Document:

EX-10.6

 Exhibit 10.6 

SECOND ADDENDUM 
 TO THE
RESEARCH AND LICENSE AGREEMENT 
 This Second Addendum to Research and License Agreement (the “Second Addendum”) is made by and between
The Technion Research & Development Foundation Ltd. (“TRDF”) and Eloxx Pharmaceuticals Ltd. (“Licensee” or “Eloxx”). 

WHEREAS, TRDF and Eloxx are parties to Research and License Agreement with an effective date of August
29th, 2013 (the “License Agreement”), as amended on November 26, 2013, January 14, 2014, June 9, 2014 and August 3, 2014 (collectively, the
“Agreement”); and 
 WHEREAS, the parties desire to continue the relationship contemplated by the Agreement and to further amend the
Agreement as set forth herein; 
 Now, THEREFORE, the parties hereby agree as follows: 

 

	1.	Unless otherwise defined herein, capitalized terms used in this Second Addendum shall have the meanings assigned thereto in the Agreement. 

 

	2.	The Parties agree to add those certain patents and patent applications described in Exhibit A(3) attached hereto, to Exhibits A(1) and A(2) of the Agreement. 

 

	3.	Except as added herein, all other terms and conditions of the Agreement shall remain in full force and effect, as relevant to this Second Addendum. 

 

	4.	This Second Addendum may be executed in counterparts, each of which shall be deemed an original and all of which together shall constitute one and the same instrument. Any signature page delivered by facsimile or
electronic image transmission shall be binding to the same extent as an original signature page. 

 IN
WITNESS WHEREOF, the parties hereby accept and agree to the terms and conditions of this First Addendum. 
  

													
	ELOXX PHARMACEUTICALS LTD.	  		  		  		  	THE TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD.

													
							
	By:	 	 /s/ Shmuel Tuvia
	  		  		  		  	By:	 	 /s/ Benjamin Soffer

	Name:	 	Shmuel Tuvia	  		  		  		  	Name:	 	Benjamin Soffer
	Title:	 	COO	  		  		  		  	Title:	 	Technology Transfer Office, Manager
	Date:	 	Jan. 21, 2015	  		  		  		  	Date:	 	

 Exhibit A(3) 
  

															
	USE OF AMINOGLYCOSIDE ANALOGS IN THE TREATMENT OF RETT SYNDROME
								
	Our
Ref
Client
Ref	 	Country	 	Earliest
Priority	 	Entry
Date	 	Filing Date
Application No.	 	Issue Date
Patent No.	 	Status	 	Assignee
								
	59529
?	 	USA
PRO	 		 		 	05-Jun-2014
62/006,028	 		 	Filed	 	Technion Research &
Development Foundation
Limited ; Eloxx
Pharmaceuticals Ltd.

																															
	 PatentNum
	  	PatentName	  	PatentStatus
Desc	  	ApplicationDate	 	  	ApplicationNum	 	  	CountryDesc	  	Patent
Date	 	  	PatentNo	 	  	PublicationDate	 	  	PublicationNum	 
	 0816
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	NP from PCT	  				  				  	N/A	  				  				  				  			
										
	 0816-00
	  	REDESIGN OF AMINOGLYCOSIDES FOR TREATMENT OF HUMAN GENETIC DISEASES CAUSED BY PREMATURE STOP MUTATIONS	  	Expired	  	 	03/04/2006	 	  	 	60/788,070	 	  	United States	  				  				  				  			
										
	 0816-01
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Expired	  	 	10/04/2007	 	  	 	PCT/IL/2007/000463	 	  	PCT	  				  				  	 	11/10/2007	 	  	 	WO 2007/113841	 
										
	 0819-02
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	25/09/2008	 	  	 	194370	 	  	Israel	  				  				  				  			
										
	 0816-03
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	25/09/2008	 	  	 	2,646,407	 	  	Canada	  				  				  				  			
										
	 0816-04
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	10/04/2007	 	  	 	07736203.6	 	  	Europe	  				  				  				  			
										
	 0816-05
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	03/10/2008	 	  	 	2009-503741	 	  	Japan	  				  				  	 	10/09/2009	 	  	 	2009-532461	 
										
	 0816-06
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	01/10/2008	 	  	 	12/285,299	 	  	United Slates	  				  				  				  			
										
	 0816-07
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	31/10/2008	 	  	 	09136/DELNP/2006	 	  	India	  				  				  				  			
										
