SUMMARY STATEMENT

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 10/25/2016

Revised Date:

Application Number: 1 R15 AI124044-01A1

Principal Investigator 

DOMASHEVSKIY, ARTEM

Applicant Organization: JOHN JAY COLLEGE OF CRIMINAL JUSTICE

Review Group: MSFB

Macromolecular Structure and Function B Study Section

Meeting Date: 10/13/2016 RFA/PA: PA16-200

Council: JAN 2017 PCC: M64A B

Requested Start: 04/01/2017

Project Title: Development of a Novel Inhibitor of Ricin: A Potential Therapeutic Leadagainst

Deadly Shiga and Related Toxins

SRG Action: Impact Score:

Next Steps: Visithttp://grants.nih.gov/grants/next_steps.htm

Human Subjects: 10-No human subjects involved

Animal Subjects: 10-No live vertebrate animals involved for competing appl.

Project Direct Costs Estimated

Year Requested Total Cost 

1 

TOTAL

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been adjusted 

to reflect any recommendations made by reviewers. If an award is planned, the costs will be calculated by 

Institute grants management staff based on the recommendations outlined below in the COMMITTEE 

BUDGET RECOMMENDATIONS section.

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1 R15 AI124044-01A1 2 MSFB 

DOMASHEVSKIY, A 

1R15AI124044-01A1 Domashevskiy, Artem 

RESUME AND SUMMARY OF DISCUSSION: This application proposes to develop new inhibitors for 

ribosome inactivating proteins such as ricin and Shiga toxin using a combination of structural 

(Crystallography and NMR), biophysical (fluorescence and calorimetry) and computational approaches. 

Given the heightened interests and importance of measures to counter bioterrorism, this project has a 

solid premise. The diverse techniques involved in this program offer an excellent training opportunity 

for undergraduate students. Particularly, the proposed research is likely to be highly impactful in the 

setting of a city university. In this resubmitted application, the applicant has done a great job 

addressing the previous critiques by providing high-quality preliminary data, thus mitigating the major 

concerns. The planned experiments are feasible, meritorious and rigorously designed, from which 

valuable outcomes can be expected. The excellent investigative team that was assembled by the 

applicant consists of nearby laboratories with all needed expertise. The host institution is also very 

supportive. Although there are a few weaknesses such as that the description of Aim 2 is a little vague, 

the validation procedures of the structures by modeling may not be justified, and that some Biosketches 

are missing, these were all considered as relatively minor concerns. In general, the panel was 

extremely positive and enthusiastic about the program. The overall impact of this application was 

deemed to be very high. 

DESCRIPTION (provided by applicant): The PI proposes a high-impact collaborative research project 

to develop new inhibitors for ribosome inactivating proteins (RIPs), such as ricin and Shiga toxin. Ricin 

toxin, produced by the castor bean plant, has a nefarious past. Ricin is a well-known homicidal poison 

and has been used in several bioterrorist attacks. Shiga toxin is a deadly product of enterobacterial 

Escherichia coli. RIPs are RNA N-glycosidases that enzymatically remove specific purine residues from 

the universally conserved sarcin/ricin loop (S/R loop) of large ribosomal RNA, causing cellular death. 

There are no effective pharmaceuticals for either ricin or Shiga toxin poisoning. The PI proposes using 

a viral protein (VPg) from turnip mosaic virus that we have shown to inhibit ricin activity in vitro. The 

goals of this proposal are: 1) Establish conditions for VPg-ricin complex formation and the rates of VPgricin interactions; 2) Determine minimum VPg peptide that effectively inhibits ricin and Shiga toxins; and 

3) Optimize conditions to enhance inhibition of these toxic proteins. This is an innovative activity. Based 

on the structural and mechanistic similarities of these toxins and previously acquired data showing that 

VPg peptides inhibit ricin activity in vitro, we believe the outcomes of this activity will provide leads for 

the synthesis of therapeutic peptides. In turn, this will serve as a catalyst for the development of 

constructively applied solutions for the inhibition of these deadly toxins. Furthermore, this proposal will 

enhance the infrastructure of research and education at John Jay College, introducing biochemical and 

biomedical research experiences to underrepresented minority and female students, who would 

otherwise lack such opportunities. This would allow them to experience a broad spectrum of 

techniques, and acquire skills such as data analysis used in modern scientific investigations, while 

developing a vast network of partnerships among scientists from national and international institutions. 

