Gametocyte-specific and all-blood-stage transmission-blocking chemotypes discovered from high throughput screening on Plasmodium falciparum gametocytes

Blocking Plasmodium falciparum human-to-mosquito transmission is essential for malaria elimination, nonetheless drugs killing the pathogenic asexual stages are generally inactive on the parasite transmissible stages, the gametocytes. Due to technical and biological limitations in high throughput screening of non-proliferative stages, the search for gametocyte-killing molecules so far tested one tenth the number of compounds screened on asexual stages. Here we overcome these limitations and rapidly screened around 120,000 compounds, using not purified, bioluminescent mature gametocytes. Orthogonal gametocyte assays, selectivity assays on human cells and asexual parasites, followed by compound clustering, brought to the identification of 84 hits, half of which are gametocyte selective and half with comparable activity against sexual and asexual parasites. We validated seven chemotypes, three of which are, to the best of our knowledge, novel. These molecules are able to inhibit male gametocyte exflagellation and block parasite transmission through the Anopheles mosquito vector in a standard membrane feeding assay. This work shows that interrogating a wide and diverse chemical space, with a streamlined gametocyte HTS and hit validation funnel, holds promise for the identification of dual stage and gametocyte-selective compounds to be developed into new generation of transmission blocking drugs for malaria elimination.

