Targeting the Translesion Synthesis Pathway for the Development of

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Targeting the Translesion Synthesis Pathway for the Development of Anti-Cancer Chemotherapeutics Dmitry M. Korzhnev*,† and M. Kyle Hadden*,‡ †

Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030, United States ‡ Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, Connecticut 06269, United States

ABSTRACT: Human cells possess tightly controlled mechanisms to rescue DNA replication following DNA damage caused by environmental and endogenous carcinogens using a set of low-fidelity translesion synthesis (TLS) DNA polymerases. These polymerases can copy over replication blocking DNA lesions while temporarily leaving them unrepaired, preventing cell death at the expense of increasing mutation rates and contributing to the onset and progression of cancer. In addition, TLS has been implicated as a major cellular mechanism promoting acquired resistance to genotoxic chemotherapy. Owing to its central role in mutagenesis and cell survival after DNA damage, inhibition of the TLS pathway has emerged as a potential target for the development of anticancer agents. This review will recap our current understanding of the structure and regulation of DNA polymerase complexes that mediate TLS and describe how this knowledge is beginning to translate into the development of small molecule TLS inhibitors.

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ubiquitinate proliferating cell nuclear antigen (PCNA),13−15 a sliding clamp protein that encircles DNA and functions as a polymerase processivity factor16 (Figure 1A). PCNA ubiquitination at sites of DNA damage (lesions) that high-fidelity replicative DNA polymerases polδ and polε do not bypass serves as the key molecular event for initialization of different modes of DDT. Monoubiquitination of PCNA by the Rad6(E2)/Rad18(E3) enzyme pair13−15 in a process that can be reversed by the deubiquitinating enzyme ubiquitin-specific protease 1 (USP1)17,18 signals switching from normal replication to the “error-prone” mode of DDT (Figure 1B). This mode of DDT is commonly referred to as translesion synthesis (TLS) and is primarily carried out by low-fidelity Yfamily (Rev1, polη, polι, polκ) and B-family (polζ) DNA polymerases.5,6,10−12 In addition, these TLS polymerases have been implicated in filling lesion-containing single-stranded DNA gaps left after replication to prevent the formation of double-stranded breaks.19−22 TLS polymerases can insert

INTRODUCTION Human cells have evolved multiple complex and coordinated pathways to repair DNA that is damaged through a variety of both endogenous and exogenous mechanisms. To maintain genome stability, these pathways, collectively termed the DNA damage response (DDR), serve to recognize distinct types of DNA damage and either activate the requisite repair pathway or guide the cell to apoptosis/senescence. Different DDR mechanisms are activated to repair double-strand breaks, excise altered nitrogenous bases and nucleotides, and repair mispaired nucleotides and intrastrand cross-links.1−4 Even with multiple repair mechanisms, the normal DNA replication machinery still encounters lesions that have evaded repair, potentially causing replication arrest and genome instability. When this occurs, the cells utilize DNA damage tolerance (DDT) mechanisms to bypass the lesion.5−7 The vast majority of DDT in eukaryotes stems from protein products of the RAD6 epistasis group of genes identified over 30 years ago from genetic studies in yeast.8,9 These genes encode a set of low-fidelity DNA polymerases,5,6,10−12 as well as ubiquitin-conjugating E2 enzymes and ubiquitin E3 ligases that © 2016 American Chemical Society

Received: April 18, 2016 Published: June 30, 2016 9321

DOI: 10.1021/acs.jmedchem.6b00596 J. Med. Chem. 2016, 59, 9321−9336

Journal of Medicinal Chemistry

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Figure 1. Role of PCNA in TLS. (A) Human PCNA in complex with p21 PIP-box peptide (yellow) (PDB 1AXC).28 Residue K164, which upon ubiquitination forms an isopeptide bond with the ubiquitin C-terminus, is highlighted in cyan. (B) Regulation of cellular DDT by PCNA ubiquitination.

possess a set of 15 known DNA polymerases that can be classified into A, B, X, and Y families:45,46 the A-family, including DNA polymerases (pols) γ (gamma),47 θ (theta),48,49 and ν (nu);50,51 the B-family, including pols α (alfa), δ (delta), ε (epsilon),52 and ζ (zeta);53,54 the X-family, including pols β (beta), λ (lambda), μ (mu), and TdT (terminal deoxynucleotidyl transferase);55,56 the Y-family,57 including pols η (eta), ι (iota), κ (kappa), and Rev1.5,6,10−12 In addition, a recently discovered primase/polymerase PrimPol belongs to the archaeo-eukaryotic primase (AEP) family,58−60 and telomerase is a reverse transcriptase.61,62 Replication of nuclear DNA is carried out by the replicative DNA polymerases polδ and polε, the primase−polα complex, and telomerase, while replication of mitochondrial DNA is solely performed by the mitochondrial replicase polγ.46 The replicative DNA polymerases are very accurate and processive;23,24 however, they generally cannot cope with sites of DNA damage. For this reason, cells possess additional DNA polymerases that are specialized for different aspects of DNA repair and damage tolerance. In humans, the vast majority of replicative bypass of DNA lesions is performed by five major TLS DNA polymerases, the Y-family polymerases Rev1, polη, polι, and polκ and the B-family polymerase polζ;5,6,10−12 however, the A- and X-family DNA polymerases (e.g., polν, polβ) may also participate in TLS across certain DNA lesions.30,51,56 Catalytic Mechanisms and Lesion Specificity of TLS DNA Polymerases. Structures for the catalytic domains of all human Y-family TLS DNA polymerases in complex with lesioncontaining DNA substrates and an incoming deoxynucleotide triphosphate (dNTP) have been determined by X-ray crystallography and highlighted in several recent reviews.11,12,63 These structures have provided important insights into their lesion specificity and catalytic mechanisms. To date, the catalytic region of the B-family TLS polymerase polζ, which is located in its Rev3 subunit, has not been structurally characterized. Despite the lack of sequence homology between the Y-family and other families of DNA polymerases, the catalytic regions of the Y-family TLS enzymes adopt a typical “right-hand” DNA polymerase architecture (Figure 2). The “palm”, “thumb”, and “finger” domains found in all DNA polymerases and a polymerase associated domain (PAD) unique to Y-family enzymes11,64,65 form a grip on the primertemplate DNA substrate (Figure 2). The “finger” domain aligns the incoming dNTP and the template base, while a set of catalytic residues that coordinate the two Mg2+ ions in the “palm” domain catalyze the formation of a phosphodiester