	 0816-08
	  	NOVEL AMINOGLYCOSIDES AND USES THEREOF IN THE TREATMENT OF GENETIC DISORDERS	  	Filed	  	 	10/04/2007	 	  	 	11173958.7	 	  	Europe	  				  				  	 	30/11/2011	 	  	 	2390255	 

																															
	PatentNum	  	PatentName	  	PatentStatusDesc	  	Application
Date	 	  	ApplicationNum	 	  	CountryDesc	  	PatentDate	 	  	PatentNo	 	  	PublicationDate	 	  	PublicationNum	 
	 1302
	  	REPAIRING FAULTY GENES BY AMINOGLYCOSIDES: IDENTIFICATION OF NEW PHARMACOPHORE WITH ENHANCED SUPPRESSION OF DI	  	NP from PCT	  				  				  	N/A	  				  				  				  			
										
	 1302-00
	  	REPAIRING FAULTY GENES BY AMINOGLYCOSIDES: IDENTIFICATION OF NEW PHARMACOPHORE WITH ENHANCED SUPPRESSION OF DI	  	Expired	  	 	18/11/2010	 	  	 	61/414,956	 	  	United States	  				  				  				  			
										
	 1302-01
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Expired	  	 	17/11/2011	 	  	 	PCT/IL/2011/000889	 	  	PCT	  				  				  	 	24/05/2012	 	  	 	WO2012/066546	 
										
	 1302-02
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Filed	  	 	16/05/2013	 	  	 	13/885,715	 	  	United States	  				  				  				  			
										
	 1302-03
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Filed	  	 	17/11/2011	 	  	 	11799501.9	 	  	Europe	  				  				  				  			
										
	 1302-04
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Filed	  	 	20/05/2013	 	  				  	Japan	  				  				  				  			
										
	 1302-05
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Filed	  	 	02/05/2013	 	  	 	2,816,789	 	  	Canada	  				  				  				  			
										
	 1302-06
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Filed	  	 	07/05/2013	 	  	 	876/MUMNP/2013	 	  	India	  				  				  				  			
										
	 1302-07
	  	AMINOGLYCOSIDES AND USES THEREOF IN TREATING GENETIC DISORDERS	  	Filed	  	 	16/05/2013	 	  	 	226390	 	  	Israel	  				  				  				  	 	.EX-10.7

 Exhibit 10.7 

THIRD ADDENDUM 
 TO THE
RESEARCH AND LICENSE AGREEMENT 
 This Third Addendum to Research and License Agreement (the “Third Addendum”) is made by and
between The Technion Research & Development Foundation Ltd. (“TRDF”) and Eloxx Pharmaceuticals Ltd. (“Licensee” or “Eloxx”). 

WHEREAS, TRDF and Eloxx are parties to Research and License Agreement with an effective date of August 29, 2013, as
amended on November 26, 2013, January 14, 2014, June 9, 2014, August 3, 2014 and January 21, 2015 (collectively, the “Agreement”); and 

WHEREAS, the parties desire to continue the relationship contemplated by the Agreement and therefore to further amend the
Agreement as set forth herein: 
 NOW, THEREFORE, the parties hereby agree as follows: 

 

	1.	Unless otherwise defined herein, capitalized terms used in this Third Addendum shall have the meanings assigned thereto in the Agreement. 

 

	2.	Licensee wishes to receive materials from the Principal Investigator under this Third Addendum, all as described in Exhibit A attached hereto which shall be added to the Agreement (the
“Materials”). 

  

	3.	TRDF has already provided Licensee the Materials, on December 2014. 

  

	4.	In consideration of the Materials provision, TRDF is entitled to a total amount of ten thousand US dollars ($10,000) (the “Payment”), VAT, as applicable on time of payment, shall be added to the
Payment. 

  

	5.	Licensee shall pay TRDF the Payment, immediately upon signing this Third Addendum and receipt of proper invoice from TRDF. 

  

	6.	Except as added herein, all other terms and conditions of the Agreement shall remain in full force and effect, as relevant to the Third Addendum. 

 

	7.	This Third Addendum may be executed in counterparts, each of which shall be deemed an original and all of which together shall constitute one and the same instrument. Any signature page delivered by facsimile or
electronic image transmission shall be binding to the same extent as an original signature page. 

 IN WITNESS
WHEREOF, the parties hereby accept and agree to the terms and conditions of this Third Addendum. 
  

					
	ELOXX PHARMACEUTICALS LTD.	 	 THE TECHNION RESEARCH & DEVELOPMENT FOUNDATION
LTD.

