Developing gametocytes (Stages I-III) are particularly vulnerable to drugs that affect hemoglobin metabolism [3]. Artemisinins, for example, inhibit both heme polymerization and the hemoglobin catabolic pathway [16]. Certain new targets attack other pathways essential to the transition from asexual to sexual stages.

Later stage gametocytes (Stages IV-V), which are more quiescent metabolically, are more likely to be affected by drugs with cytotoxic properties. Some 8-aminoquinolines (8AQ) including primaquine may directly exert toxicity against parasite mitochondria, inhibiting metabolism through interference with the electron transport functions of ubiquinone in the respiratory chain [17]. Certain 8AQ metabolites may also kill by inducing oxidative stress within cells through the spontaneous generation of superoxides [18,19]. Toxicity of several drugs, including atovaquone, may relate to their activity against the dihydroorotate dehydrogenase complex of mitochondria, which functions in the biosynthesis of pyrimidines [3]. The array of targets available for inducing parasite-specific toxicity continues to expand and will be discussed in detail in later sections.

Diverse mechanisms are involved in interrupting transmission after gametocytogenesis, some operating along the various stages of extrinsic incubation from exflagellation to the formation of sporozoites, or even by damaging the salivary glands of vector mosquitoes [3]. Pyrimethamine has been shown to directly damage ookinetes and along with proguanil, inhibit dihydrofolate reductase [3]. New opportunities for interference with gametogenesis and sporogony have arisen, particularly with regard to proteases and protein kinases. Many of the molecular mechanisms involved in the transition through sporogony remain to be determined.

Drugs that are also highly effective against asexual parasites would be most attractive for incorporation into front-line therapies. Alternatively, a transmission blocker can be incorporated as part of a combination or used as a follow-on drug as part of a course of treatment that would both treat clinical infections and prevent the transmission of parasites to others.

Epoxomicin is naturally derived from certain strains of Actinomycetes bacteria, originally evaluated for its anti-tumor properties [50]. Its active moiety appears to be an α9,β9-epoxyketone. It can also be synthesized from spirodiepoxides [51]. It acts as a proteasome inhibitor, exploiting the expression of genes that encode cysteine proteases and the proteasome throughout gametocytogenesis [52]. The proteasome is a complex of nuclear proteases responsible for degrading and recycling unnecessary or damaged intracellular proteins.

A broad in vitro screening of potential proteasome inhibitors revealed that 100 nM of epoxomicin, was found to reduce P. falciparum gametocytes by 77% within 24 h. Complete kill of gametocytes was achieved after 72 h, even at a dose of 10 nM [52].

This inhibition of proteasome activity appears to have a toxic affect, and does not lead to resumption of development if inhibition is removed. Specifically, epoxomicin interacts with the β5 and β2 subunits of the P. falciparum proteasome. While these active sites are conserved, the amino acids surrounding them are distinct in P. falciparum, suggesting targets for novel inhibitors that preferentially block P. falciparum enzymes. Epoxomicin did not appear to adversely affect the viability of mouse (3T3) or human (A549) cell lines, offering promise for future in vivo investigations.

Epoxomicin also affects the morphology of developing gametocytes, greatly restricting the circumference of parasites exposed to this compound. Mature stage V gametocytes, however, are not inhibited from undergoing exflagellation in vitro.

Another proteasome inhibitor evaluated, N-acetyl-l-leucyl-l-leucyl-l-methioninal (ALLN), was also effective at killing gametocytes, but only at higher concentrations (100nM) [52].

Methylene Blue (MB) is a heterocyclic aromatic compound with a wide array of uses, including as a bacteriologic stain. Medicinally, it is most commonly used as an antidote to reverse methemoglobinemia in cases of cyanide poisoning. MB was one of the first synthetic drugs ever used against malaria [53]. It acts by inhibiting disulfide reductases and heme detoxification [54].

Methylene Blue given in combination with amodiaquine or artesunate at a daily dose 10 mg/kg for three days, exhibited strong gametocytocidal activity against P. falciparum in vivo [55]. Unlike the standard artesunate-amodiaquine (AS-AQ) controls, both MB-AS and MB-AQ achieved complete clearance of all sexual stages. It appears to act against both older gametocytes and those still developing. In terms of PCR-confirmed clinical and parasitological efficacy, MB-AQ outperformed the AS-AQ controls 95% to 82%. As a well-tolerated and highly efficacious gametocytocide, MB shows great potential as a component of prospective combination therapies against malaria.

Dyhydroacridine-dione administered at a dose of 0.39 mg/kg was found to inhibit sporogonic development in An. dirus [42,43]. (WR-250547) It appears to be effective at preventing sporozoites from entering salivary glands even up to eight days after administration. This drug is not currently under development as an antimalarial, despite its promising sporontocidal properties.