nucleotides across a range of DNA lesions; however, their accommodating active sites make TLS enzymes highly mutagenic with the rates of nucleotide misincorporation on undamaged DNA of 10−1−10−4; orders of magnitude higher than 10−6−10−8 for the replicative DNA polymerases polδ and polε.23,24 PCNA can be further polyubiquitinated via the formation of K63-linked ubiquitin chains by Ubc13/Mms2 (E2) and HLTF or SHPRH (E3) enzymes,25−27 and this signals switching to the “error-free” mode of DDT that occurs by a template switching (TS) mechanism that utilizes a newly synthesized daughter strand in the sister chromatid as the template to copy over DNA lesions (Figure 1B).7 The DDT pathways are critical for normal cell survival and maintenance of genome stability in the presence of DNA damage; however, these pathways also enable rapidly dividing cancer cells to tolerate DNA damage induced by genotoxic chemotherapeutics, limiting the efficacy of first-line cancer treatment.5,29,30 Platinum-based (cisplatin, carboplatin, oxaliplatin) and other genotoxic agents, which function by forming DNA adducts that block replication in proliferating cancer cells, are the main component of first-line chemotherapy regimens used against many types of human malignancy.31,32 The primary DDT mechanism through which cancer cells can tolerate chemotherapy-induced DNA damage is mutagenic Rev1/polζ-dependent TLS.33−36 In this process, DNA adducts formed by genotoxic agents are bypassed by a combination of TLS DNA polymerases (polη, polι, polκ, Rev1, and polζ), which are organized in a multiprotein TLS complex on a monoubiquitinated PCNA.13−15 Error-prone TLS increases the survival of cancer cells following genotoxic chemotherapy, contributes to enhanced mutagenesis in tumors, and has been linked to the onset of chemotherapy resistance.37−42 With this in mind, targeted inhibition of the TLS pathway is emerging as a new therapeutic strategy to enhance the efficacy of first-line chemotherapy and avert acquired resistance to genotoxic drugs.37,43,44 Herein, we review the current understanding of the structure and regulation of mammalian TLS DNA polymerases acting in “error-prone” DDT, discuss possible strategies for TLS inhibition, and describe recent advances and future prospects for the development of small molecule inhibitors of TLS as anticancer agents.

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STRUCTURE AND REGULATION OF TLS DNA POLYMERASES DNA Polymerases in Human Cells. To efficiently replicate nuclear and mitochondrial DNA, mammalian cells 9322



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Figure 2. Catalytic architecture of Y-family TLS polymerases. The catalytic domain of human Y-family TLS DNA polymerase polη in ternary complex with the incoming dCTP nucleotide (magenta) and primer-template DNA (orange−yellow) that contains a cisplatin adduct (blue spheres (PDB 4EEY).67 The “thumb”, “palm”, “finger”, and “little finger” (or PAD) domains are shown in different colors. The inset details a magnified view of the active site. A comprehensive list of the available structures of catalytic domains of eukaryotic TLS DNA polymerases can be found in table 1 in ref 12.

bond between the 3′-OH end of the DNA primer and dNTP αphosphate.11,12,63 Unlike replicative DNA polymerases, Yfamily TLS enzymes have smaller “thumb” and “finger” domains and form less contacts with dNTP and DNA,11,12,63 resulting in more spacious active sites capable of accommodating distorted lesion-containing DNA substrates. This allows Yfamily enzymes to incorporate nucleotides across a wide range of DNA lesions, making them less processive and selective against incorrect incoming nucleotides and resulting in “errorprone” activity. Additionally, unlike replicative DNA polymerases, all Y-family TLS enzymes and the B-family TLS polymerase polζ are devoid of 3′ → 5′ exonuclease (proofreading) activity,11,66 which also contributes to their low fidelity.23,24 While sharing with all other DNA polymerases the same general catalytic mechanism, Y-family TLS enzymes possess unique structural features, allowing them to replicate across various types of DNA damage.11,12,63 There is a considerable redundancy in TLS DNA polymerase functions, and a given DNA lesion can be copied over by multiple TLS enzymes, albeit with different efficiencies and accuracies. Nevertheless, there is also a significant diversity in active site structures and detailed mechanisms of TLS polymerases, with different TLS enzymes specialized on a bypass of distinct types of DNA damage (cognate lesions).5,6,10−12 The prominent example is accurate and efficient bypass of the most common UV-induced DNA lesion, TT cyclobutane pyrimidine dimers (TT-CPDs), by polη.68−71 TT-CPDs can also be copied over by other TLS enzymes in a less accurate, more mutagenic manner;72,73 therefore, loss of polη activity in xeroderma pigmentosum variant (XPV) patients results in hypersensitivity to sunlight and predisposition to skin cancer.69,74 Additionally, polη is proficient in nucleotide incorporation opposite intrastrand G− G cross-links caused by cisplatin (cisPt-GG),67,75−77 and a number of other lesions.6 Another Y-family TLS polymerase, polκ, can also insert nucleotides opposite various lesions,6 as well as extend the aberrant DNA primer terminus;78,79 most notably, it can efficiently and accurately bypass N2-dG adducts such as those formed by benzo[a]pyrene (BaP-G) present in tobacco smoke.80−82 Polι is the least accurate TLS enzyme related to polη,23,24,83 which uniquely utilizes Hoogsteen pairing of the incoming nucleotides with the template bases and often misincorporates dGMP opposite T bases.84,85