PUBLIC HEALTH RELEVANCE: Ricin and related Shiga toxins are potentially lethal poisons, which as 

of yet, have no effective pharmaceutical treatments or preventions. The global problem of these 

poisons is an increasing threat to public health, directly relating to the mission of the National Institutes 

of Allergy and Infectious Diseases. Ricin, astoundingly easily acquired, is an ideal toxin for bioterrorism, 

while the widespread transmission of E. coli poisoning is responsible for many severe human illnesses 

and several fatalities. In the future, it may be possible to utilize these toxins for their public health 

benefit, by repurposing them to attack specific human and animal diseases. 

CRITIQUE 1 

Significance: 2 

Investigator(s): 2 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 R15 AI124044-01A1 3 MSFB 

DOMASHEVSKIY, A 

Innovation: 2 

Approach: 2 

Environment: 2 

Overall Impact: Ribosome inactivating proteins (RIPs) are an important class of cytotoxin. RIPs, such 

as Ricin A-chain (RTA) and Shigella toxin, are N-glycosidases that modify rRNA thereby deactivating 

ribosomes. RIPs are extremely toxic and there is significant interest in strategies to inhibit their action. 

To date there is neither an effective vaccine nor a therapeutic agent available to reverse the effects if 

Ricin once exposed. The PI has previously discovered a peptide inhibitor of RTA, called VPg, that binds 

to RTA with nM affinity. Here the PI plans to further characterize the parameters of VPg binding by RTA 

and determine the minimum VPg peptide that binds and inhibits RTA. Resulting VPg constructs will be 

tested in vivo by a collaborator, Dr. O’Brien. Structural information on the complex will be obtained by 

either X-ray crystallography or NMR, for which good collaborations are established. Residues that 

participate in RTA-VPg binding will be probed by in silco analysis to find mutants of VPg that bind RTA 

more tightly. These mutations will then be validated in vitro (and in cell-based assays?). On the whole 

this is a strong proposal with only a few weaknesses. The undergraduate environment is excellent, very 

supportive of the PI, and serves a large population of URMs. 

1. Significance: 

Strengths 

 Strong scientific premise. 

 RIPs are an important class of toxins for which there are no current treatments or vaccines. 

Weaknesses 

 None stated. 

2. Investigator(s): 

Strengths 

 The PI discovered that VPg inhibits PAP and has all the necessary expertise (along with the 

collaborators) to complete the proposed studies. 

 The PI has a solid publication record relevant to the proposed studies. 

Weaknesses 

 None stated. 

3. Innovation: 

Strengths 

 Employing peptide inhibitors of RIP is innovative. 

Weaknesses 

 Not really a weakness but all approaches, including site-directed mutagenesis, are standard. 

4. Approach: 

Strengths 

 Strong preliminary data showing that VPg binds RTA with high affinity. 
 

 

 

 

 

 

 

1 R15 AI124044-01A1 4 MSFB 

DOMASHEVSKIY, A 

 A rigorous and well thought out approach is provided, with good alternative strategies outlined 

should problems arise. 

 Proposed experiments are accessible to undergraduates and the PI provides a strong 

mentoring statement. 

 Use of ITC and fluorescence anisotropy is a plus. 

Weaknesses 

 Lack of RTA-VPg crystals (or preliminary NMR data). 

 It is unclear how successful the creation of VPg constructs with higher affinity for RTA will be, 

given that the interaction is already quite strong (~50 nM). The proposed mutagenesis strategy 

will more likely reveal residues that do not contribute to affinity significantly. Modification of 

these residues may help in generating a peptide with better properties for use as a drug. 

5. Environment: 

Strengths 

 The PI or collaborator labs have access to all the necessary equipment. 

 A strong list if collaborators. 

 Institute is very supportive and has a high URM representation. 

Weaknesses 

 Many of the experiments proposed will be performed outside of the PI lab. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 

Resubmission: 

 The PI was receptive to previous comments/suggestions. 

