Note for: Potential Strategies to Target Protein-Protein Interactions in the DNA Damage Response and Repair (DDR) Pathways

Note for: Potential Strategies to Target Protein-Protein Interactions in the DNA Damage Response and Repair (DDR) Pathways

doi: 10.1021/acs.jmedchem.7b00358

This paper focus on protein-protein interaction of protein in DNA damage response and repair pathways.

1.       How to select the target PPI site base on its function

2.       Functional rational and biochemical feasibility to identify a PPI inhibitor

26 PPI discuss in this paper – include BER, MMR, NER, NHEJ, HR, TLS and ICL

Why focusing on PPI in DDR

-        Targeting protein in DDR is not easy --> many DDR proteins are unconventional drug targets (example of conventional drug target – cell surface -- G-protein coupled receptor, ion-channel or conventional enzyme – protease/kinase)

-        Drawback for DDR – assay is difficult for screening

-        Some important mlcs in DDR are large – difficult to handle (purify as large scale screening)

-        Instability of DDR enzymes – make it more difficult to develop assay

-        DDR proteins

o   scaffolding protein

o   enzyme

-        Good point of PPI inhibition

o   PPI assays easier than enzymatic assays

o   PPI inhibitors can be used to target broader range of proteins – like non-enzymatic proteins

o   PPI on particular protein is very selective due to proteins are multifunctional

o   PPI can be design at the level of PTM (post-translational modification)

Therapeutic rational for targeting DDR pathway

-        Many molecules in DDR are redundancy

-        One molecule can play role in many DDR pathways

-        Functional redundancy is therapeutically very important – it can eliminate therapeutic effect of inhibitor (bc there is another molecule to back up the mechanism)

Environmental DDR: how to avoid side effect

-        DNA damage are naturally generated in everyday life

o   Base deamination

§  Base mismatch

o   Nucleoside hydrolysis

§  Apurinic/apyrimidinic/abasic (AP)

o   Oxidative damage

§  Guanine oxidation -- oxoG

o   UV damage

§  Cyclopyrimidine dimer

-      Naturally or man-made DNA damage – DDR is for therapeutic DNA damage

-     The question raises whether disrupting DDR should be absolutely avoided (due to therapeutic DNA damage) or allowed at certain level to improve effectiveness and not toxic

Cancer-therapy resistance

DPC – DNA-protein cross-linking

-        Lesion size and difficulty of the repair – might be important factors for selection of the DDR pathways

Cell-cycle-, replication-, and cell proliferation-dependent DDR

-        Cancer divide rapidly and uncontrolled

-        S-phase is important for duplicating the genetic materials

-        We can focus DDR at the S-phase or later to selectively kill cancer cells

-        Targeting at the transcription level can be a good strategic, however, we need to find which DDR-related transcription (etc., TC-NER) is used by cancer, esp. during S-phase – but it may not effective due to there might be another backup mechanism to compensate the transcriptional activity.

Mutagenic DDR

-        Secondary cancer can be generated from DNA damage by chemotherapy

-        Targeting DDR related to mutagenicity is of interested (to prevent secondary cancers)

Polypharmacology approach for DDR targeting

-        Target multiple target to improve the effectiveness

-        The target should not be epistatic

-        must be complement – synthetic lethality? --> mlc in DDR is redundance

-        inhibiting a molecule that acts in multiple DDR pws --> good strategy

How to justify and proceed with PPI inhibitor project

PPI drugable for cellular fn?

-        biochemically and functionally druggable

-        biochemically --> disrupting the physical interation

-        functionally --> show the phenotype

Assay right for PPI inhibition?

-        Direct binding (e/g/, NMR, SPR, ITC, thermal stabilization) does not mean PPI inhibition – I think he means one to one interaction (PPI + targeted protein)

-        The partner protein must be known to validate the PPI – the reliable assay must be validated

-        Important to confirm the interaction inhibition of the full-length PPI in cells (e.g., co-IP FRET, Proximity Ligation Assay (PLA) )

Structure-based strategy reliable?

-        Be aware of induced fit which the 3D structure might miss

Hit compound can be optimized?

