*5.2.2.4. Forward is back*

Compared with the fruitless target-based genomic approach, traditional whole-cell assays are more effective in antibiotic discovery. Just because it is not necessary to worry about cell permeability of a novel scaffold in the development process if whole-cell assays are used. As most of the existing libraries have already been used to screen for antibacterial drugs, libra‐ ries with new chemical diversity are extremely important in this approach. Sometimes, look for libraries that don't belong to antibacterial development areas may be useful. In fact, most pharmaceutical companies of other therapeutic areas have invested considerable resources in synthesizing small molecule libraries [59]. Candidates with a strong hit in a whole-cell an‐ tibacterial assay should be tested in the right animal model early in development, because In vitro experiment results are not always reliable. For example, Antimicrobial drug target type II fatty acid synthesis (FASII) is reported to be essential for their efficacy against infections caused by multiresistant Gram-positive bacteria. But another study showed that Streptococ‐ cus agalactiae and S. aureus could take up sufficient unsaturated fatty acids from human se‐ rum to obviate the essentiality of FAS II enzymes in vivo [72].

#### *5.2.2.5. Focus on spectrum*

Antibacterial spectrum is a major consideration when selecting a target for lead optimiza‐ tion. Permeability and target distribution determine the pectrum [73]. That is to say, the drug candidates should possess two properties at the same time: one is penetrating the cell and evading efflux pump systems, another is retaining potent activity at the molecular tar‐ gets. However, since almost all targets of the antibacterials in clinical use are present in all bacteria, the antibacterial drug spectra are determined largely by the ability of permeability. Therefore, some compounds are just Gram-positive organism-selective and have no effect against Gram-negative pathogens which have a second membrane acting as a permeability barrier [74; 75]. Efflux pump inhibitors (EPIs) have been explored for broadening the anti‐ bacterial spectrum and overcoming bacterial resistance. Although no clinically useful drugs have come out, extensive efforts have been made to test the effectiveness of EPIs across a range of in vitro and in vivo assays, especially the compound MC-207,110 [76].

'Broader is better' is the rule of antibacterial activity spectrum. But developing the agents with a narrower spectrum may be helpful in treating some special antibiotic resistant patho‐ gens or the non-multiplying bacteria. One human squalene synthase inhibitor blocked staphyloxanthin biosynthesis in vitro, resulting in colorless bacteria which became more sensitive to killing by human blood and innate immune clearance [77]. Rifampicin is a standard antibiotic used for clearance of non-multiplying tuberculosis. Monoclonal antibod‐ ies (Mabs) have also become potential agents for narrow-spectrum antibacterial therapy. In clinical experiment C. difficile Mab combination MDX-066 and MDX-1388, which targets and neutralizes two main C. difficile toxins, can reduce the recurrence of C. difficile infec‐ tion [78; 79]. A microbiologic diagnosis should be made before using these kinds of antibiot‐ ics for therapy. Such genus-selective agents may have the benefit of leaving more of the endogenous microfloraun unattacked compared with conventional antibiotics.