Catalytic activity of Rev1 is limited to dCMP incorporation across G-templates and abasic sites,86−90 while the B-family polymerase polζ primarily specializes in extension of the distorted primer-template following sites of DNA damage,33−36,53,54 but can also incorporate nucleotides across certain DNA lesions.91−94 Two-Step Rev1/polζ-Dependent TLS. The replicative bypass of the vast majority of DNA lesions requires a coordinated action of several TLS DNA polymerases in a two-step process of Rev1/polζ-dependent TLS.33−36 An analysis of TLS across various DNA lesions in mammalian cells suggests that this mechanism is operational for all examined DNA lesions (except TT-CPD), including cisPtGG and BaP-G adducts.33,34 In the first step of Rev1/polζdependent TLS, an “inserter” TLS enzyme incorporates the nucleotide(s) opposite a DNA lesion; this step is typically performed by polη, polι, or polκ,33,34 although for some lesions nucleotide insertion is carried out by Rev186−90 or polζ.91−94 In the second step, the “inserter” polymerase is replaced by an “extender” TLS enzyme, which extends the aberrant primertemplate distorted by a DNA lesion; cell-based data revealed that this step is exclusively carried out by a master “extender” Bfamily TLS polymerase polζ,33,34 although polκ has also demonstrated the ability to extend primer-templates in vitro.78,79 DNA polymerases acting in Rev1/polζ-dependent TLS are organized as a multiprotein TLS complex stabilized by the two central scaffold proteins: PCNA and Rev1. While the limited catalytic activity of Rev1 as a dCPM transferase is considered dispensable,95,96 its “second” function97 to mediate important protein−protein interactions within the overall complex is critical for Rev1/polζ-dependent TLS.5,6,10−12 TLS Regulation by Protein−Protein Interactions. To prevent mutagenic TLS enzymes from copying undamaged DNA and recruit them only when it is necessary to rescue replication stalled at DNA lesions or seal lesion-containing DNA gaps left after replication, cells have evolved a sophisticated network of protein−protein interactions (PPIs) for regulating TLS function. These PPIs mediate localization of TLS DNA polymerases at replication sites, fine-tune assembly of TLS enzyme complexes, and regulate polymerase switching in response to DNA damage.5,6,10−12,53 Beyond their catalytic domains, TLS DNA polymerases possess accessory motifs, domains, and subunits that mediate intermolecular interactions 9323



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Figure 3. Domains and structure of TLS polymerases. (A) Domain organization of human Y-family DNA polymerases Rev1, polη, polι, and polκ.5,6,10−12 (B) Subunit and domain arrangement of the B-family TLS DNA polymerase polζ.53,98−101 (C) Possible configurations of a multiprotein complex acting in Rev1/polζ-dependent TLS at the nucleotide insertion (left) and primer-template extension (right) steps.102 (D) Available structures of the accessory modules of human TLS DNA polymerases, including the Rev1−BRCT domain (PDB 2EBW), Rev1−CT domain in complex with polη−RIR motif (PDB 2LSK),103 polι−UBM (PDB 2KHU),104 and polη−UBZ (PDB 2I5O),105 Rev7 in complex with the Rev7BD region of Rev3 (Rev7/3) (PDB 3ABD),106 and polD2 in complex with the N-terminal domain of polD3 (PDB 3E0J).107 More related structures are available in the Protein Data Bank, such as human Rev1−CT complexes with polκ−RIR108 and polD3−RIR motifs,102 or triple Rev7/ 3:Rev1−CT and quadruple Rev7/3:Rev1−CT:polκ−RIR complexes.109,110 A more comprehensive list of the available structures of regulatory domains of eukaryotic TLS DNA polymerases can be found in table 1 in ref 12 (plots A−C are reproduced from Pustovalova et al.102).

with one another, with the sliding clamp PCNA, and with DNA (Figure 3A,B). Some of these interactions are relatively weak and competitive with one another, providing the means for a multiprotein TLS complex to adjust its configuration and select appropriate TLS enzyme(s) for the bypass of a given DNA lesion. This complex likely adopts different configurations at different types of DNA damage, at replication forks and postreplicational gaps, and at “insertion” and “extension” steps of Rev1/polζ-dependent TLS (Figure 3C) with different subsets of possible interaction realized at a given time. The structures of these accessory modules and an in-depth understanding of their intermolecular interactions are only now beginning to emerge (Figure 3D). PCNA-Mediated Interactions. Ubiquitination of the sliding clamp PCNA is a central event in TLS regulation, creating new binding sites that modulate affinities of PCNA for TLS enzymes. The exchange of replicative to TLS DNA polymerases at replication forks stalled at DNA lesions is triggered by Rad6/

Rad18-dependent monoubiquitination of PCNA at residue K164.13−15 TLS polymerases polη, polι, and polκ interact with PCNA via noncanonical PCNA interacting protein box (PIPbox) motifs111−114 that are also found in many proteins involved in DNA replication and DDR16 (Figures 1A, 3A). By contrast, Rev1 does not have a PIP-box and utilizes its BRCA1 C-terminus (BRCT) domain for PCNA binding115,116 (Figure 3A,D). In addition, all Y-family TLS enzymes have ubiquitin binding UBM (Rev1, polι) or UBZ (polη, polκ) domains that enhance their interaction with monoubiquitinated PCNA117 (Figure 3A,D). Because ubiquitin-binding domains are missing in replicative DNA polymerases polδ and polε, UBM and UBZ domains of the Y-family TLS enzymes facilitate the replicative DNA polymerase to TLS DNA polymerase switch following PCNA ubiquitination via an affinity driven competition.118 PCNA and ubiquitin binding domains are required for onepolymerase bypass of the polη cognate lesion TT-CPD and for 9324



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subunits (that are also accessory subunits of the replicative DNA polymerase polδ), represents a more efficient and processive form of polζ that exhibits an order of magnitude greater polymerase activity than the Rev3/Rev7 complex.53,98−101 In addition to Rev3/Rev7 interactions, the foursubunit polζ complex is stabilized by interactions between polD2 and the C-terminal region of Rev3, which contains an iron−sulfur 4Fe−4S cluster and a zinc-finger domain,98,140 and between polD2 and the N-terminal domain of PolD3107 (Figure 3C). The four-subunit polζ complex acquires PIP-box and RIR motifs in the C-terminal part of polD3 subunit (Figure 3C) that enhance polζ interactions with PCNA and Rev1, helping displace an “inserter” TLS enzyme with the “extender” polζ upon Rev1/polζ-dependent TLS.102 Overall, these studies suggest that PPIs of the accessory subunits of polζ play important roles in both modulating the enzymatic activity of polζ and mediating polymerase switching events.