-        if modified must consider ADMET property

Target in DDR

BER

-        SSBs is generated by DNA-damaging therapies (alkylating drugs and IR) – BER might be the mechanism for resistance of cancers to therapies

-        BER mechanisms

o   Short patch

§  Cell-cycle-independent

§  Some anticancer drugs (e.g., 5-FU modified base and short-patch is used to remove artificial base)

o   Long patch

§  Upregulated in G1/S-phase

§  The mechanisms shared with the regular DNA replication – favored S-phase

§  This mechanism might be active in the cancer cells – due to the rapidly growth (entering S-phase more often)

-        Things to be considered;

o   which chemotherapeutic drugs are used to treat cancer,

o   which pws the cancer cells use to detoxify the DNA damaging drug

§  Short patch

§  Long patch

-        Dissecting the mechanism

o   11 glycosylases – used in base removal – redundancy

o   Thus, strategy to design PPI inhibitor – should cover the redundancy

o   Targeting the scaffold protein, like XRCC1 (interact with several BER enzymes)

o   Must design which region on XRCC1 should be targeted for inhibition

-        XRCC1-APE1

o   This PPI enhanced the activity of APEI

o   Thus might be good target for inhibition

o   Deletion of the first 35 AA – lost the PPI

§  We can design inhibitor based this sequence

-        XRCC1-Polb

o   Polb(-/-) sensitizes cells to oxaliplatin and temozolomide – meaning polb is important to repair DNA-damage induced by these two drugs

o   Crystal structures of Polb are available thus the PPI site can be determined

-        XRCC1-REV1

o   It is the TLS component – backup BER component

o   Functional assay could be using the reporter reactivation assay – plasmid + MMS --> substrate for BER

MMR

-        Forming an unannealed loop

-        Activation-induced cytidine deaminase (AID) -->using in Ig class switching – MMR substrate

-        Inhibition of MMR – might affect the immune system

-        MMR is required for the toxicity of some anticancer DNA damaging drugs

-        Deficiency of MMR -- therapeutic resistance

-        Observation – MMR contributes to apoptosis -- > defective of MMR -- > enable cancer survival

-        MSH2-MSH6

o   MSH2-MSH6 can trigger apoptosis

o   Mutation in these two protein cause cancer (if I understand correctly – must find the inhibitor which stabilizes the MSH2-MSH6)

-        MLH1-PMS2

o   Same strategy as MSH2-MSH6

NER

-        General concept of NER

o   Excise the damaged portion (25-30 nucleotide) – resolve the distortion

-        

         NER is indispensable for removal of DNA interstrand cross-link and unhooking of an ICL

-        Thus, targeting NER sensitizes cancer cells to drugs that cross-link DNA

-        NER mechanism

o   Global genome NER (GG-NER)

§  Sensing helix distortion

§  Few study regarding this mechanism

§  Active in undifferentiated cells

§  Therefore, perfect for targeting cancer

o   Transcription-coupled NER (TC-NER)

§  RNA-pol III detects damages during transcription

-        Functional assay for NER

o   Using DNA duplex containing NER substrate lesion – analyze the excision by gel electrophoresis or comet

o   Using UV-damaged reporter plasmid are scalable and practical for screening purposes -- > it generates signals either by NER and TLS

o   Using hairpin DNA duplex containing a lesion for NER in the cells -- > recovered DNA analyzed by qPCR; TLS leaves the lesion that prohibits the PCR

-        XPA-RPA

o   There is the information on PPI-defective XPA mutations

o   This could lead to designing the assay for PI inhibitor

-        XPA-ERCC1

o   There is information on PPI-defective XPA mutations

o   This could lead to designing the assay for PI inhibitor

-        XPF-ERCC1

o   Essential for the first incision step of NER

o   Inhibition of this PPI sensitizes cancer cells to DNA-damaging drugs, esp the one generates ICL

-        XPG-TFIIH

o   Mutation in either protein reveals the PPI

NHEJ

-        Two subpathways – hard to diiferentiate

o   Canonical NHEJ

§  KU70/80 – DNAPK – MRN

§  Get rid of DNAPK sensitizes cells to DSB-generating agents

§  Mechanistically mutagenic on the DSB ends

§  However, can incorporate nascent RNA as template for recovering the deleted sequences

o   Microhmology-mediated non classical NHEJ

§  MRN is used in both cNHEJ and aNHEJ – but decision is unclear

o   Some evidence showed there might be equilibration between conventional and alternative NHEJ.

o   Blocking either NHEJ pw cause equilibrate adjustment, thus, it should take into consideration