The authors present a novel methodology for high-throughput screening of Plasmodium transmission-blocking compounds, employing bioluminescent gametocytes of the human P. falciparum parasite. They report on the screening of a large number of compounds (~120.000) and on the identification of a few dozen hits either with gametocyte-specific activity or with activity against both sexual and asexual parasite forms. This is a novel approach to the identification of compounds with Plasmodium transmission-blocking potential that deserves being reported to the community. However, I do have a few concerns that the authors should address before publication in Communications Biology can be considered.
Please revise citation formats. For example, the WHO's World Malaria Report 2021 is cited as "2020 1997" (line 60) and a WHO resolution is cited as "Organization 2021" (line 63). Referencing softwares do not always display citations in the most correct manner and care should be exerted to individually inspect each citation for its appropriateness.
Line 67: although widely employed, the term "malaria infections" is incorrect. Malaria is a disease, not an organism. Either state that "Malaria symptoms are…" or that "In Plasmodium infections, …".
Lines 67-75: when providing these numbers (line 71), the authors should specify whether they are talking about P. falciparum specifically or about all human-infective Plasmodium species. There are a few grammar / semantic incorrections throughout, which the authors should address. For example, in Line 78 "malaria acute phase" should be "the acute phase of malaria" and the term "insurgence" (which means "insurrection") is incorrectly used (the authors probably mean "appearance"). In Lines 106 and 126, "To this am" should be "To this end". In Lines 125-126, "paramount important" should be "paramount importance". Line 130, "microscopic examination" should be "light microscopy examination". Line 136, "half 384 well plate" should be "half of a 384-well plate". Line 145, the authors probably mean "successful" instead of "successive". Lines 208-209, "This procedure gave a collection of 371 clusters whose 205 were singletons" should probably be "This procedure gave a collection of 371 clusters, 205 of which were singletons". And so on… Paragraph Lines 82-90 makes several statements, none of which is referenced. Please cite appropriate references. Line 95: it is unclear whether "half of which in only two screenings" refers to the "5 million compounds that were screened against the asexual stage parasites" or the 300.000 compounds that were "tested against P. falciparum gametocytes". Likely the latter, but that is exactly the opposite of what the sentence, as it stands, says.
Lines 97-100: the authors write "A variety of HTS gametocyte assays were used so far relying on multiple detection techniques: fluorescent/luminescent reporters, parasite enzymes activity assays, gametocyte ATP content determination, uptake of redox sensitive dyes and the assessment of the motility of male gamete flagella" without providing a single reference. This is totally unacceptable. Each and every one of these reported techniques must be backed by at least one reference.
Lines 100-103: sentence "Beyond assay signal detection procedure incompatible with HTS, the sensitive and specific acquisition of the above assay readouts imposes to purify gametocytes from uninfected erythrocytes, or to cultivate and/or treat them in the absence of human serum" is poorly written and needs to be rephrased. It also needs referencing.
Line 113: the acronym "CNCCS" appears without any definition of what it stands for.
Lines 126-130: the authors state that "… the timing of addition of N-acetyl glucosamine (NAG), used to clear residual asexual stages after appearance of stage I gametocytes, was optimized. A three-day treatment with 50 mM NAG, followed by an additional five days of cultivation, yielded cultures with about 2% gametocytaemia of early-stage V gametocytes, as confirmed by microscopic examination of Giemsa-stained blood smears" and point towards Fig. 1A to substantiate this statement. Fig. 1A does not serve this purpose, unless the authors clearly state that this single image is representative of the entire sample. "hours" should be denoted by "h" throughout and not by either "hours" (e.g. Line 132) or "h" (e.g. Line 137) indistinctly.
Line 139: the term "CV%" must be defined and explained. The authors cannot assume all the readers know what this acronym stands for or what this measurement means. Without an explanation for this, Fig. 1C is unintelligible.
Lines 139-140: why are the data of CV% values for the positive and negative controls not shown? Again, without a proper explanation of the meaning of "CV%", the information that both were "less than 10%" is meaningless. The authors should understand that not all the readers will be as versed on this topic as they are, and that explanations of less obvious terms and their meaning must be provided for clarity. Fig. 1 legend: acronyms must be defined the first time they appear. "RBC" is used without any prior definition.
Lines 158-159: "present at that time" begs the question "when?". This is not clear and the reader is not obliged to know when the assay was performed and what changed in terms of the number of compounds in this library since then.
Lines 189-191: the authors state that "the amount of compounds that confirmed active in both lines (top right field), was similar to the amount of poorly active compounds in both lines (bottom left field". First, it is not clear from looking at Fig.3 B that this would be the case. Second, why do the authors consider this surprising, i.e., what would they expect to see? Third, can the authors clarify the choice of 40% inhibition for definition of the quadrants in the plot on Fig. 3B? Line 211: "the activity data on the three parasite strains". These are not three different strains of parasite, but rather 1 transgenic line of strain Pf3D7 and 2 transgenic lines of strain PfNF54. The authors should mention the work by Azevedo et al. employing bioluminescent P. berghei parasites for screening transmission-blocking compounds (PMIDs 28348156, 31752986, 32038528) in their Discussion.
Reviewer #3 (Remarks to the Author): In this paper the authors screened Plasmodium gametocytes against a large compound library. Their premise is that more drugs are needed to prevent the spread of malaria to mosquitos from infected people.
The authors propose the novelty of this paper is 1) the assay using gametocytes and 2) discovery of drugs that block human to mosquito transmission.
From the citations in the methods, it is unclear if this is a new method for screening gametocytes. It also leads me to question if people would be willing to take a medicine to prevent mosquito infection.
From the article, it is not clear if any of these hits will achieve this purpose. Will the 9 compounds that made it through the funnel actually fit this purpose?
The findings are really about the pipeline, not really about the hits themselves. What are the prospects of these hits?

Needs english language editing
Reviewer #1 (Remarks to the Author): The research article "Novel gametocyte-specific and all-blood-stage transmission-blocking chemotypes from high throughput screening on Plasmodium falciparum gametocytes " reported the establishment of a simple, sensitive and robust HTS assay funnel for the identification/screening of anti-gametocyte compounds including a set of counter-screenings and validation assays. With these tools authors set out to identify bona fide gametocyte active chemotypes and investigated the selectivity of them against the human host and the asexual stage parasites. The whole work is interesting and authors have covered mostly all the stages in transmission blocking.