Tipranavir, a nonpeptidic HIV protease inhibitor was found to be both gametocytocidal and inhibitory of gametocytogenesis at clinically relevant doses (EC₅₀, 12 to 21 μM) [59]. Several other protease inhibitors evaluated in the same trial were also found to be inhibitory, including: darunavir, saquinavir and lopinavir.

The 9-anilinoacridines were originally developed as anti-cancer agents [60]. Analogs bearing 3,6-diamino substitutions on their acridine rings are particularly active in inhibiting DNA topoisomerase II and the formation of β-hematin in asexual parasites of P. falciparum [61]. However, this 3,6-diamino substitution is less effective against sexual stages. The most effective moiety against asexual stages was 3,000-fold less effective against gametocytes. The most gametocytocidally active of an array of thirteen analogs evaluated in vitro against P. falciparum was a 1′-CH₂NMe₂-9-anilinoacridine (IC₅₀, 0.82-2.1 μM) against the KT1 and KT3 strains, respectively [62].

Riboflavin has been found to be toxic to P. falciparum gametocytes in vitro, by possibly interfering with the digestion of hemoglobin during gametocytogenesis. It appears to be effective against both immature and mature gametocytes. Riboflavin also potentiates activity against asexual parasites synergistically with drugs including mefloquine, pyrimethamine and quinine [63], suggesting that it may warrant consideration as a component of a combination therapy.

Neem is a product derived from Azadirachta indica, a tree native to India. The repellent and insecticidal properties of neem and its derivatives have long been known [64], but also appears to be active against the sexual stages of malaria parasites [65]. The activity of neem, a complex mixture of substances, is attributed to several component limonoids, a class of oxygenated terpenoids, specifically: azadirachtin, gedunin and nibolide [66].

A 50 mg/kg dose of Neemazal®, an azadarachtin-enriched extract of neem seeds, was found to inhibit the development of parasites in An. stephensi mosquitoes allowed to feed on rodents infected with P. berghei [67]. Neemazal® did not exhibit any observable sporontocidal activity.

Neem leaf extracts have also been shown to be active against the sexual stages of P. falciparum in vitro [68], killing more than 90% of both developing and mature gametocytes at a concentration of 2.5 μg/mL [69]. Azadirachtin, in particular, appears to act by preventing exflagellation through inhibition of cytoskeletal activity and interference with microgametocyte endomitosis [70].

While promising, significant technical challenges limit the feasibility of using neem and its components as drugs. Limonoids tend to have minimal bioavailability and suffer from a short metabolic half-life [71]. Gedunin appears to be poorly absorbed in the digestive tract [72]. Once these technical challenges are overcome, questions of safety and interaction with other drugs will remain.

Proteolysis is likely to be important both for the remodeling associated with morphological changes during gametocyte formation as well as gamete formation and the invasion of mosquito midgut walls by ookinetes. Thus, proteases may offer a target of drug intervention via inhibitors. In P. berghei, for example, both exflagellation center formation can be blocked by 1,10 phenanthroline (1mM) as well as the cysteine/serine protease inhibitors TPCK and TLCK at concentrations of 75 and 100mM [77]. These latter inhibitors also seem to affect ookinete formation while 1,10 phenanthroline has no effect. It may also be worth investigating other protease inhibitors that may have similarly specific effects on certain stages of P. falciparum.

Two papain-like cysteine proteases, falcipain-2 [78] and falcipain-3 [79] are present in the food vacuole of trophozoites and young gametocytes and appear to be involved in hemoglobin digestion [80,81]. In late stage gametocytes, only falcipain3 appears to be expressed, and appears to support oocyst development as indicated by the suppressive activity of E64d, a membrane-permeant protease inhibitor [82]. Thus, cysteine proteases appear quite promising as potential targets for the inhibition of gametocytogenesis and sporogony.

Protein kinases are involved in the modification of other proteins via phosphorylation. Mitogen-activated protein kinases (MAPKs) are activated by diverse extracellular factors in a protein kinase cascade and appear to be involved in the regulation of the cell cycle [83].

In P. falciparum, the novel MAPK Pfmap1 phosphorylates threonine-tyrosine (TXY) sequence motifs as in most eukaryotic MAPKs [84] while Pfmap2 is active against threonine-aspartic acid-tyrosine (TDY) motifs [85]. Pbmap2, the P. berghei equivalent of Pfmap2 has been shown to be involved in the regulation of male gametogenesis [86,87], suggesting a similar role for Pfmap2. The divergence of Pfmap2's sequence motif affinity from the typical mammalian pattern offers particular promise as a drug target because of the potential for parasite-selective drug activity which may be associated with low human toxicity.