											
						
	By:	 	 /s/ Silvia Norman
	 		 	By:	 	 /s/ Rita Bruckstein
	 	
	Name: Silvia Norman	 		 	Name: Rita Bruckstein	 	
	Title: CEO	 		 	Name: Research Authority	 	
	Date: 27/01/15	 		 	Date: 9/2/2015	 	

 Exhibit A 

Supply of the compounds: NB84, NB122, NB124 and NB127, each in the amount of 100 mg 

  
 2EX-10.8

 Exhibit 10.8 

Execution Copy  
 FOURTH ADDENDUM

 TO THE RESEARCH AND LICENSE AGREEMENT 

This Fourth Addendum to Research and license Agreement (the “Fourth Addendum”) is made by and between the Technion Research and
Development Foundation Ltd. (“TRDF”) and Eloxx Pharmaceuticals Ltd. (“Licensee” or “Eloxx”). 

Whereas, TRDF and Eloxx are parties to a Research and License Agreement with an effective date of August 29th 2013 (the “License Agreement”), as amended on November 26th, 2013 (“First Amendment”), January 14th, 2014 (“Second Amendment”), June 9th, 2014 (“Third Amendment”), August 3rd 2014 (“First Addendum”), January 21st, 2015 (“Second Addendum”) and February 9th 2015 (“Third Addendum”) (collectively, the “Agreement”); and 

Whereas, according to Section 1.26 to the License Agreement, the first Research Period has ended on September 30th, 2014 (“First Research
Period”) and the First Research Plan was completed respectively (“First Research Plan”);and 
 Whereas, the parties desire
to extend and continue the Research Period and the Research for a second year; and 
 Whereas, the parties desire to continue the relationship
contemplated by the Agreement and therefore to further amend the Agreement as set forth herein; 
 NOW, THEREFORE, the parties hereby agree as
follows: 
  

	1.	Unless otherwise defined herein, capitalized terms used in this Fourth Addendum shall have the meaning assigned thereto in the Agreement. 

 

	2.	The parties wish to extend the Research Period for a second year, commencing upon the expiration of the First Research Period, on October 1st, 2014 for twelve (12) months until September 30th, 2015 (“Second
Research Period”). The extension shall be on the same terms and conditions as contained in the Agreement unless otherwise agreed in this Fourth Addendum. 

 

	3.	Exhibit D to the Agreement is hereby replaced with a new Exhibit D attached hereto (“Second Research Plan”). 

  

	4.	The parties wish to set the terms tor the funding for the performance of the Second Research Plan during the Second Research Period, and replace section 2.2.1 of the License Agreement, as follows: 

 

	 	a)	Licensee shall fund the Research to be performed during the Second Research Period under the Second Research Plan in the total amount of fifty thousand US Dollars ($50,000) in accordance with the following schedule:

  

	 	1)	First installment of twenty five thousand US Dollars ($25,000) shall be paid upon signing this Fourth Addendum. 

  

	 	2)	Second installment of twenty five thousand US Dollars ($25,000) shall be paid upon completion of the Second Research Plan and no later than October 1st 2015.

 Execution Copy 
  

	 	b)	V.A.T as applicable on time of payment shall be added to each installment. 

  

	 	c)	TRDF shall issue a proper invoice for each installment. 

  

	5.	Except as amended herein, all other terms and conditions of the Agreement shall remain in full force and effect. 

  

					
	ELOXX PHARMACEUTICALS LTD. DEVELOPMENT	 	   THE TECHNION RESEARCH & FOUNDATION
LTD.

											
						
	By:	 	 /s/ Silvia Norman
	 		 	By:	 	 /s/ Mano Medalsi
	 	
	Name: Silvia Norman	 		 	Name: Mano Medalsi, CPA	 	
	Title: CEO	 		 	Title:	 	
	Date: 24/4/15	 		 	Date: 29/4/15	 	

  

 Exhibit D 

 Submitted to Eloxx Pharmaceuticals LTD 

Research Plan 

(Continuation for the second year 1.10.2014-30.092015) 

Title 
 Development of
Aminoglycose-Based Drug for Treatment of Human Genetic Diseases and Many Forms of Cancer Caused by Nonsense Mutations 

I.     Brief Summary of the First Year’s Research 

The two specific aims were: To provide Eloxx with all the important lead compounds developed by us in appropriate quantities (as agreed and
signed) for the validation of our previously published data, along with the detailed protocols of the synthesis and biological evaluations performed in our labs (Aim #1); Further modifications of the developed leads and/or development
of new lead structures for improved efficacy as potential drugs (Aim #2). The main achievements include: 
 (1) We successfully
synthesized all the lead compounds, provided them to Eloxx, and the company has successfully validated with these compounds the entire biological and toxicity data previously reported by us. 