localization of polη, polι, polκ, and Rev1 at DNA damage induced foci.113,115,117−121 Rev1-Mediated Interactions. The primary role of Rev1 in mutagenic Rev1/polζ-dependent TLS is as a scaffold protein that mediates both assembly of the multipolymerase TLS complex and recruitment of the “extender” polymerase polζ to sites of DNA damage.5,6,10−12,53 Beyond the ubiquitin-binding UBM motifs, Rev1 possesses two unique interaction modules that are critical for functional Rev1/polζ-dependent TLS, the N-terminal Rev1−BRCT and the C-terminal Rev1−CT domains (Figure 3A,D). Deletion or mutation of either modules leads to a phenotype similar to that of Rev1-deficient cells, which is characterized by increased sensitivity to DNA damage and a significantly reduced mutation rate.35,96,122−124 In fact, UV-sensitive nonmutable (or reversionless) phenotype conferred by rev1−1 mutation that maps to the Rev1−BRCT domain led to identification of the REV1 gene in yeast genetic screens in the early 1970s.125 The Rev1−BRCT domain mediates its interactions with PCNA by utilizing the binding site on the PCNA surface that also interacts with the PIP-box motifs.115,116 In addition, the Rev1−BRCT domain and preceding ∼20 amino acid residues interact with 5′ phosphorylated recessed DNA primer-template junctions, presumably to mediate Rev1 recruitment to postreplicational single-stranded DNA gaps.126 The Rev1−CT domain is a fourhelix bundle module (Figure 3A,D) that mediates Rev1 interactions with other TLS DNA polymerases, including polη, polι, polκ, and polζ, and plays an indispensable role in Rev1/polζ-dependent TLS.102,103,108−110,127−133 In spite of its relatively small size (11 kDa), Rev1−CT utilizes two independent interaction interfaces to simultaneously bind the accessory Rev7 subunit of the “extender” TLS polymerase polζ, and a Rev1-interacting region (RIR)130 from one of the “inserter” polymerases, polη, polι, or polκ.102,103,108−110,131−133 RIR-motifs have also been identified in polD3 that may serve as an accessory subunit of polζ102 (Figure 3B,C) and in other DDR proteins such as XRCC1, which is involved in base excision and single-strand break repair.134 These findings point to a key role of Rev1−CT/RIR PPIs in mediating TLS enzyme selection for a lesion bypass and polymerase switching, as well as coordination of TLS and other DDR pathways. Multisubunit polζ Assembly. The structure, subunit arrangement, and architecture of the master “extender” TLS DNA polymerase polζ are the most enigmatic aspects of Rev1/ polζ-dependent TLS.53,54 The complex of catalytic Rev3 and accessory Rev7 subunits constitutes the minimal unit required for polζ function.91 Rev7 (Mad2B) is a 24 kDa HORMA (Hop1, Rev7, and Mad2) domain106,135,136 that interacts with a short Rev7-binding (Rev7BD) motif of Rev391,106 (Figures 3B,D). Rev7 undergoes significant conformational change upon Rev3 binding, which locks Rev7 in a closed conformation and creates a Rev7 binding site for the Rev1−CT domain (Figure 3D).106 Recent discovery of a second Rev7BD motif in human Rev3137 (Figure 3A) and observation that human Rev7 undergoes dimerization in solution138 suggests a possibility that the Rev3/Rev7 assembly may contain two copies of Rev7 associated with one another. Beyond mediating interactions with Rev7 via the two Rev7BD motifs,91,106,137 the functional role of the nearly 2300 residue long N-terminal region preceding the catalytic domain of human Rev3139 remains to be established. Recently, several groups have shown that a foursubunit polζ (Figure 3B,C), which consists of Rev3/Rev7 and a complex of two additional polD2 (p50) and polD3 (p66)

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TLS AND CANCER TLS in Onset and Progression of Cancer. TLS provides an essential means to complete DNA replication and maintain genomic stability following DNA damage.5−7,29,30 Defects in TLS result in genomic instability and predisposition to cancer, as evidenced by loss of polη function resulting in light sensitive XPV syndrome.69,74 Disruption of polζ activity in Rev3deficient cells results in increased frequency of chromosome aberrations,141−143 and a conditional loss of Rev3 in mice leads to enhanced spontaneous tumorigenesis.144 On the other hand, functional TLS is intrinsically mutagenic, with the majority of DNA damage induced and spontaneous mutations introduced in the genome attributed to Rev1/polζ-dependent TLS.35,39,40,124,145−147 For this reason, TLS has been implicated as a causative factor for the development of cancer.148−150 Furthermore, TLS has been identified as a mechanism through which cancer cells become resistant to genotoxic first-line chemotherapy.37−42 Thus, TLS contributes to a mutator phenotype of cancer cells characterized by mutation frequency orders of magnitude greater than that of normal tissue.151 The increased intrinsic mutation rate in cancers is further elevated by genotoxic chemotherapy, resulting in accumulation of random mutations and rapid development of tumor heterogeneity. The selection of subpopulations of drug-resistant cells evolved as a result of mutagenesis underlies the chemotherapy resistance acquired by relapsed tumors.37,38 Many studies that link TLS to the onset of chemotherapy resistance have focused on the mechanisms through which Rev1 and the polζ subunits Rev3 and Rev7 contribute to cisplatin-induced mutagenesis and acquired resistance in several forms of human cancers.37−42 Initial studies exploring the requirements of TLS for cisplatin-mediated mutagenesis focused on the essential role of polζ subunit Rev3 in this process in human fibroblasts.39 A reduction in Rev3 mRNA expression in human fibroblasts resulted in a significant decrease in both the DNA mutation rate and development of resistance associated with cisplatin treatment. Studies in ovarian cancer cell lines demonstrated that knockdown of Rev1 with shRNA produced similar results.40 A decrease in Rev1 expression significantly reduced the ability of cisplatin to generate drug-resistant variants in the surviving population while concomitantly increasing the sensitivity of these cells to cisplatin treatment. More recently, several research groups have expanded these studies to explore Rev1/Polζ-dependent TLS in cellular and 9325



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Figure 4. Small molecule inhibitors of Y-family polymerase catalytic activity.

among a number of possibilities in the extensive TLS interaction network (Figure 3). Another strategy being utilized to inhibit TLS is through the development of small molecules that target the activity of Rad6/Rad18 and USP1 enzymes, which are crucial for PCNA ubiquitination/deubiquitination and regulate switching between normal DNA replication and TLS.13−15,17,18 Decreasing the level of PCNA monoubiquitination by inhibiting the Rad6/ Rad18 complex may prevent TLS enzymes from gaining access to DNA and thus lead to TLS inhibition (although in human cells some TLS activity may occur without PCNA ubiquitination153,154). The development of small molecules that inhibit the deubiquitinase USP1 have also demonstrated the ability to enhance sensitivity to cisplatin in cell culture,155,156 suggesting that this strategy may also result in targeted adjuvant agents for first-line chemotherapy. USP1 inhibition results in constitutive PCNA ubiquitination, which may lead to more frequent access of TLS enzymes to DNA and the inability of replicative polymerases to resume and complete DNA replication.155 It is important to note that USP1 inhibition may also affect other DDR pathways because, beyond PCNA, it also deubiquitinates DDR proteins such as FANCD2 and FANCI, which are involved in interstrand cross-link repair.17,18

