**4.8. Anti-retroviral peptides**

**Source Source organism Compound Mechanism of anti-retroviral action Reference**

Grapes and berries Resveratrol Inhibits tat-induced HIV transactivation.

Unidentified compounds

220 Trends in Basic and Therapeutic Options in HIV Infection - Towards a Functional Cure

compounds

A

didehydro-cortistatin

Arthropods Bee venom Melittin Binds to various HIV surface

Tanshinone II A Inhibits host cellular machinery

retrovirals

and inhibits entry

Griffithsin / Grifonin-1 Binds to various HIV surface

Fascaplysin Inhibits host cellular machinery

cells

Cyanovirin-N Binds to various HIV surface

Ratjadone-A Inhibits host proteins involved in

envelope

entry

Scorpion venom Kn2-7 Interacts with HIV envelope and inhibits

and inhibits entry

Leptomycin-B Inhibits the viral protein rev which helps

Scytovirin [92]

nuclear export of HIV mRNA

in nuclear export of HIV mRNA

components and disintegrates the

Cnidarian proteins Various anti-HIV activities including

responsible for HIV transcription

Inhibits HIV entry [82]

Also possess cystostatic effects on CD4 T-cells similar to hydroxyl urea

Neutralizes the CYP3A4 in the GI tract and increases the bioavailabity of anti-

components including gp41 and gp120

Most potent inhibitor of tat-dependent HIV transcription identified till date

responsible for HIV transcription. Also inhibits HIV reverse transcriptase

inhibition of HIV protease, inhibition of HIV entry and cytotoxicity of infected

components including gp41 and gp120

[81]

[83]

[84]

[85, 86]

[47, 87]

[89, 90]

[91]

[93]

[94]

[95]

[96]

[88]

*Salvia miltiorrhiza* (Chinese medicinal

*Pelargonium sidoides* (German medicinal

*Griffithsia* species (Red

*Fascaplysinopsis reticulata*

Cassiopia *Andromeda*

*Galaxura filamentosa* (Red algae) *Litophyton arboreum* (Soft coral) Bacteria *Nostoc ellipsosporum*

(Cyanobacterium)

*Scytonema varium* (Cyanobacterium)

*Sorangium cellulosum* (Myxobacterium)

*Streptomyces* species (Actinomycete)

**Table 4.** Anti-retroviral compounds isolated from natural sources

*Corticium simplex* (Sponge)

Grapefruit juice Unidentified

plant)

plant)

algae)

(Sponge)

(Jelly fish)

Marine species Eukaryotic organisms are known to secrete antimicrobial peptides as a part of their innate immunity, which have a broad spectrum of activity against various pathogenic microbes [97]. These peptides are usually short sequences of 12 - 20 amino acids, but some of them may also be around 40 amino acids long. Apart from the inherent anti-retroviral peptides produced by the human body, numerous studies have identified several peptides from natural and synthetic sources that are capable of inhibiting HIV replication [98]. With the FDA approval of the fusion inhibitor peptide enfuvirtide, coupled with the growing problem of resistance to anti-retroviral drugs, much interest is being shown in the development of novel anti-retroviral peptides. Currently, there are nearly a thousand HIV inhibiting peptides identified and new members are being added on a daily basis [99].

The anti-retroviral peptides identified till date are predominantly from natural sources ranging from bacteria to plants to primates. Also a significant number of these peptides are derivatives of HIV itself, which structurally mimic the viral substrates and competitively inhibit the various replicatory processes. On the other hand, synthetic production using techniques such as phage display, offer the scope of combination of related or unrelated peptides to achieve maximal anti-retroviral effect. Most of the anti-retroviral peptides inhibit one or more of the phases of HIV replication. Majority of the peptides act extracellularly to inhibit HIV attachment or its fusion while others inhibit the intracellular phases of replication such as reverse tran‐ scription, transcription and integration. Apart from the direct administration of peptides for therapy, these agents could also be administered in their complementary DNA form which on recombination with the host cell genome and subsequent expression, makes the target cell resilient to HIV infection (subsequently discussed). Certain peptides possess the unique property of cell penetration and hence being evaluated for their possible role in targeted nanotechnological and cellular delivery techniques [99, 100].

