Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication initiation and the primosome are still largely underexplored targets.
Protein Target | Inhibitor | Species * | Ref. |
---|---|---|---|
DnaA | Bisindole derivatives | Eco | [41,112] |
DnaB | Myricetin | Eco | [113] |
Galangin | Kpn | [114] | |
Coumarin-based | Ban Sau |
[115] | |
Triaminotriazine derivatives | Eco Sau Pau |
[116] | |
DnaG | Para-phenyl substituted tetrazoles | Eco | [117] |
Benzo[d]imidazo[2,1-b]imidazoles Benzo[d]pyrimido[5,4-b]furans Pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidines |
Eco | [118] | |
Tilorone Doxorubicin Suramin |
Ban | [119] | |
Doxorubicin Suramin Ellagic acid |
Mtb | [39] | |
Anthracyclines and aloe-emodin | Mtb | [120] | |
Daunorubicin derivatives | Mtb | [121] | |
3-[1-benzyl-5-chloro-2-(ethoxycarbonyl)-4-(trifluoromethyl)-1H-indol-3-yl]propanoic acid 3-[1-benzyl-7-chloro-2-(ethoxycarbonyl)-5-(trifluoromethyl)-1H-indol-3-yl]propanoic acid |
Mtb | [122,123] | |
9-fluorenone-based derivatives | Sau Ban Bth |
[124] | |
Dequalinium analogues | Sau | [125] | |
Phenolic monosaccharides derived from Polygonum cuspidatum | Eco | [126] | |
Bicyclic 10-membered macrolide (Sch 642305) from Penicillium verrucosum |
Eco | [127,128,129] |
Target | Species * | Type | Advantages | Disadvantages | Ref. |
---|---|---|---|---|---|
DnaA | Eco | Cell-based (dnaA219rnhA reporter strain) | Compounds can cross bacterial membrane | Cannot distinguish compounds with specificity for DnaA | [133] |
Eco | Minichromosome-based (GFP reporter) | Compounds can cross bacterial membrane | Inhibition of plasmid-based oriC not always repeatable with chromosomal oriC | [134] | |
Eco | Cell-based (pBR322-DARS2 and hda mutant reporter strains) | Compounds can cross bacterial membrane | Cannot distinguish compounds with specificity for DnaA | [135] | |
Eco | Filter binding assay ([α-32P]ATP) | Could be converted for HTS | Requires radioactive labelling and scintillation counter | [41] | |
Spy | In silico (molecular dynamics simulation) | Inexpensive, can be used for pre-screening | Requires additional in vivo efficacy conformation | [136] | |
Eco | Cell-based (SF53 reporter strain) | HTS format (384-well plates) | Cannot distinguish compounds with specificity for DnaA | [137] | |
DnaB | Kpn | ATPase assay (molybdophosphoric acid complex) | Could be converted for HTS | Low throughput | [40] |
Kpn | Helicase activity assay (FRET 1) | Could be converted for HTS | Low throughput | [40] | |
Sau | ATPase assay (molybdophosphoric acid complex) | HTS format (96-well plates) | Requires helicase activity assay for confirmation | [115] | |
Eco Sau Ban Pau |
Helicase activity assay (FRET 1) | HTS format (96-well plates) | Cannot distinguish compounds with specificity for DnaB | [115,116,138,139,140] | |
Kpn | dNTP dissociation (fluorescence) | Could be converted for HTS | Requires helicase activity assay for confirmation | [114] | |
viral and bacterial | Time-resolved FRET 1 (Tb3+, Eu3+) | HTS format (Up to 1536-well plates) | Optical interference from compounds | [141] | |
Eco Bst |
ATPase assay (NADH) | Could be converted for higher throughput | Low throughput | [113,132] | |
Bst | Helicase activity (radioactive label not specified) | Can be used for inhibitors of DnaB/G interaction | Low throughput, requires radioactive labelling | [132] | |
UvrD | Eco | Helicase activity (SYTOX stain) | Microfluidic flowcell format, could be used for DnaB | Low throughput | [142] |
DnaB/ DnaG |
Bst | Reverse yeast three-hybrid (β-galactosidase) | Allows screening of potential antimicrobial peptides | Low throughput, for screening of peptides only | [143] |
Eco | SPA 2 ([3H]CTP) | HTS format (96-well plates) | Requires radioactive labelling and scintillation counter | [118,138] | |
DnaG | Eco | Thermally denaturing HPLC (260 nm) | Can distinguish between de novo synthesis and elongation | Low throughput | [113,144] |
Mtb Ban Sau |
Pyrophosphatase assay (molybdophosphoric acid complex) | HTS format (384-well plates) | Cannot distinguish compounds with specificity for DnaG | [39,119,120,125] | |
Eco Mtb |
Primase activity ([3H]NTP, [α-32P]ATP) | Can be used for inhibitors of DnaB/G interaction | Low throughput, requires radioactive labelling, cannot distinguish compounds with specificity for DnaG | [123,126,128] | |
Eco | SPR 3 competition assay | Can be used for inhibitors of DnaG/SSB interaction | Cannot distinguish compounds with specificity for DnaG | [117] | |
Eco T7 |
STD 4 and 2D NMR (Imax, 15N-1H HSQC 5) | Could be used for inhibitors of other proteins | Requires pooling of compounds for initial screens | [117,122] | |
Eco | Primase/Replicase activity assay (PicoGreen) | Can be used for inhibitors of DnaG/SSB interaction, HTS format (384-well plates) | Cannot distinguish compounds with specificity for DnaG | [145,146] | |
All | Any species | Molecular docking (AutoDock Vina, Glide, Molecular Operating Environment) |
Can give indication of mechanism of action | Requires additional in vivo efficacy conformation | [117,118,121,122,136] |
Any species | SPR 3 | Sensitive, can determine binding kinetics | Expensive, need to control for buffer effects | [147,148] | |
Any species | Mass spectrometry (AS-MS 6, LC-ESI-MS 7) |
Sensitive, can pool compounds to increase throughput | Requires multiple rounds for inhibitor ranking | [147,149] | |
Any species | Thermofluor (SYPRO Orange & ANS stain) | HTS format (384-well plates) |
Optical interference from compounds | [147,150] | |
Any species | DSF-GTP 8 (GFP) | HTS format (96-well plates), can be used in mixed samples, can test target access |
Optical interference from compounds | [151,152] |