animal models of B-cell lymphoma, lung, and prostate cancer.37,43,44 Depletion of either Rev3 or Rev1 (via shRNA) sensitized B-cell lymphomas to cisplatin treatment in vitro and in vivo.37 In addition, reduced expression of Rev1 resulted in a corresponding decrease in the incidence of DNA mutations and acquired resistance for the first-line chemotherapeutic cyclophosphamide in a murine model of Burkitt’s lymphoma, which correlated to sustained tumor regression and an increase in overall survival.37 Similar results were seen in vitro and in vivo when Rev3-deficient nonsmall cell lung cancers (NSCLC) were treated with cisplatin.43 Rev3 suppression promoted enhanced efficacy of cisplatin in vivo and significantly increased survival rate. Finally, codelivery of Rev1/Rev3 specific siRNA and a cisplatin prodrug encapsulated together in a biodegradable nanoparticle completely abolished tumor growth in a murine xenograft model or prostate cancer.44 Taken together, these data highlight the potential therapeutic effect associated with disruption of TLS in cancer cells and provide important context for developing small molecules that target this process. Targeting TLS for Cancer Therapy. Several distinct strategies exist for developing small molecule inhibitors of TLS as anticancer agents. The first strategy is to identify inhibitors of TLS enzyme catalytic activity; however, different DNA polymerases share the same general catalytic mechanism11,12,63 and one may anticipate that such inhibitors will lack the necessary selectivity. This broad spectrum of activity is clearly exemplified by the replicative DNA polymerase inhibitors based on the nucleoside scaffold.152 The Y-family TLS enzymes demonstrate significant structural diversity in their active sites, which enables them to accommodate and bypass different types of DNA damage.11,12,63 In addition, structural differences between the TLS and replicative DNA polymerases discussed above suggest that the development of specific inhibitors of catalytic activity for a given TLS enzyme may be possible. A second strategy is to target essential PPIs mediated by regulatory domains and subunits of TLS DNA polymerases that provide access to DNA or mediate assembly of their active complexes. The fact that deletion/mutation of key regulatory domains such as Rev1−CT and Rev1−BRCT domains results in increased cell sensitivity to DNA damage and reduced mutagenesis suggests that this is a valid approach to inhibit TLS.35,96,122−124 Primary concerns of this strategy include the inherent difficulty in identifying and developing small molecule inhibitors of PPIs and choice/validation of a target interaction

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SMALL MOLECULE INHIBITORS OF TLS As noted above, there are multiple families of DNA polymerases (A, B, X, and Y) that are associated with cellular DDR pathways. Although A- and X-family polymerases may participate in TLS across certain types of DNA damage,30,51,56 here we primarily focus on small molecule inhibitors and potential strategies for inhibition of the Y-family polymerases (Rev1, polη, polι, and polκ) and the B-family polymerase polζ known to be essential for TLS and other proteins involved in regulation of these TLS enzymes in human cells. Small Molecules that Target TLS Polymerase Activity. Researchers at the National Institutes of Health have reported the development and implementation of a novel, broadly applicable assay to identify inhibitors of DNA polymerase enzymatic activity in real time.157 This assay is a primer extension assay based on a tripartite synthetic nucleotide containing the following: (A) a template strand labeled at the 5′-end with a BHQ-2 quencher, (B) a 3′-end primer strand complementary to the template, and (C) a reporter strand complementary to the 5′-end of the template labeled at the 3′9326



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Figure 5. Fungal-derived natural product inhibitors of Y-family DNA polymerases.

end with a rhodamine dye.157 Incorporation of a nucleotide by an active DNA polymerase at the 3′-end of the primer sequence displaces the fluorescent reporter, which reduces its proximity to the quencher and results in a fluorescent signal. The assay was miniaturized and validated in a 1536-well format and several small molecules known to inhibit DNA polymerase β or other DNA-processing enzymes were evaluated for their activity against polι and polη. Compounds 1 (aurintricarboxylic acid, IC50 values = 75−99 nM) and 2 (ellagic acid, IC50 values = 62− 81 nM) were identified as potent, nonselective inhibitors of both TLS DNA polymerases (Figure 4). Researchers at the Oregon Health and Science University applied the DNA-strand displacement assay described above to screen approximately 16K bioactive small molecules for their ability to inhibit polκ.158 Secondary validation studies of the screening hits identified 3 (MK-886, IC50 = 13 μM), 4 (candesartan cilexetil, IC50 = 9.2 μM), and 5 (manoalide, IC50 = 3.4 μM) as moderate inhibitors of polκ (Figure 4). Additional in vitro studies demonstrated the ability of each of these compounds to inhibit TLS by evaluating their ability to prevent polκ-mediated TLS past acrolein-induced γ-HOPdG (γhydroxy-1,N(2)-propano-2′deoxyguanosine) lesions at a level comparable to their inhibition of polκ enzymatic activity (IC50 values = 5.6−14 μM). In addition, 4 enhanced UV-induced cellular toxicity, suggesting its ability to inhibit TLS in cultured cells; however, it demonstrated comparable inhibitory activity against both polη and polι (IC50 values = 11.2 and 6.2 μM, respectively).158 Interestingly, follow-up studies for 3 demonstrated that it was approximately 6-fold more potent against polι than the other Y-family polymerases.159 Preliminary computational docking of 3 with the Y-family polymerases suggested two potential binding pockets conserved across polη, polκ, and polι, with a third potential binding pocket distinct to polι.159 Mechanistic analysis of the kinetics with which 3 inhibits polι was inconclusive, suggesting a potential mixed-type inhibition of the TLS polymerase. Several fungal-derived natural products have been recently characterized as nonselective inhibitors of human TLS