Applications from Foreign Organizations: 

Not Applicable (No Foreign Organizations) 

Select Agents: 

Acceptable 

Resource Sharing Plans: 

Unacceptable 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 R15 AI124044-01A1 5 MSFB 

DOMASHEVSKIY, A 

 None provided 

Authentication of Key Biological and/or Chemical Resources: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 2 

Significance: 2 

Investigator(s): 4 

Innovation: 2 

Approach: 2 

Environment: 3 

Overall Impact: This is a resubmission of a R15 application that seeks to develop new antitoxins for 

ricin and Shiga toxin using viral protein VPg, making the significance of this proposal very high. The 

previous proposal was criticized for lack of key preliminary binding data, no discussion of other 

biophysical methods such as NMR and X-ray, and poorly outlined molecular dynamics simulation. All of 

these weaknesses have been adequately addressed. Strong preliminary data show actual binding of 

VPg to and RTA and inhibition of RTA making this project entirely feasible. Prior postdoctoral 

experience, publications and preliminary data indicate the PI is capable of carrying out the project. The 

judicious choice of experts to round out areas of weakness (NMR with Dr. Greenbaum; X-ray with Dr. 

Jeruzalmi; and molecular modeling, docking and scoring algorithms with Drs. Shaneen Singh and 

Emillio Gallicchio) is also a big plus. The proposed research is clearly written, rationales are outlined 

and the methodology clearly laid out for easy participation by undergraduate students, with many 

opportunities for the students to interact with these collaborators. Given the large population of 

underrepresented groups potentially being exposed to a strong undergraduate educational 

environment, given that conditions for forming a stable RTA-VPg complex have been worked out, and 

given the physiological relevance of RTA-VPg complex, I am very enthusiastic for the R15 proposal. 

1. Significance: 

Strengths 

 Premise is sound: ~50nM high affinity binding between VPg and RTA and ability to inhibit RTA 

enzymatic activity does provide an avenue to develop novel inhibitors. 

 No currently known inhibitors of ricin makes this work significant. 

Weaknesses 

 None stated. 

2. Investigator(s): 

Strengths 

 Has extensive training and expertise in area proposed. 
 

 

 

 

 

1 R15 AI124044-01A1 6 MSFB 

DOMASHEVSKIY, A 

 Areas of weakness are supplemented nicely with expertise in X-ray crystallography (Dr. 

Jeruzalmi); NMR (Dr. Greenbaum); molecular modeling, docking and scoring algorithms (Drs. 

Shaneen Singh and Emillio Gallicchio). 

Weaknesses 

 None stated. 

3. Innovation: 

Strengths 

 Work to use peptides derived from VPg as inhibitors is novel. 

Weaknesses 

 None stated. 

4. Approach: 

Strengths 

 Rigorous application of sound biophysical tools are a strength: outlined NMR experiments to do 

assignments and monitor and calculate changes in chemical shift perturbation (with 

Greenbaum), stopped flow kinetics (in Goss lab) and ITC (in Latham lab). 

 Has relevant clones in hand. 

 Will be able to test developed antitoxins (with O’Brien). 

 Uses knowledge gained to expand to small molecule discovery using virtual screening and 

molecular docking. 

Weaknesses 

 Some details in the approaches missing. Minor. 

5. Environment: 

Strengths 

 Environment at JJC is very supportive and a large population of URMs stand to greatly benefit 

from this proposal’s success and therefore potentially will have a high impact on exposure of 

students to state of art research. 

Weaknesses 

 None stated. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Not Applicable (No Biohazards) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 R15 AI124044-01A1 7 MSFB 

DOMASHEVSKIY, A 

Applications from Foreign Organizations: 

Not Applicable (No Foreign Organizations) 

Authentication of Key Biological and/or Chemical Resources: 

Not Applicable (No Relevant Resources) 

Budget and Period of Support: 

Recommended budget modifications or possible overlap identified: 

CRITIQUE 3 

Significance: 3 

Investigator(s): 2 

Innovation: 5 

Approach: 2 

Environment: 1 

Overall Impact: This is a well-written proposal from a new investigator who is an Assistant Professor at 

John Jay College. The PI proposes to explore the interaction of the VPg protein from the turnip mosaic 

virus with the A chain of the ricin toxin (RTA). The long term goal is to define the molecular details of 

VPg binding and inhibition of ricin and Shiga toxin so that novel peptide-based inhibitors might be 

developed for these toxins. The experimental plan, especially in Aim 1, is extremely well described as 

are areas of involvement for student researchers at John Jay College. The PI has previously described 

the interaction of VPg with another ribosome inactivating protein (PAP) and is clearly capable of 

conducting the binding and in vitro activity studies with RTA. In this amended application the PI has 

now included clear preliminary data demonstrating that VPg binds and inhibits RTA. He has also made 

other changes in response to the previous review that have strengthened the study. One area of 

concern is that the studies in Aim 2 are a bit outside of the PI’s direct expertise and are not described in 

sufficient detail. Nevertheless, the project as a whole is meritorious, will expose students to research, 

and will strengthen the research environment at John Jay College – in short – it meets the goals of the 

R15 program quite well. 