Similar advantages exist with another group of P. falciparum-specific, NIMA-related protein kinases, Pfnek2 [88], Pfnek 3 [89] and Pfnek2 [90]. Disruption of nek-2 and nek-4 genes in P. berghei models interferes with DNA replication during meiosis and prevents ookinetes from developing [88,90]. Pfnek3 appears to be a specific activator of the MAPK Pfmap2 [89]. Pfnek3 is expressed in late asexual intraerythrocytic stages as well as in gametocytes.

A calcium-dependent protein kinase (CDPK4) appears to have important role in male gametogenesis and in the maturation of fertilized zygotes [91].

Yet another pair of kinases, MAP2 and NEK4, identified through proteome analysis and investigated through targeted gene disruption [92]. The male gametocytic proteome is particularly rewarding in such searches due to the abundance of distinct proteins involved in flagellar motility and genome replication. MAP2 appears to have a sex-specific role in male gamete formation while NEK4, an FG-specific NIMA-related kinase, appears to be involved in zygote development and meiosis [92].

Thus, protein kinases appear to offer an abundant source of potential targets for transmission-blocking drugs.

Peroxiredoxins are antioxidant enzymes that protect macromolecules from reactive peroxides and the oxidative stress they impose on sensitive malaria parasites. Certain peroxiredoxins may also be involved in the activation pathways of certain MAP kinases [93].

Gene disruption of the 2-Cys peroxiredoxin TPx-1 in turn disrupts gametocytogenesis in P. berghei, making it a useful target for a prospective drug [94]. As has been demonstrated with other molecules, P. falciparum-specific analogs of TPx-1 are likely to show similar activity. Sequestration of parasites, more typical of P. falciparum infections than in rodent malarias, may exacerbate oxidative stress over what circulating parasites experience, thus increasing their sensitivity to interference with peroxiredoxins [94]. Disruption of TPx-1 did not appear to affect asexual parasite development or male gametogenesis [94].

Exflagellation of male gametocytes, triggered in part by the temperature decrease and rise in pH associated with the midgut environment of a mosquito, is mediated by a cascade of factors including a calcium-dependent protein kinase and cGMP [95]. Disruption of a P. falciparum gene (PfPDEδ) expressing one of four known cyclic dinucleotide phosphodiesterases (PDE), a key component of the cGMP signaling pathway, interferes with gametogenesis [96]. Similar disruption of guanylyl cyclase, another mediator of cGMP, had no such effect [96]. Three other P. falciparum PDE genes remain to be evaluated for similar effects and the role of cGMP in other phases of development may warrant further investigation.

Osmiophilic bodies are granular organelles associated with late stage macrogametocytes of many Apicomplexa [97,98] Their presence appears to be correlated with the emergence of Stage V macrogametocytes from the erythrocytic membrane in the mosquito midgut after the ingestion of infective blood. The main protein associated with osmiophilic bodies, Pfg377, is expressed solely in female gametocytes and appears to play a critical role in this mechanism, as targeted gene disruption reduces the efficiency of female gamete emergence both in vitro and in vivo [99].

Studies of mutant clones suggest that the P. falciparum gene Pf11-1 appears to code for a glutamic acid-rich protein that has a similar and perhaps more general role in the rupture of erythrocytic membranes to allow the escape of gametes [100]. Disruption of these mechanisms would thus inhibit sporogony and transmission.

Transcriptome analyses [101,102] of parasites undergoing differentiation into gametocytes in vitro have been used to reveal a wide array of proteins that are upregulated in the sexual stages. Some of these proteins appear to functionally important in gametocytogenesis, making them potentially useful as targets for drug intervention, although in most cases, their exact role in differentiation and the molecular mechanisms by which they operate require further clarification. Such proteins include:

Pfs16 and Pfg27 are both upregulated during the differentiation of trophozoites to gametocytes prior to the appearance of the subpellicular microtubule network that distinguishes the State II, crescent-shaped gametocytes from earlier forms, suggesting a possible role in inducing these morphological changes [102]. In vitro studies suggest that Pfs16 is over-expressed during the first 24 h of culture leading to the production of mature gametocytes [102]. A definitive causal link has not yet been established, however, between this gene or its products and gametocyte maturity [102].

Pfgig is a gene encoding for a yet to be identified protein involved in the transition from asexual stage parasites to sexual stages. Its silencing has a six-fold suppressive effect on gametocyte production and its complementation causes the upregulation of Ps16 [110]. Pfgig is transcribed abundantly in schizonts but no mRNA is detected in gametocytes, suggesting that commitment to sexual stages may occur early in the life cycle of malarial parasites [110].