polymerases (Figure 5). Two structurally related compounds isolated from the fungus Penicillium dalae were characterized as inhibitors of several vertebrate Y-family DNA polymerases (human polκ and polη; mouse polι).160 Penicilliols A (6) and B (7) (Figure 5) were identified as noncompetitive inhibitors with moderate activity (approximate IC50 values 20 and 40 μM, respectively). Interestingly, these natural products were inactive against all other vertebrate and invertebrate DNA polymerases and other DNA metabolic enzymes at concentrations up to 100 μM.160 Talaroflavone (8) and 1-deoxyrubralactone (9) were isolated from a fungal strain that was originally collected from a Japanese sea algae and identified as modest inhibitors of vertebrate DNA polymerases (IC50 values approximately 80 μM).161 In contrast to the penicilliols, these natural products inhibited vertebrate polβ and polλ more potently than the Yfamily polymerases, with IC 50 values against polβ of approximately 10−20 μM. A cultured fungal strain from a sea salt pan in Australia produced 3-O-methylfunicone (10) as a modest inhibitor of polη (IC50 = 50 μM), polι (IC50 = 34 μM), and polκ (IC 50 = 12.5 μM). 162 Natural product 10 demonstrated slight inhibition of several B-family polymerases (approximately 30−40% inhibition at 100 μM). Finally, three structurally related azaphilones (11−13) from a seaweedassociated fungus (Penicillium pinophilum) were moderate inhibitors of both B-family and Y-family polymerases (IC50 values = 49−92 μM) that demonstrated antiproliferative effects in several human cancer cell lines (GI50 values = 50−99 μM). Small Molecules that Disrupt TLS Protein−Protein Interactions. Targeting Protein−Protein Interactions. Although PPIs play an essential role in cellular processes, targeting them with “drug-like” small molecules for therapeutic purposes is difficult. Most often, this stance is attributed to the large, flat surface area at the PPI interface (1000−3000 Å2), which precludes a small molecule from disrupting PPIs to a degree sufficient to perturb the protein complex.163,164 Studies have shown that the entire surface area of the PPI interface is oftentimes not critical; rather, small sets of residues, termed hot spots, contribute the majority of binding free energy for the 9327



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Figure 6. Protein−protein interaction hot spots in the TLS multiprotein complex. (A) Human PCNA/polη-PIP binding interface.114 Residues L704, F707, and F708 (magenta) of polη-PIP, 704-MQTLESFF-708, fit in the binding pocket on the PCNA surface formed by residues marked in blue; additional binding pocket (Q-pocket) accommodates side chain of M701 of the PIP-motif. (B) Hot spots on the human Rev1−CT/Rev7110 and Rev1−CT/polη−RIR103 binding interfaces. Key residues involved in interactions are labeled.

two α-helices and an N-terminal β-hairpin and account for the majority of intermolecular contacts that stabilize the Rev1− CT−RIR complex (Figure 6B). Rev1−CT−Rev7/3 PPIs are mediated by a combination of hydrophobic contacts and intermolecular hydrogen bonds, all of which involve the Rev1− CT face opposite of the binding site for RIR motifs.108−110,131−133 Binding interactions are mediated by Rev1−CT residues from the beginning of α-helix H3 and the H2−H3 loop and from the spatially adjacent region encompassing the end of α-helix H4 and the flexible Cterminus of the domain (Figure 6B). This region includes a well-characterized hydrophobic pocket at the Rev1−CT− Rev7/3 interface that holds potential as a hot spot for small molecule inhibitor development. PCNA/PIP-Box PPI. Researchers at St. Jude Children’s Hospital performed a high-throughput screen of approximately 38K compounds to identify small molecules with the ability to disrupt PCNA/PIP-box peptide interactions. From this screen, 3,3′,5-triiodothyronine (T3, 14), a potent thyroid hormone, was identified as a modest inhibitor of this PPI with an IC50 value of approximately 3 μM (Figure 7).168 To enhance selectivity for PCNA/PIP-box PPIs, a structure-based approach was employed to develop a related analogue, 15 (T2AA), which did not exhibit appreciable thyroid hormone activity but maintained the ability to disrupt the PCNA/PIP-box PPI (IC50 approximately 1 μM) (Figure 7). The determination of a

entire interface.165,166 These hot spots generally occupy an area comparable to the size of a small molecule and can adopt multiple conformations, suggesting they hold intriguing potential as small molecule binding sites. The identification and targeting of these hot spots has allowed for a significant increase in the development of small molecule inhibitors of a variety of PPIs, many of which exhibit drug-like properties and have entered clinical trials.163,167 These advances in targeting PPIs highlight the increasing knowledge with respect to developing small molecules against these structures while also providing a template for the successful development of future compounds. PCNA serves as a binding platform for a number of proteins involved in DNA replication and DDR.16 The majority of partner proteins that interact with PCNA do so via canonical PIP-box motifs defined as Qxx(M/L/V)xx(F/Y)(F/Y). By contrast, noncanonical PIP-box motifs, in which the conserved Q is replaced by an M, K, or R residue, are found in Y-family TLS polymerases polη, polι, and polκ111−114 (Figure 3A). The PIP-box is also present in the C-terminus of polD3 that may serve as an accessory subunit of polζ. The binding site for PIPbox motifs located in the interdomain region of PCNA has subsites that can accommodate side chains of key residues of the PIP-box motif28,114 and represent a potential hot spot that can be targeted with small molecules (Figure 6A). It is important to note that PCNA/PIP-box interactions are essential not only for TLS but also for other DDR mechanisms and processive DNA replication;16 therefore, disruption of this PPI may prove less selective for cancer cells when compared to other TLS PPIs. Other examples of PPI hot spots that potentially can be exploited for the design of small molecule TLS inhibitors are binding pockets at the interfaces of Rev1−CT−RIR motif (Figure 6B, right) and Rev1−CT−Rev7/3 complexes (Figure 6B, left). The results of structural and mutational studies of the polκ, polη, and polD3 RIR motifs identified the importance of an FF pair within the RIR for binding the Rev1−CT domain.102,103,108,109,130,132,133 The side chains of these Phe residues (F531 and F532 in human polη103,130) interact with a binding pocket on Rev1−CT formed by residues from the first