(2) We have generated a new series of N1-alkyl derivatives on the disaccharide scaffold and
demonstrated their improved activity in comparison to parent compounds. Encouraged, the best disaccharide derivative NB146 was further converted to the corresponding trisaccharide derivative, NB147. Both these new leads (NB146 and NB147) were
provided to Eloxx for further assessment their biological tests. 
 (3) By developing a simple and robust synthetic scheme for the
modification of the developed leads as poly-ester derivatives, we have produced two such new derivatives Bz-G418 (a prototype structure for the
proof-of-concept) and Bz-NB124 (along with their parent structures) and provided them to Eloxx for further assessment their biological tests. 

In addition, two workshops have organized by the PI during this grant period with Eloxx together with a professional in CF, Prof S. Rowe (UAB,
USA) and the professional in Hurler syndrome, Prof. D. Bedwell (UAB, USA) to critically discuss our recent developments and future progresses. 

II.     Proposed Working Plan for the Second Year Research 

Specific Aims 

Aim # 1: Rational Design and Synthesis of New Readthrough Drugs. Our design principles integrate the insights from the
first year’s achievements in our lab along with the insights in other labs in order to construct new classes of compounds by a structure-based approach. Several sets of structurally distinct chemical compounds will be included in the initial
library and promising lead compounds will be further refined for better cell permeability and prolonged in-vivo action. The synthesis will use state-of-the-art strategies for the assembly of complex carbohydrate, along with the convenient up-to-date analytical
techniques. 
 Aim #2: Chemical Modification of NBs for Improved Cell Permeability and Oral Bioavailability. We will
continue the project from the first year on the polyester derivatives of NBs and this strategy will be further expanded with polyamide derivatives and with the special modifications of NBs as pro-drugs for
better blood-brain-barrier (BBB) permeability. 
 Aim #1- (a) rational design of pseudo-disaccharide
scaffolds: Based on our previous results with development of lead structures of NB-series, we propose to modify these compounds as well as to extend our study to more diverse structures with
potentially improved suppression activity and lower toxicity. We will probe the pseudo-disaccharides of set1 (Fig. 1) as the minimum basic structures. The rationale in selecting them is based on our observations with previous leads exhibiting
C6’-OH, and that further modifications by inserting either unsaturation at ring I (glucosamine ring, compounds 1-9) or the 6’,7’-diol (compounds
10-11) are expected to improve their specificity 
  
 

 
  

 Timor Baasov 
  

 
 towards the cytoplasmic A-site, leading to enhanced readthrough and reduced
toxicity. This expectation is supported by: (i) the aminoglycoside (AG) sisomicin that exhibits a similar unsaturated ring between C4’- C5’ at ring I displays better antibacterial activity than similar
4,6-disubstituted AGs with a saturated ring I, like gentamicin, tobramycin and amikacin. The superior antibacterial activity of sisomicin has been attributed to its specific binding mode1: while a typical saturated ring I with a chair conformation stacks on G1491 through CH-p interactions, the unsaturated ring I of sisomicin stacks with a
partially planar conformation through p -p interactions with G1491 and achieves a closer fit within the binding pocket; (ii) while sisomicin is active
only against bacteria, 6’-hydroxysisomicin possesses promising antiprotozoal activity2: a comparison of stacking interactions between G1491 and ring I of 6’-hydroxysisomicin and
geneticin in the protozoal A-site clearly favors the former (Fig. 2A). 6’-hydroxysisomicin can adopt a lower energy conformation within the protozoal A-site,
leading to the entropically favorable burying of the hydrophobic region of ring I with optimized p-p stacking on G1491. (iii) Our recently solved crystal
structure of G418 bound to a model of the Leishmania ribosomal A-site supported the above notions3: the ring I of G418 is fixed into the Leishmania A-site due to a stable pseudo pair with G1408 (Fig. 2B) and the additional H-bonds O3’

 and

 on the opposite site of the pseudo pair (Fig. 2C), leading to an unfavorable stacking of ring I with the A1491. [Note that one of the major differences between the protozoal and Leishmanial A-sites is the nucleotide at position 1491 (E. coli numbering): G1491 in most protozoa and A1491 in Leishmania; therefore the Leishmanial ribosome shares greatest similarity with human ribosomes, with only a single
nucleotide differentiating the AGs putative binding sites (U1409 in Leishmania vs. C1409 in human]. Therefore, the deletion of only C4’-OH (1-9, R1=OH, Fig 1) with simultaneous introduction of C4’-C5’ double bond, can make ring I relatively free to move within the binding pocket and form
stronger n-n stacking with A1491. 
 The rationale for the selective attachment of (S)-4-amino-2-hydroxybutanoyl (AHB) at the N1 position of set1 (R=AHB) is based on the highly positive impact of this
group (for example in NB84) compared to their parent structures (for example NB74), respectively4-8. Therefore, we will continue this approach in new
designs. Finally, in all set1 structures we preserved the C6’-OH function as a crucial motif for favorable H-bond interactions between ring I of AG and G1408
(Fig. 2B). We anticipate that the deletion of C4’-OH or C3’,C4’-hydroxyls with a simultaneous introduction of unsaturation on ring I will make the ring relatively “free” to move within
the binding pocket for better pseudo-pair interaction with G1408 and improved p-p stacking with A1491 (Fig. 2C). The recent comparative x-ray data of G418 and 6’-hydroxysisomicin in complex with the bacterial and protozoal A-sites support this expectation1-2 (Fig. 2A). In order to form a pseudo pair with 6’- hydroxysisomicin, the G1408 residue in the protozoal A-site shifts toward the deep/major groove compared
with the position of A1408 in bacterial A-site, while ring I rotates approximately 13o around O4- C1’ glycosidic bond between rings I and II; allowing the
formation of a stable pseudo-pair between the ring I of 6’- hydroxysisomicin and G1408, along with the simultaneous strong p-p stacking of
ring I with G1491. 
  
 

 
  

 Timor Baasov 

 

 While these data support the likelihood that the compounds 1-9 of set1 structures (Fig. 1) with 6’-OH and unsaturation at ring I may perform better than previous leads with the saturated ring I, we want to introduce
additional structural motifs at the C6’- region that would further increase this expectation. We plan to replace the CH2OH (and/or
CH(CH3)OH) at 6’-position with the vicinal 6’,7’-diol (compounds 10-11, Fig.1). The replacement of
C6’-OH with 6’,7’-diol is expected to generate stronger H-bond interactions of ring I with A site residues G1408 and C1409 resulting in better
pseudo-disaccharide scaffold of set1 (Fig. 3). Following important points in Fig. 3 are of note. First, unlike our solved structure of G418 bound to Leishmania A-site (Fig. 2B and 2C) in which ring I of
G418 makes two H-bonds with G1408, the structure of G418 bound to the entire yeast ribosome recently reported by Yusupov and co-workers9, shows only one H-bond between O6’ and G1408 nitrogen (Fig. 3). Modelling ring I of the 6’,7’-diol 10 onto this structure of Yusopov,
clearly shows that O7’-OH can make additional H-bond with the oxygen of C1409. 

To test this issue, we initially synthesized compound 10 (Fig. 1) as a single diastereomer at C6’ with unknown configuration (the
complete assignment of the absolute configuration of 10 at C6’, along with the synthesis of its C6’- distereomer are underway). Preliminary in-vitro suppression tests demonstrated that 10
exhibits superior readthrough to that of 12, and paromamine (Fig. 4). Interestingly, installation of 6’,7’-diol on the paromamine scaffold (10) dramatically increases its in-vitro
readthrough activ ity. It is of note that the observed activity increase in 10 is far higher than the impact of the chiral (R)-6’- methyl group in 12, which was to date our most
favorable pharmacophore at C6’- regon7,8. With compound 10 we are already one step ahead! Further installation of chiral
(R)-6’-methyl group and/or N1-AHB group on compound 10 will likely provide additional improvement of this performance, and these potentials will be tested in
the subsequent steps. 
  
 

 
 In addition to 10 we also synthesized the simplest representative of set1 structures,
compound 1 (R=R1=R2=H, Figs. 1 and 2). Preliminary comparative in-vitro suppression tests
demonstrated that 1 exhibits superior readthrough to that of its parent paromamine and is very similar to that of compound 12 (Fig. 4). These data suggest that the anticipated p-p stacking interaction of the unsaturated ring I of 1 with A1491 is much stronger than the CH-p interactions of the saturated ring I of paromamine with A1491.
The data in Fig. 4 also suggests that the advantage of such stacking interactions in 1 (paromamineg1) is similar to that of
(R)-6’-methyl group in 12 (paromamineg12), and thus identifies unsaturation at ring I as a potential new pharmacophore. 