Both PfPuf1 and PfPuf2 bind RNA and, according to a microarray analysis, are upregulated in gametocytes [109]. A subtraction library analysis suggests that PfPuf1 also appears to be upregulated by sporozoites [114].

PfPuf2 regulates translation in early stage gametocytes, promoting sexual development and sex differentiation [109]. Disrupting the activity of PfPuf2 increases gametocytogenesis and causes distortions in the sex ratio favoring males.

Pfpeg-3, also known as Pfmdv-1 [107] is produced in Stage I gametocytes and present on the gametocyte plasma membrane and other membranes in the parasitophorous vacuole and in the infected erythrocyte. Interference with this protein leads to sex-specific reductions in mature gametocytes, favoring female gametocytes [107]. It has been suggested that disrupted nutrient transport might be responsible for this effect in strains of parasites with a Pfmdv-1 defect [107]. Less is known of Pfpeg-4, other than that it appears in the highest concentrations in Stage II gametocytes [102].

Each of these proteins offer promise as targets for drugs that might disrupt the early stages of gametocytogenesis and skew sex ratios. Expression of HMGB2, a high mobility group protein that binds DNA (but is not sequence-specific) and is conserved in all malaria species, peaks in the gametocyte stage, although translation of mRNA seems to be delayed until parasites have differentiated into ookinetes [111]. Disruption of the locus encoding HMGB2 in P. yoelii through gene knockout has been shown to severely impair the formation of oocysts [111]. HMGB2 apparently helps regulate transcription of genes critical for the completion of sporogony.

Adherence to endothelial tissue beds allows gametocytes to develop in situ without facing the hazards associated with passage through the spleen. This accounts in part for why young gametocytes rarely circulate in peripheral blood and why mature infective gametocytes usually appear only after the asexual parasitemia has peaked.

Host receptors and parasite ligands involved in the adhesion and sequestration of gametocytes in the vascular endothelium offer potential targets for potential transmission-blocking vaccines [115]. Specific host receptors involved in the general sequestration of infected erythrocytes include: CD36 (a glycoprotein), vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and thrombospondin (TSP) [116]. All but VCAM-1 have been found to bind to the parasite ligand PfEMP-1 [117].

These molecules might also be targeted by immunotherapies or moieties that specifically bind or otherwise inhibit them. Thus, agents that interfere with the surface antigens expressed on young, sequestering gametocytes could impede normal gametocyte development in P. falciparum.

Some parasite ligands, particularly those associated with the sequestration of Stage II and IV gametocytes in bone marrow, have yet to be characterized although putative host receptors include CD49c, CD164, CD166 and ICAM-1 [118]. It may be that ligand candidates or their associated markers could be identified indirectly through genomic analysis [115]. Some of the more promising targets include:

Interference with or reversal of adhesion might be accomplished with conjugates incorporating monoclonal antibodies or segments of peptide ligands, similar to approaches suggested for prospective treatments of severe malaria involving blockage of cerebral capillaries [121,122].

One clinical study [121] attempting to use 400 mg/kg high-titer antibodies to reverse cytoadherence of erythrocytes infected with asexual stages failed to affect adherence, possibly because only one-fifth of the concentration of antibodies necessary to reverse adherence in vitro was administered to patients. It has been suggested that excessively high levels of antibody (2X the normal presence in peripheral blood) may be necessary to successfully reverse sequestration in the systems thus far evaluated [122]. It is not clear how well the sexual stages of Plasmodium would respond to such an approach. Immunoliposomes have also been suggested as a possible mode of drug delivery for monoclonal antibody conjugates targeting adhesion molecules [123].

Molecules involved in adhesion may also play other roles in gametocyte development and sporogony that offer alternative mechanisms of interference. For example, the expression of lectin adhesive-like proteins (PbLAP1, 2, 4 and 6) have been shown to be critical for oocyst maturation and sporozoite formation in P. berghei [124,125]. This expression occurs from female nucleus-derived mRNA prior to the expression of proteins from male-derived sequences, likely during the transition from gametocyte to ookinete [125]. P. falciparum-specific analogues (PfLAP 1 and 4) of these molecules have also been shown to be necessary for successful sporozoite infection of mosquito salivary glands [126].

An array of LAP-related PfCCP adhesion proteins (PfCCP1, PfCCP2, PfCCP3) occur co-dependently in the parasitophorous vacuole of the gametocyte plasma membrane of P. falciparum [127]. They are expressed in Stages II-V of gametocytogenesis as well as during the early part of gametogenesis. Disruption of PfCCP2 and PfCCP3 genes interrupts sporogony by interfering with the transition of sporozoites from oocysts to mosquito salivary glands [126]. PfCCP4 has been found to co-localize and form a complex with the gametocyte specific surface antigen Pfs230, but does not appear to be essential for sexual stage development [127].