Figure 7. Small molecule inhibitors of PCNA/PIP-box PPIs. 9328



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∼10−30 μM). Finally, compound 20 (0.5 μM) inhibited Rad6B-mediated ubiquitination of histone H2A in MDA-MB231 lysates, demonstrating its ability to inhibit Rad6B in cell culture.172 To date, the ability of this class of compounds to inhibit ubiquitination of PCNA or other aspects of TLS have not been reported. USP1/UAF1. USP1 is a deubiquitinase enzyme (DUB) that has demonstrated the ability to deubiquitinate PCNA in cells17,18 and has been implicated as a potential therapeutic target for enhancing the efficacy of first-line genotoxic chemotherapeutics. The catalytic activity of USP1 is minimal prior to complexation with USP associated factor 1 (UAF1).173 Researchers at the University of Delaware recently performed a screen on ∼10K small molecules with known biological activity to identify compounds that inhibit USP1/UAF1 enzymatic activity.174 Secondary validation of hit compounds in an orthogonal USP1/UAF1 inhibition assay identified five compounds with promising activity. Pimozide (21, IC50 = 2 μM), GW7647 (22, IC50 = 5 μM), flupenthixol (23, IC50 = 7 μM), and trifluoperazine (24, IC50 = 8 μM) as reversible inhibitors, while rottlerin (25, IC50 = 8 μM) was an irreversible inhibitor (Figure 9). Of these, 21 and 22 are the most selective for the USP1/UAF1 complex and were characterized further. Both compounds were identified as noncompetitive inhibitors of the catalytic activity of the complex, but neither functioned by disrupting the USP1/UAF1 PPI. Combined administration of either compound with cisplatin significantly enhanced (2−4fold) the antiproliferative activity of the platinating agent in cisplatin-resistant human NSCLC cell line H596, but they had no effect in cisplatin sensitive cells. Finally, both compounds, alone and combined with cisplatin, increased the level of UbPCNA in HEK293T cells, providing evidence of their antiUSP1/UAF1 activity in cell culture.174 A parallel small molecule screen of approximately 400 K compounds identified hit compound 26 as a modest inhibitor of USP1/UAF1 (IC50 = 7.9 μM) (Figure 9).155,156 A subsequent medicinal chemistry effort was undertaken to optimize both the potency and selectivity of the hit scaffold.156 Modifications to the o-trifluoromethyl phenyl ring demonstrated that while substitutions at the 2-position were welltolerated, incorporating functional groups at either the 3- or 4position abolished the inhibitory activity of the scaffold. The most active compounds were generated when the 2-position substitution was an iso-propyl moiety. Replacement of the quinazoline core with a pyrimidine was well-tolerated and reduced the molecular weight and lipophilicity.156 Replacement of the thiophene with a phenyl ring was well-tolerated, and substitutions to the ring had minimal effects on potency. Ultimately, a 4-position 1,2,3-triazole was determined as most advantageous due to the enhanced metabolic stability and cellular permeability of lead analogue ML323 (27, Figure 9), which demonstrated high potency against USP1/UAF1 (IC50 = 0.076 μM), moderate stability (T1/2 = 15 min in rat liver microsomes), and promising in vitro physicochemical properties.156 The extensive pharmacological characterization of 27 showed that it is a reversible inhibitor of the USP1/UAF1 complex is inactive against other DUBs, related proteases, and kinases.155 Combined treatment of H596 cells with cisplatin and 27 resulted in a 3-fold increase in levels of Ub-PCNA compared to either monotherapy and cotreatment significantly enhanced the antiproliferative effects of cisplatin in these cells [GI50 = 486 (cisplatin alone) and 59 (cisplatin + 27) nM]. Finally, treatment with 27 led to a 31% decrease in the ability of

cocrystal structure of 15 bound to PCNA demonstrated that the small molecule binds PCNA at the PIP-box cavity.169 In addition, 15 prevented PCNA/polη PPIs in a concentrationdependent fashion (IC50 approximately 20 μM) and sensitized HeLa and U2OS bone osteosarcoma cells to cisplatin treatment.169 Subsequent to these findings, a structure−activity relationship (SAR) study on the 14/15 scaffold was undertaken to identify compounds with improved potency and anti-TLS activity.170 Overall, removal of the iodine at the 3′-position of the “left-side” phenyl improved activity and replacing the primary alcohol with substituted amides provide the most active compounds, exemplified by compounds 16 and 17 (IC50 values = 1.5 and 1.3 μM, respectively) (Figure 7). Neither compound demonstrated antiproliferative effects in U2OS cells up to 15 μM when administered alone; however, both enhanced the sensitivity of U2OS cells to cisplatin when coadministered with the platinating agent.170 An irreversible inhibitor of PCNA/PIP-box PPIs based on the scaffold of 15 was also recently reported.171 This compound, termed T2Pt (18), was designed to preferentially alkylate Met40 and His44 of PCNA, two residues essential for the PCNA/PIP-box PPI (Figure 7). Compound 18 demonstrated minimal activity in clonogenic survival assays when administered as a single agent; however, it was able to sensitize cells to cisplatin at a modest concentration (30 μM). Small Molecule TLS Inhibitors that Target Other Proteins in the Pathway. Rad6B. As noted above, monoubiquitination of PCNA by Rad6/Rad18 enzymes signals switching from normal replication to TLS following DNA damage.13−15 For this reason, small molecules that interfere with human Rad6B and/or Rad18 function may hold promise as potential TLS inhibitors. A computational lead identification approach was utilized by researchers at Wayne State University to identify the first small molecule inhibitors of Rad6B function.172 Conserved E2 residues responsible for stabilizing the E2-ubiquitin thioester intermediate were utilized to generate a four-point pharmacophore model based on key hydrogen-bond donor and acceptor points. This pharmacophore model was screened against a prefiltered subset of the ZINC database to identify the substituted diamino-triazine core as a potential lead scaffold for inhibition of Rad6B function, and a small series of triazine analogues was synthesized and evaluated for their ability to inhibit Rad6B. Preliminary SAR for these compounds suggested that while substitution at either phenyl ring was not required for inhibition of Rad6B, incorporation of a nitro functionality at the para-position of the ester-linked phenyl ring enhanced activity and selectivity for Rad6B when compared to other E2 enzymes.172 The two most active triazines, 19 and 20 (Figure 8), demonstrated potent inhibition of Rad6B-mediated ubiquitination at 25 nM in a biochemical assay and decreased cellular proliferation and migration of MDA-MB-231 breast cancer cell lines (GI50 values

Figure 8. Small molecules that inhibit Rad6B. 9329



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Figure 9. Small molecule inhibitors of USP1/UAF1.

demonstrating their up-regulation in a variety of human cancers.180,181 Small molecule inhibitors of each of these enzymes have demonstrated the ability to sensitize human cancer cells to a variety of first-line genotoxic agents, albeit at modest concentrations.176−178 In general, extensive SAR studies for these scaffolds against polβ or polλ have not been explored; therefore, the number of small molecule polymerase inhibitors available is minimal. To the best of our knowledge, most of these compounds have not been evaluated for their ability to inhibit TLS polymerases. As noted above, the structural architecture of the catalytic domain is similar across all DNA polymerases; however, the active site of Y-family TLS polymerases is more spacious, which is important for their function in copying across distorted lesion-containing DNA substrates but is also the primary cause of their low fidelity. Moving forward, it will be important to determine whether SAR studies on these or new scaffolds can take advantage of the difference in active site arrangement and size to develop compounds that selectively target the TLS polymerases. In addition to targeting the catalytic activity of TLS polymerases, the recent mapping of key interactions between various proteins of the TLS complex has identified several modules of TLS polymerases that mediate important PPIs as potential targets for TLS inhibitor development. To date, the PIP-box binding site on PCNA is the only PPI relevant to the TLS pathway for which small molecule inhibitors have been reported.169−171 A potential caveat in regards to small molecules that disrupt PCNA/PIP-box PPIs is that this particular interaction is essential for multiple DDR mechanisms and processive DNA replication,16 not just TLS; therefore, selectivity concerns may arise for these compounds. By contrast, some PPIs mediated by subunits and domains of Rev1 and polζ (see above) are unique to the TLS pathway and provide a better opportunity to develop small molecules as selective inhibitors of TLS enzymes. Interfaces that contain small molecule hot spots that hold promise as more selective

