*3.1. Human Immunodeficiency Virus (HIV)*

HIV is a lentivirus of the Retroviridae family. HIV targets immune cells, and reverse transcribes its single-stranded RNA (ssRNA) genome, integrating into the host chromosomal DNA. The virus uses high antigenic diversity and multiple mechanisms to avert recognition by the human immune system thus posing a challenge to host defences and treatment [28].

Various assays have been developed and used to identify molecules with anti-HIV activity. Some of the assays includes structure-based design of a small molecule CD4-antagonist with broad

spectrum anti-HIV-1 activity [29], structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1 [30], and identification of HIV inhibitors by high-throughput (HTP) two-step infectivity assay [31]. The HTP assay has been used on myxobacterial-derived molecules with success due to the ability to screen a large number of molecules in a short period.

Sulfangolids, the first sulfate esters containing a series of secondary metabolites produced by several strains of *Sorangium cellulosum*, were isolated together with the structurally related macrolide kulkenon (**5**) [32]. Sulfangolid C (**1**), soraphen F (**2**), epothilon D (**3**), and spirangien B (**4**), showed impressive activity, with EC50 values in the nM range with a selectivity index value greater than 15 (SI > 15) in the high-throughput two-step infectivity assay [31]. Despite the impressive antiviral activity of **5**, the SI is low because of toxicity. A search in the SciFinder database revealed dozens of analogues of soraphen and epothilone [33]. It may be promising to screen the large number of analogues in the soraphen and epothilone families to attempt to encounter more potent representatives with better SI values. The soraphens have been reported as acetyl-CoA carboxylate transferase inhibitors [34], while the epothilones stabilise microtubuli of macrophages in a similar manner as Taxol® without showing taxol-like endotoxin activity [35,36]. In fact, the FDA-approved anticancer drug, Ixabepilone®, is an epothilone B derivative [37]. Metabolites **3** and **4** are reported to decelerate the phosphorylation and degradation of inhibitor of kappa Bα (IkBα) [36,38]. The compounds identified as preliminary hits for anti-HIV included **1**–**5** (Table 1, Figure 2) [31]. Rhizopodin (**6**), from *Myxococcus stipitatus* was identified as interesting in the two step HTP assay, likely because of its mode of action [31]. HIV cell-to-cell transmission is the primary route of HIV infection in naive cells in vivo. Actin filaments are known to be essential for virological synapse formation, therefore, virus synapses are interfered by **6**, which is a known actin inhibitor. Disorazol, tubulysin and stigmatellin variants were also reported to have mild anti-HIV activity [31]. Thiangazole (**7**), phenalamide A1 (**8**), and phenoxan (**9**) isolated from two strains of *Polyangium* sp. and *Myxococcus stipitatus* strain Mx s40 were reported to have anti-HIV activity (Figure 2) [39]. They all revealed high activity by suppressing HIV-1-mediated cell death in the MT-4 cell assay with EC50 values of **9** and **8** in the nanomolar range, whereas thiangazole (**7**) had an impressive EC50 value in the picomolar range, making it a possible lead compound for anti-HIV therapy (Table 2, Figure 2) [39]. In another assay involving measuring ATP levels as a parameter of cell viability of TZM-bl cells aetheramide A (**10a**) and aetheramide B (**10b**) isolated from the recently described genus *Aetherobacter*, inhibited HIV-1 infection with IC50 value of 0.015 and 0.018 μM, respectively [40–42]. Concurrently, the aetheramides were reported to be moderately antifungal and cytotoxic [41]. The chemical structures of **10a** and **10b** are rare, containing a polyketide moiety and two amino acid residues, thus forming a new class of antivirals [40–42]. This discovery of new antivirals from the recently described myxobacteria genus, *Aetherobacter*, represents an example of the enormous biosynthetic capabilities of myxobacteria and their importance to drug discovery efforts [42]. Ratjadon A (**11**), an α-pyrone metabolite isolated from *Sorangium cellulosum* (strain Soce 360), was reported to inhibit HIV infection by blocking the Rev/CRM1-mediated nuclear export pathway [43,44]. The CRM1-Rev complex is an attractive target for the development of new antiviral drugs because the nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1 [44]. Despite **11** being reported to exhibit a strong anti-HIV activity, it has a low selectivity due to toxic effect. The low SI value limits the potential use of **11** as a therapeutic drug. More studies with derivatives of **11** need to be done. It is important to observe that different assays were used to screen for anti-HIV compounds. There is a need therefore for a standardised method to be able to adequately compare the anti-HIV activity of those compounds that have been identified as preliminary hits. Equally important is an evaluation of the mechanism of action on viruses in comparison to the mechanism of action on bacteria or fungi. Investigation for synergism between the identified anti-HIV compounds for possible use at a lower concentration to improve the selectivity index of the metabolites should be looked into in the future. Even the active metabolites that cannot realistically be further

developed as drug candidates because they are too toxic, could be used as biochemical tools to attain a better understanding of the invasion mechanism of HIV, or for development of synthetic analogues that mimic these compounds without causing toxicity.

**Figure 2.** Myxobacterial-derived compounds with activity against human immunodeficiency virus (HIV).


**Table 1.** Preliminary anti-HIV hits from a high-throughput two-step infectivity assay [31].

\* Control, <sup>1</sup> Molecular weight, EC50: effective concentration; CC50: cytotoxic concentration; \*\* Selectivity Index = CC50/EC50. EC50 is the concentration of a drug or metabolite which induces a response halfway between the baseline and maximum after a specified exposure time or gives the desired effect to 50% of test subjects. While SI is a comparison of the amount of a drug or metabolite that causes the desired effect to the amount that causes death or toxicity. Metabolites with a low EC50 and a high SI values are good drug candidates.

**Table 2.** Anti-HIV-1 activities of compounds derived from myxobacteria by MT-4 cell assay [40,41].


\* azidothymidine Control, <sup>1</sup> Molecular weight, <sup>2</sup> Toxicity (Tx) is the lowest toxic concentration (nM) of the compound in the MT-4 cell assay, <sup>3</sup> Antiviral efficacy (AE) is given as the lowest effective concentration (μM) of the compound at which 100% prevention of the virus-mediated cytopathogenicity was observed in the MT-4 cell assay, \*\* Selectivity Index = Tx (nM)/AE (μM).
