Table 1.

Summary of anti-virulence targets in N. gonorrhoeae.

Anti-Virulence Target	Function	Target Validation			Available Inhibitors	Inhibitor Studies	References
		In Vitro Testing	Structural Studies	In Vivo Models
Bacterial cell wall maintenance and modification
EptA	Catalyzes the addition of pEtN onto lipid A of the OM.	Loss of EptA increases susceptibility to killing by PMNs, macrophages, CAMPs and human serum.	Full structure solved of N. meningitidis homologue (98% identity, PDB accession code 5FGN).	Reduced survival rates of eptA mutant in mouse and human models.	N/A	N/A	[83,108,109,110,111,112]
Lst	Catalyzes the addition of N-acetyl-neuraminic acid onto lacto-N-neotetraose of LOS. Primary mechanism for resistance to human complement.	Loss of Lst increases susceptibility to killing by PMNs and human serum.	Full structure of N. meningitidis homologue apo form (92% identical, PDB accession code 2YK4 1) and with structural donor sugar analogs or products solved (PDB accession code 2YK5, 2YK6, and 2YK7 1).	Reduced survival rates of lst mutant in mouse models.	FHD1119G and Leg5,7Ac2	Increased serum sensitivity. Significantly reduced duration and burden of infection in mouse vaginal colonization model.	[113,114,115,116,117,118,119]
NgACP and SliC	Essential for survival against lysozyme.	Loss of NgACP and SliC increased susceptibility to human lysozyme. NgACP loss significantly reduced survival in PMNs.	Mature NgACP structure has been solved (PDB accession code 6GQ4). Structure of SliC homologue in Pseudomonas aeruginosa (MliC) solved (23.3% identity, PDB accession code 3F6Z 1).	Reduced survival rates of sliC mutant in mouse models.	N/A	N/A	[120,121,122,123]
PatB	Catalyzes O-acetylation of N-acetyl-muramic acid.	Increased sensitivity to lysozyme in human sera or lysozyme purified from human PMNs.	Structure of PatB homologue in Staphylococcus aureus (OatA C-terminal catalytic domain) has been solved (15% identical, PDB accession code 6VJP 1).	N/A	Compound 89224	Treatment reduced bacterial growth by 90%. Inhibitor binding studied using microtiter plate-based fluorometric assay.	[124,125,126,127,128,129,130,131]
LtgA	Catalyzes cleavage of N-acetyl-muramic acid-β-1,4-N-acetylglucosamine to form PG monomer fragments during cell growth.	Reduction in PG monomer release. Loss of LtgA in N. meningitidis has a detrimental effect on bacterial cell growth, division, and separation.	Structure of N. meningitidis homologue (97% identical, PDB accession code 6FPN 1).	NmLtgA mutant cleared quicker than wild-type and reduced cytokine induction in mouse model.	Bulgecin A	Inhibited LgtA activity and had a synergistic effect with β-lactams.	[75,132,133,134,135,136,137]
Anaerobic survival
AniA	Reduces nitrite to nitric oxide. Essential for anaerobic growth.	Loss of AniA reduces anaerobic growth and biofilm formation.	Soluble domain structure solved (PDB accession code 1KBW, 1KBV, 5TB7, and 5UE6).	Immunization with a truncated form of AniA generates protective antisera in a mouse model.	C7-3	Significantly inhibited enzyme activity and gonococcal growth under anaerobic conditions. Inhibitor binding studied using molecular docking and biolayer interferometry. A patent has been approved for C7-3 and its derivatives.	[138,139,140,141,142]
Efflux pump
MtrCDE	Selective efflux of antimicrobials resulting in increased resistance to penicillins, macrolides and extended spectrum cephalosporins.	Loss of the MtrCDE pump results in increased susceptibility to penicillin, ceftriaxone, azithromycin, tetracycline, and solithromycin in the WHO clinical panel of multidrug-resistant (MDR) strains.	Full structures of MtrD and MtrE solved (PDB accession code 4MT1 (MtrD), 4MT0 (MtrE), 6VKS (MtrD from strain CR103 in complex with ampicillin) and 6VKT (MtrD from strain CR103 in complex with erythromycin). Full structure of MtrC homologue (MexA) in P. aeruginosa solved (43% identity, PDB accession code 1VF7 1).	Loss of the MtrCDE pump reduced gonococcal survival and increased penicillin susceptibility to therapeutic levels in mouse models.	Phenylalanine arginine β-naphthylamide (PaβN)	Untested in N. gonorrhoeae, derivatives have been halted due to high host cell toxicity.	[46,99,143,144,145,146,147,148]
Protein folding pathways
Mip	Catalyzes the cis–trans isomerization of peptide bonds directly preceding a proline residue.	Loss of Mip decreased gonococcal survival within macrophages and PMNs.	Full structure of Legionella pneumophila homologue solved (44.8% identical, PDB accession code 1FD9 1).	N/A	PipN3 and PipN4	Compounds inhibited enzyme activity and reduce gonococcal survival in neutrophils.	[149,150,151]
DsbA/DsbB	Catalyzes formation of disulfide bonds in OM proteins involved in virulence.	Loss of DsbA1 in N. meningitidis affects tfp function, reducing colonization and competence.	Full structures of N. meningitidis homologues DsbA1 (97% identical, PDB accession code 3DVW and 3A3T 1) and DsbA3 (93% identical, PDB accession code 3DVX and 2ZNM1) solved. Full structure of E. coli DsbA/B complex homologue solved (28.7% identical, PDB accession code 3E9J 1).	N/A	Phenylthiazole, benzofuran, phenylthiophene or phenoxyphenyl derivatives	Untested in N. gonorrhoeae.	[152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169]
Adhesion and invasion
Type IV pili	Essential for adhesion/colonization, horizontal gene transfer, twitching motility.	Tfp mutants lacking PilE are unable to adhere to human epithelial cells, are non-motile and are incompetent.	Tfp structure has been solved (PDB accession code 5VXX, 1AY2, 2HIL and 2HI2).	In the human male model of infection, men inoculated with a gonococcal pilE mutant developed watery urethral discharge or were asymptomatic.	Compound B	Prevents pilus elongation.	[170,171,172,173,174,175,176,177,178,179,180,181,182,183]
					Phenothiazines	Inhibited Na+-pumping NADH:quinone oxidoreductase. Tests with N. meningitidis reduced bacteremia and increased survival in a mouse model.
					Compound G2, carbamazepine and methyldopa	Inhibits tfp binding to host receptor CR3 on primary cell line. Carbamazepine and methyldopa are re-purposed drug (FDA-approved anti-convulsant and high blood pressure medication, respectively).
Mannose-binding (Opa) proteins	Required for adherence to host epithelial cells.	Opa-less bacteria do not adhere to Chinese hamster ovary cells.	Structure of Opa60 has been solved (76% identity, PDB accession code 2MAF 1).	Gonococci recovered from human models are always Opa positive, even if the inoculum was Opa negative.	ConA and α-methyl D-mannoside (Mannosides)	Compounds reduced gonococcal adherence to primary cervical epithelial cells and urethral epithelial cells.	[184,185,186,187,188,189,190]

¹ PDB identification and percentage identity obtained using the NCBI BLASTp query of the PDB database [191,192,193].