Aim #1- b) rational design of pseudo-trisaccharide scaffolds: It should be noted that
the set1 structures (Fig. 1) are not expected to possess significant PTC suppression activity (Fig. 4); rather they will be used as scaffolds for the construction of new efficacious structures by attaching various sugar and/or acyclic
appendages. For a systematic study, the same modifications will be performed on all pairs of set1, with the aim to reach the best lead compound. As a ring III we have developed a new structure of (S)- 5’’-amino-5’’-methyl-D-ribose, and its advantage over 5”-aminoribose was evident in our previous leads7,8. Therefore, we will use this novel ring III for the generation of new pseudo-trisaccharides (set2-set3, Fig. 5). Encouraged by the preliminary data obtained with 10 (Fig. 4), we will
scrutinize the stereochemistry at C6’ of 10 and will assemble the corresponding pseudo- 
  
 

 

  
 6 

 Timor Baasov 

 

 
trisaccharides 13 and 14 (set2, Fig. 5), along with all the variations of pseudo-trisaccharides with unsuterated ring I (compounds
15-22, set3, Fig. 5). In addition, based on the observed strong impact of the vicinal 6’,7’-diol in 10 (Fig. 4), this variable (6’,7’-diol) will further be added to the
planned pseudo-trisaccharides of set3 structures in Fig. 5, to yield additional sets of compounds incorporating the vicinal 6’,7’-diol and unsaturation at ring I. 

Aim #2. a) Modification of lead structures for improved cell permeability and oral bioavailability: Because
AGs are highly charged, water soluble compounds, they have poor intestinal absorption and are usually injected. In addition, AGs are short-lived in the circulatory system, rapidly eliminated by glomerular filtration in the kidney. They also exhibit
poor permeability into eukaryotic cells, which requires higher dosages that often cause harmful side effects that limit their use in therapy. In order to overcome these limitations we designed different sets of compounds. One of such sets (Fig. 6)
considers that all hydroxyls of the AG are replaced by esters. The rationale is to increase the lipophilicity and cell-permeability of the drugs, improve in-vivo half-life and oral bioavailability. These
changes should provide important therapeutic benefit over treatment with the initial lead alone. 
 The suggested modifications on
NB124 and G418 (Fig. 6) to yield the corresponding per-benzoate esters (OBz, 51 and 52, respectively) are based on the following observations. (i) In our most recent comparative study of our
lead compounds using an extended repertoire of CF cell lines, reporters, assays and animal models, comp. NB124 was able to most effectively rescue CFTR function, was superior to gentamicin, exhibited favorable pharmacokinetics, and was less
cytotoxic than gentamicin in the explant model of ototoxicity10; (ii) the benzoate esters 51 and 52 are chemically more stable than the corresponding simple alkyl asters (like
acetate, isobutirate, isopropionate); (iii) the poly-esters 51 and 52 can easily be prepared in good overall yields from the corresponding AGs in three steps (Boc2O, Et3N, H2O/MeOH; BzCl, Py, 4DMAP; TRA, CH2Cl2); (iv) to test our hypothesis we initially used 52 as a prototype derived from commercial G418. 
  

 
 Preliminary tests of 52 against its parent G418 demonstrated that 52 lacks any readthrough
activity both in cell free (see the data in the 1st year research report) assays and HEK293 cells stably expressing UGAC/CGAC dual luciferase (Fig. 7A) even when the incubation time was extended
up to 72 hrs. Interestingly, when we tested the antibacterial activities of G418 and 52, we found that while the activity of 52 was similar to that of G418 against various G- and G+ WT bacteria, the MIC values of 52 were significantly lower than that of G418 in bacterial strains harboring plasmids of AG resistance determinant enzymes (see the data in the 1st year research report). Encouraged, we argued that HEK293 cells, being engineered cell lines, might not contain the required esterases to hydrolyze the benzoate esters in 52. Indeed, using
MEFs derived from homozygous Idua-W402X mice11, we found that the activity of 52 significantly exceeds that of G418 at all incubation times and concentrations tested. At a dose of only 13.6
micromolar, treated cells had already reached the glycosaminoglycan (GAG) levels found in WT cells (Fig. 7B). These encouraging data with 52 prompted us to prepare compound 51 (see the data in the 1st year research report), which is currently under similar tests in Idua-W402X MEFs (by our collaborator D. Bedwell at Univ. of Alabama, Birmingham) and CF cell lines (primary human bronchial
epithelial, HBE cells, G542X/delF508, S. Rowe at the UAB). In-vivo tests in the Idua-W402X mice (D. Bedwell) will follow these experiments. 