The advantages of therapeutic peptides include their high specificity for the site of action, rapid break down into harmless amino acids which are eliminated easily and hence possess less toxicity and adverse effect profile [98]. However, this rapid breakdown of peptides by the peptidases could also be a drawback since they could be cleared off before they exert their action. Currently, synthetic peptides incorporated with D-isomers of amino acids (instead of the naturally occurring L-isomers) or with added non-peptide moieties, overcome this drawback and possess extended half life [101]. Peptides are highly antigenic and elicit the production of antibodies. Hence the extracellular acting peptides face the problem of antibody mediated clearance. Although the intracellular acting peptides are devoid of the antibody problem, availability of efficient delivery systems into the cell and degradation by the intracellular peptidases are the challenges they face. Poor oral bioavailability, inability to cross biological barriers and high production cost are further problems to be addressed prior to approval for therapeutic use [100, 102]. The FDA however, has approved enfuvirtide, this approval has offered hope for the success of other anti-retroviral peptides currently undergo‐ ing clinical trials [103]. With advancements in nanotechnology and cellular delivery systems, peptide therapy might become a reality for HIV infection in due course of time.

#### **4.9. RNA based therapeutics**

RNA based therapeutic strategies exert their action between the phases of transcription and translation. Numerous RNA based techniques have been developed which are classified by their mechanism of action. They include; inhibitors of messenger RNA (mRNA) translation (antisense oligonucleotides), the agents of RNA interference (RNAi), catalytically active RNA molecules (ribozymes) and RNAs that bind proteins and other molecular ligands (aptamers) [104, 105] (Table-5). These techniques can be utilized for the treatment of any viral disease by engineering specific, complementary inhibitory RNA particles to the viral transcription components. Among these techniques, RNAi and to a lesser extent antisense oligonucleotides, have been tried out to inhibit retroviral replication. As these techniques can also target the host cellular processes, they are being exploited in strategies to increase the resilience of target cells to HIV infection, which is discussed later [106].

RNAi is an endogenous mechanism which involves the down regulation of mRNA activity during transcription and post transcription phases using short double stranded RNA (dsRNA) called micro RNA (miRNA), which are about 20-30bp long. The identification of this regulatory process has provoked the interest of controlling unwanted viral replication using exogenously administered specific sequences of short dsRNA. The exogenously administered agents of RNAi therapy include small interfering RNA (siRNA) and short hairpin RNA (shRNA). These agents act on the post transcript mRNA and either cause direct sequence specific cleavage when there is a perfect sequence complementarity match, or lead to translational repression and degradation of mRNA when the interfering RNA sequence is of limited complimentarity to the targeted mRNA. As the siRNA get cleared off after their action, their effects are only transient and need repeated administration similar to agents of chemotherapy. On the other hand the action of shRNA is similar to gene therapy, as they get expressed on promoters and cause long term effects. Various viral and non-viral delivery mechanisms and active targeting strategies have been developed to deliver these active agents into the target cells [107, 108].

Systems employing siRNA and shRNA to target the gene products of tat, rev, nef, env, vif and pol have been designed and evaluated for efficacy [47, 106]. However, the use of this technol‐ ogy against viral replicatory processess is threatened by the genetic variation exhibited by HIV. Very simple mutations allow HIV to escape from the action of both siRNA and shRNA [109, 110]. Four possible solutions are being tried to tackle the problem of these escape mutants. The first attempt is to expand the RNAi technique to simulataneously inhibit multiple HIV mRNA targets similar to the concept of multidrug use. Recent studies have demonstrated that concurrent inhibition of HIV mRNA with three different shRNAs can prevent viral escape *in vitro* [111, 112]. In the second possible solution, inhibitory RNAs with a complete match to the most commonly encountered viral escape sequences are being designed. When used along with the inhibitory RNA of the wild type virus, these could prevent a majority of the mutants from escape [113]. The third solution involves identifying novel, genetically conserved sequences of HIV which do not usually undergo mutation. Targeting these stable sites would favour the success of RNAi [114]. Finally, RNAi techniques are also being designed to restrict the 'genetically more stable' host factors that help in HIV replication (discussed later under strategies to enhance target cell resilience to HIV infection).

Apart from the disadvantages posed by the virus per se, RNAi technology has numerous setbacks. The short lived action of siRNA warrants its repetitive administration thereby compounding the treatment cost. The overexpression of shRNA can result in cell death and hence needs strict dose titration. RNA is immunogenic and is rapidly neutralized by antibodies in circulation, hence effective vectors are needed for the *in vivo* administration of RNA. Even after successful intracellular delivery, the administered RNA moieties are easy targets for degradation by cytoplasmic ribonucleases. Engineered RNA with modifications in sugar moieties, nucleotides or their backbone are found to possess improved cytoplasmic stability and are being evaluated for their superiority and efficacy [115].

Due to the numerous setbacks, almost all RNAi techniques that exclusively inhibit the viral targets have stagnated at the level of pre-clinical testing and none have entered into clinical trials. A recent technique which involves the simultaneous use of three shRNAs to specifically inhibit three corresponding targets of HIV, has been found to be safe and effective and is hopeful to enter phase I trials in the near future [111].