-        MRN

o   Play the central role in both cNHEJ and aNHEJ

-        KU70-KU80

o   Core initiator of cNHEJ

o   Blocking KU70-KU80 might activate aNHEJ – not good

-        XRCC4-LIG4

o   Key platform for the final ligation step of cNHEJ

-        XRCC4-XLF

o   Mutagenesis characterization of XLF in XRCC4 reveals this PPI is required for bridging the DSB ends

-        53BP1

o   NHEJ promoter/HR suppressor

o   There are several domains which can be dissected for the interaction partners as well as functions

o   Thus, we can design PPI inhibitor regarding above mentioned

HR

-        Using sister-chromatid as a template

-        Available at S-phase

-        Thus, it is of interested due to cancer cells pass through the S-phase more often than the normal cells

-        Depleted HR – cause aNHEJ is activated which raising concern

-        Gamma-H2Ax foci formation can be formed due to DSB, SSB, replication stress, or in absence of DNA damage

-        Phosphorylated-ATM and 53BP1 are more-specific DSB markers

-        CTIP-NBS1

o   CtIP PPI has been characterized as an event initiating the HR pathway

o   DNA end-resection exclusively in the S-G2 phase

-        EXO1

o   5’-end exonuclease

o   Functional redundancy with DNA2 nuclease – therefore, not a good target

-        RAD51-BRCA2

o   Detailed mechanism of the interplay between BRCA2, RAD51 and ssDNA is not completely understood

-        BLM-TopoIIIalpha

o   Crystal structure of BLM is not available

 

TLS

-        Characterized by two step process

o   Nucleotides are inserted over the damaged template strand

o   DNA is extended from the inserted nucleotides

-        Occur during DNA replication, thus, this mechanism is active in S-phase

-        RAD18-RAD6

o   RAD18 is E3 ubiquitin ligase which ads Ub to PCNA at K164 9monoubiquitin) – upstream initiator of TLS

-        REV1-monoubiquitinated proteins

o   PPIs of REV1 UBM to a monoubiquitinated protein are essential for TLS – can be a therapeutic target

-        REV1-RIR

o   REV1 C-terminus can interact with Poln and Polk – meaning it might regulate the switching between these two Pols

o   Many proteins contain RIR-motif including Pols

-        REV3-REV7

o   REV3L is huge protein, hard to purify, thus, cannot perform biochemical test

o   But crystal structure of REV7 is available

ICL

-        Complex and time-consuming

-        REV1 and PolZeta have been identified as essential for ICL repair

-        FA pathway coordinates several steps in ICL

-        Several components of the FA pathway are shared with other DDR pathways

-        FA is active after S-phase

-        Cellular toxicity to an ICL drug (e.g., cisplatin) is often used as a surrogate functional assay for ICL repair inhibition – but not reliable due to no drugs are 100% specific to for ICLs lesion + resistance to a drug is often dependent of DDR (might be other mechanisms involve)

-        Comet assay measures DNA damage based on the DNA integrity, it measures unhooking but not removal of an ICL.

-        Direct measurement of ICL species in nuclear DNA is possible by LC-MS/MS

-        FANCD2-FANCI

o   Complex is upstream initiator of FA pathway and they are triggered by their monoubiquitinations

-        MUS81-EME1

o   MUS81 is an endonuclease, forms a complex with noncatalytic EME1 protein, and can cleave a DNA fork in vitro.

-        SLX4-XPF

o   SLX4 is a multidomain scaffold protein, organizes many important proteins for ICL repair.

-        SLX4-SLX1

o   PPI for the endonucleolytic process of ICL repair, esp, for HJ resolution

DPC (DNA-protein crosslink repair)

-        Cisplatin can generate this type of lesion (DNA-transcription factor, or DNA-histone)

-        Thus, DPC is part of the effect from cisplatin treatment

Future perspective

-        Challenge in targeting the DDR is cancer specificity

-        DDR pws are the fundamental basis for maintenance of normal cells and not unique to cancer (less specificity)

-        Epigenetics controlling DDR and PTM controlling DDR – might offer a good target

-        Mapping PTM upregulation in cancers and analyzing their role in DDR pws – also their effect on chemotherapeutic response, potential PPI targets for cancer-selective chemotherapeutic enhancement

-        Conventional enzyme – inhibit the activity

-        Nonconventional enzyme – inhibit PPI (some enzymes play role as scaffolding protein)