1. Significance: 

Strengths 

 There are few effective therapies for ribosome inactivating toxins. 

 Ricin toxin has been identified as a possible bioterrorism agent. 

 This work could lead to the development of therapeutic inhibitors for RIPs including Shiga toxin, 

which is a contributor to food-borne illness. 

Weaknesses 

 None stated. 

2. Investigator(s): 

Strengths 

 The PI has previously described VPg binding and inhibition of PAP and is well qualified to 

conduct the studies described in Aim 1. 
 

 

 

1 R15 AI124044-01A1 8 MSFB 

DOMASHEVSKIY, A 

 The PI has been engaged in training students in research. 

 The array of collaborators should provide the necessary expertise to contribute to the areas 

where the PI has less experience (Aim 2). 

Weaknesses 

 While the collaborators appear to have the necessary experience, it was hard to discern this 

without having their biosketches included. 

3. Innovation: 

Strengths 

 The use of peptides to inhibit RIPs is novel. 

Weaknesses 

 The research proposed uses standard approaches and is not very innovative. 

4. Approach: 

Strengths 

 The premise of the study is very clear and solid. 

 The experiments described in Aim 1 are very detailed and rigorous. 

 Considerable attention is given to describe expected outcomes and alternative approaches. 

 The new preliminary data demonstrating interaction of RTA and VPg indicates that these 

studies aimed at defining this interaction will yield useful information. 

 The PI has included additional biophysical methods (NMR, X-ray crystallography) to more 

completely define the RTA-VPg interaction, and has identified collaborators to assist with this 

work. 

Weaknesses 

 The molecular docking experiments do not seem to be very well justified. They are proposed to 

“confirm” the X-ray and NMR studies which seems to be somewhat odd. If the PI is successful 

in generating a high-resolution co-crystal structure of RTA and VPg, then I do not see the need 

to conduct docking after the fact. 

 Unlike in Aim 1, the studies in Aim 2 using NMR and X-ray crystallography are not well 

described. For instance, attention is given to some issues relating to generating crystals but little 

information in provided on how the structures will be determined. 

5. Environment: 

Strengths 

 The PI and his collaborators have access to all the necessary equipment to do this work. 

 The institution is well positioned to benefit from this R15 project. 

 The high representation of URM students and the plan to provide support for these students to 

participate in the research. 

Weaknesses 

 None stated. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 R15 AI124044-01A1 9 MSFB 

DOMASHEVSKIY, A 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 

 The work proposed with full ricin and Shiga toxin will be conducted in an approved facility with 

the appropriate controls in place. 

Resubmission: 

 The PI has done an excellent job in responding to the previous critiques. In particular the 

inclusion of new preliminary data has alleviated the major concern. 

Applications from Foreign Organizations: 

Not Applicable (No Foreign Organizations) 

Select Agents: 

Not Applicable (No Select Agents) 

Resource Sharing Plans: 

Unacceptable 

 No resource sharing plan was included. 

Authentication of Key Biological and/or Chemical Resources: 

Unacceptable 

 No mention of how the various peptides or proteins will be authenticated was included in this 

section. 

Budget and Period of Support: 

Recommend as Requested 

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS' 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES: 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

Footnotes for 1 R15 AI124044-01A1; PI Name: Domashevskiy, Artem 
 

 

 

 

 

 

 

 

 

1 R15 AI124044-01A1 10 MSFB 

DOMASHEVSKIY, A 

NIH has modified its policy regarding the receipt of resubmissions (amended applications). 

See Guide Notice NOT-OD-14-074 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD14-074.html. The impact/priority score is calculated after discussion of an application by 

averaging the overall scores (1-9) given by all voting reviewers on the committee and 

multiplying by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review meeting 

or calculated into the overall impact score. Some applications also receive a percentile 

ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring.