutathione 2.1.4), (v) (Sectionprodrugs 2.1.3 cleaved), (iv) prodrugsby glucuronidase cleaved by (Section enzymes 2.1.5), overexpressed (vi) prodrugs in cancer cleaved cells by ( Sectionprostate-spe- 2.1.4), (v)cific prodrugs antigen (PSA) cleaved or by PSMA glucuronidase (Section 2.1.6), (Section (vii) 2.1.5 antibody–drug), (vi) prodrugs conjugates cleaved (Section by prostate- 2.2), specific(viii) antibody-directed antigen (PSA) or enzyme PSMA (Section prodrug 2.1.6 therapy), (vii) (Section antibody–drug 2.3), (ix) conjugates gene-directed (Section enzyme 2.2), (viii)prodrug antibody-directed therapy (Section enzyme 2.4), (x) prodruglectin-directed therapy enzyme-activated (Section 2.3), (ix)prodrug gene-directed therapy (Sec- en- zymetion 2.5), prodrug and (xi) therapy protease-targeting (Section 2.4), (x)chimeras lectin-directed (Section enzyme-activated 3). prodrug therapy (Section 2.5), and (xi) protease-targeting chimeras (Section3). 2. Prodrugs 2. Prodrugs 2.1. Targeting by Selective Cleavage of Prodrugs in the Microenvironment of Cancer Cells 2.1. Targeting by Selective Cleavage of Prodrugs in the Microenvironment of Cancer Cells The metabolism in cancer cells involves a high rate of anaerobic glycolysis resulting The metabolism in cancer cells involves a high rate of anaerobic glycolysis resulting in in an overproduction of lactic acid and carbonic acid. Since the acid protons of these acids an overproduction of lactic acid and carbonic acid. Since the acid protons of these acids are are exported into the extracellular medium, intracellular pH of cancer cells typically is exported into the extracellular medium, intracellular pH of cancer cells typically is higher (pHhigher 7.4 versus(pH 7,4 7.2 versus in normal 7.2 in cells). normal [8,11 cells).,12]. As [8,11,12]. a consequence As a consequence of the proton of transport the proton the microenvironmenttransport the microenvironment surroundingcancer surrounding cells has cancer a pH cells of 6.8 has in a contrast pH of 6.8 to in the contrast is estimated to the pHis estimated of blood pH 7.4 of [13 blood]. A high7.4 [13]. rate A of high glycolysis rate of glycolysis without oxygen without supply oxygen causes supply hypoxia causes insidehypoxia cancer inside cells cancer [8,11 cells,14,15 [8,11,14,15].]. The ensuing The rise ensuing in the rise intracellular in the intracellular level of reactive level oxygenof reac- speciestive oxygen (ROS), species such (ROS), as hydrogen such as peroxide,hydrogen formedperoxide, during formed hypoxia during conditionshypoxia conditions may be employedmay be employed as