polη to bypass UV-induced lesions in a cell-based assay, providing evidence that inhibition of USP1/UAF1 may prevent completing TLS. A group of researchers at Harvard Medical School have also identified and characterized small molecule inhibitors of USP1/ UAF1 following a screen of approximately 150K small molecules.175 The initial hit identified through this screen, 28 (C527), demonstrated submicromolar activity against USP1/ UAF1 (IC50 = 0.88 μM) and comparable activity against several other DUBs (Figure 9). Efforts to generate improved inhibitors ultimately resulted in a slightly modified analogue 29 (SJB2043), which demonstrated enhanced activity against USP1/ UAF1 (IC50 = 0.54 μM). To date, additional studies detailing the ability of these compounds to directly inhibit TLS has not been reported.175

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DISCUSSION AND FUTURE DIRECTIONS The recent identification of TLS as a driving force responsible for the development of acquired resistance to first-line genotoxic chemotherapy has increased interest in the development of small molecule TLS inhibitors as potential anticancer agents. Several potential drug targets are present within the multiprotein TLS complex, including catalytic sites of TLS DNA polymerases, protein−protein interactions between key scaffolding components of the TLS complex, as well as enzymes that regulate the ubiquitination state of PCNA. Drug discovery efforts aimed at each of these potential targets are still in the early stages; however, preliminary results suggest that small molecule TLS inhibitors hold promise as adjuvant agents for genotoxic first line chemotherapy. The early stage development of small molecule anticancer chemotherapeutics that inhibit the catalytic activity of human DNA polymerases has primarily focused on compounds that selectively target either polβ or polλ, two X-family polymerases that are important for several DDR mechanisms.152,176−179 The interest in these two particular polymerases results from studies 9330



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ACKNOWLEDGMENTS We thank Dr. Irina Bezsonova for discussions and help with preparation of the manuscript graphics. We thank Dr. Graham Walker and his laboratory members for helpful discussions. This work was supported by UCHC startup funds to D.M.K. and a Connecticut Institute of Clinical and Translational Science (CICATS) pilot to D.M.K. and M.K.H. Research in the Hadden lab is supported by the NIH/NCI (CA190617), the American Cancer Society (RSG-13-131-01), the NSF (1515808), and the University of Connecticut Research Foundation.

small molecule binding sites include PPIs mediated by Rev1− CT and Rev1−BRCT domains (Rev1−CT/RIR motif, Rev1− CT/Rev7, Rev1−BCRT/PCNA) and potentially a number of other PPIs involving interaction modules of four-subunit polζ (Figure 3). Several important considerations for the future development of TLS inhibitors as anticancer agents should be taken into account. First, it will be important to determine whether small molecules that inhibit TLS demonstrate greater selectivity for cancer cells. In cancer cells that harbor genetic mutations, TLS activity may be an important part of maintaining cellular proliferation and genomic stability; however, detailed studies exploring the status of key TLS components across a wide range of human cancers have not been explored. Similarly, it will be essential to determine whether inhibition of TLS in normal cells has detrimental side effects. Advancing small molecule TLS inhibitors into xenograft or allograft models of human cancer will not only provide key preliminary information with respect to their in vivo activity, but these studies will also provide key evidence as to their selective toxicity toward tumor cells and what additional effects are manifest upon TLS inhibition in normal tissues. In conclusion, the results from early studies focused on the development of small molecule TLS inhibitors have proven promising; however, their continued preclinical evaluation is essential to determine the full potential of such compounds as targeted adjuvant anticancer agents.

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ABBREVIATIONS USED DDR, DNA damage response; DDT, DNA damage tolerance; TLS, translesion synthesis; PCNA, proliferating cell nuclear antigen; PrimPol, primase/polymerase; dNTP, deoxynucleotide triphosphate; PPIs, protein−protein interactions; PAD, polymerase associated domain; BRCT, BRCA1 C-terminus; UBM, ubiquitin binding motif; UBZ, ubiquitin binding zinc-finger; Rev7/3, Rev7 in complex with the Rev7BD region of Rev3; TT-CPDs, thymine−thymine cyclobutane pyrimidine dimers; XPV, xeroderma pigmentosum variant; NSCLC, nonsmall cell lung cancers; PDB, Protein Data Bank; SAR, structure−activity relationship; γ-HOPdG, γ-hydroxy-1N(2)-propano-2′deoxyguanosine; USP1, ubiquitin-specific protease 1; UAF1, USP associated factor 1

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*For D.M.K.: phone, 1-860-679-2849; fax, 1-860-679-3408; Email, [email protected]. *For M.K.H.: phone, 1-806-486-8446; fax, 1-860-486-6857; Email, [email protected]. Notes

The authors declare no competing financial interest. Biographies Dmitry M. Korzhnev is an Assistant Professor of Molecular Biology and Biophysics at the University of Connecticut Health Center since 2010. He completed a postdoctoral training in structural biology and biomolecular NMR spectroscopy with Lewis Kay at the University of Toronto (2001−2010) and with Martin Billeter in the Swedish NMR Center at Göteborg University (2000-2001). He received his Ph.D. in Biophysics (1999) and M.S. in Applied Physics and Mathematics (1995) from Moscow Institute of Physics and Technology under the supervision of Alexander Arseniev. M. Kyle Hadden received his Ph.D. in Pharmaceutical Sciences from the Medical University of South Carolina under the supervision of Thomas A. Dix. His dissertation research focused on the preparation and characterization of peptide-based neurotensin analogues as potential antipsychotic agents. He performed postdoctoral studies with Brian S. J. Blagg at the University of Kansas, where he designed, synthesized, and evaluated Hsp90 inhibitors for the treatment of human malignancy. Dr. Hadden joined the Department of Pharmaceutical Sciences in the School of Pharmacy at UConn in 2009, where he is currently an Associate Professor of Medicinal Chemistry. His research focuses on identifying and developing small molecules as either probes or potential therapeutics for a variety of molecular targets implicated in cancer onset and progression. 9331



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