 
 

 

  
 7 

 Timor Baasov 

 

 

 
 Aim #2 b) Modifications for improved capacity to pass the blood-brain barrier
(BBB). While we have been successful in identifying promising nonsense suppression agents (NB84) for the Idua-W402X mutation and partially alleviating the primary GAG storage defect associated with MPS
I-H within a variety of tissues (including brain), we have been unable to completely restore GAG levels in the mouse model, even in a long-term, 28-week drug-treatment12,13. Even though the newly proposed sets of compounds might prove better than the previously examined leads (especially the structures with elevated lipophilicity like 51) for treatment of MPS
I-H, we propose to also test a brain-targeted chemical delivery system (CDS)14 which is expected to produce a more robust therapeutic response. The
approach is based on the attachment of a dihydropyridine moiety as a carrier for delivering drugs through the BBB (Fig. 8). This site-selective delivery will reduce the exposure of the body to high drug levels and consequently increase their
therapeutic index. Since NB84 has already been proven to significantly moderate disease progression in-vivo in our MPS I-H mouse model ,12 we will now synthesize the new derivative of NB84, the compound NB84-CDS (Fig. 11). We (our collaborator at UAB, D. Bedwell) will then determine whether
delivery of NB84-CDS through the BBB is better than NB84 in a long term study using established biochemical and immunohistochemical assays as previously
described12. 
 REFERENCES 

 

	(1)	Kondo, J., Koganei, M., and Kasahara, T. (2012) Crystal Structure and Specific Binding Mode of Sisomicin to the Bacterial Ribosomal Decoding Site. ACS Med. Chem. Lett. 3,9,
741-744. 

	(2)	Kondo, J., Koganei, M., Maianti, J. P., Ly, V. L., and Hanessian, S. (2013) Crystal Structures of a Bioactive 6’-Hydroxy Variant of Sisomicin Bound to the Bacterial and Protozoal Ribosomal Decoding Sites.
ChemMedChem 8, 733–739. 

	(3)	Shalev, M., Kondo, J., Kopelyanskiy, D., Jaffe, C. L., Adir, N., and Baasov, T. (2013) Identification of the molecular attributes required for aminoglycoside activity against Leishmania. Proc. Natl. Acad. Sci.
U. S. A. 110, 13333–13338. 

	(4)	Nudelman, I., Rebibo-Sabbah, A., Cherniavsky, M., Belakhov, V., Hainrichson, M., Chen, F., Schacht, J., Pilch, D. S., Ben-Yosef, T., and Baasov, T. (2009) Development of
novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations. J. Med. Chem. 52, 2836–2845. 

  
 8 

 Timor Baasov 

 

	(5)	Nudelman, I., Glikin, D., Smolkin, B., Hainrichson, M., Belakhov, V., and Baasov, T. (2010) Repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (G418) with enhanced suppression of
diseases-causing nonsense mutations. Bioorg. Med. Chem. 18, 3735–3746. 

	(6)	Rebibo-Sabbah, A., Nudelman, I., Ahmed, Z. M., Baasov, T., and Ben-Yosef, T. (2007) In vitro and ex vivo suppression by aminoglycosides of PCDH15 nonsense mutations
underlying type 1 Usher syndrome. Hum. Genet. 122, 373–381. 

	(7)	Kandasamy Atia-Glikin, D., Belakhov, V. and Baasov, T., J. (2011) Repairing faulty genes by aminoglycosides: Identification of new pharmacophore with enhanced suppression of diseases-causing nonsense mutations.
Med Chem Comm 2, 165–171. 

	(8)	Kandasamy, J., Atia-Glikin, D., Shulman, E., Shapira, K., Shavit, M., Belakhov, V., and Baasov, T. (2012) Increased selectivity toward cytoplasmic versus mitochondrial ribosome confers improved efficiency of
synthetic aminoglycosides in fixing damaged genes: A strategy for treatment of genetic diseases caused by nonsense mutations. J. Med. Chem. 55, 10630– 10643. 

	(9)	De Loubresse, N. G., Prokhorova, I., Holtkamp, W., Rodnina, M. V., Yusupova, G., and Yusupov, M. (2014) Structural basis for the inhibition of the eukaryotic ribosome. Nature 513, 517-522. 

	(10)	Gunn, G., Dai, Y., Du, M., Belakhov, V., Kandasamy, J., Schoeb, T. R., Baasov, T., Bedwell, D. M., and Keeling, K. M. (2014) Long-term nonsense suppression therapy moderates MPS
I-H disease progression. Mol. Genet. Metab. 111, 374-381. 

	(11)	Keeling, K. M., Wang, D., Dai, Y., Murugesan, S., Chenna, B., Clark, J., Belakhov, V., Kandasamy, J., Velu, S. E., Baasov, T., and Bedwell, D. M. (2013) Attenuation of Nonsense-Mediated mRNA Decay Enhances In Vivo
Nonsense Suppression. PLoS One 8, 4, e60478. 

	(12)	Gunn, G., Dai, Y., Du, M., Belakhov, V., Kandasamy, J., Schoeb, T. R., Baasov, T., Bedwell, D. M., and Keeling, K. M. (2014) Long-term nonsense suppression therapy moderates MPS
I-H disease progression. Mol. Genet. Metab. 111, 374-381. 

	(13)	Keeling, K. M., Wang, D., Dai, Y., Murugesan, S., Chenna, B., Clark, J., Belakhov, V., Kandasamy, J., Velu, S. E., Baasov, T., and Bedwell, D. M. (2013) Attenuation of Nonsense-Mediated mRNA Decay Enhances In Vivo
Nonsense Suppression. PLoS One 8, 4, e60478. 

	(14)	Bodor, N., and Buchwald, P. (2008) Retrometabolic drug design: Principles and recent developments. Pure Appl. Chem. 80, 1669-1682. 

III. Proposed Budget (in US Dollars) and Time Frame 
  

													
	  	  	The proposed task	  	Time Frame	  	Budget
Requested	 	  	Remarks	  	Notes
	 Aim # 1
	  	Establishment of config. In 10 and synthesis of 13.	  	 6-12 months
	  	$	15,000	 	  	One postdoc/student dedicated.	  	 PI T. Baasov

		  	Synthesis of 15 and preliminary biochem. Analysis of 10,13 and 15.	  	 6-12 months
	  	$	15,000	 	  	One postdoc/student dedicated.	  	
						
		  		  	Total Aim #1:	  	$	30,000	 	  		  	
		  		  	  
	  	  
	  
	 	  		  	
						
	 Aim # 2a
	  	Synthesis of NB84-esters and NB124-esters	  	 8-12 months
	  	$	10,000	 	  	One postdoc/student dedicated.	  	
						
	 Aim # 2b
	  	Synthesis of NB84-CDS and its Preliminary biochemical analysis	  	 6-12 months
	  	$	10,000	 	  	One postdoc/student dedicated.	  	
		  		  	Total Aim #2:	  	$	20,000	 	  		  	
		  		  	  
	  	  
	  
	 	  		  	
		  		  	Grand total for the Aims # 1&2:	  	$	50,000	 	  		  	
		  		  	  
	  	  
	  
	 	  		  	

  
 9 

 Timor Baasov 

 

 NOTE the budget does not include ex vivo (MEFs) and in vivo studies required for the project completion
and only can be done with additional funding of collaborators labs (Bedwell and Rowe at UAB)! 
 IV. Detailed Budget (in US
Dollars) 
 Personnel: 
  

													
	Role in project	  	 	  	% Time	 	  	Salary	 
	 1.
	  	 Lab. Assistant
	  	 Lab. Assistant
	  	 	50	 	  	 	12,000	 
	 2.
	  	 Postdoctorant
	  	 Researcher
	  	 	50	 	  	 	12,000	 
		  		  		  				  	  
	  
	 
		  	 Total:
	  		  				  	 	24,000	 

 Supplies: 
  

					
	 1. Chemicals, absolute & deuterated solvents
	  	 	18,000	 
	 2. Lab. equipment, glassware, plastic ware
	  	 	2,000	 
	 3. Biochemicals
	  	 	2,000	 
	 4. In vitro and ex vivo assay kits
	  	 	2,000	 
	 5. Chromatography material for various purifications
	  	 	1,000	 
	 6. NMR and Mass Spectrometry tests
	  	 	1,000	 
		  	  
	  
	 
	 Total:
	  	 	26,000	 
		  	  
	  
	 
	 Grand Total:
	  	$	50,000	 

 Budget Justification: 

The requests for laboratory assistant and postdoctorant for the duration of the grant period are in recognition of the amount of work required in this project.
Considerable effort will be expended in the syntheses of various lead compounds discussed in the proposal, their structure determination and analysis, assays for their activity. The request for materials, supplies, and chemicals/biochemicals is an
important part for a successful development and completion of the project. 
 V. Investigators’ Curriculum Vitae 

 

									
	Surname:	  	 Baasov
	  		  	First name:	  	 Timor

	Birthdate:	  	 January 3, 1954
	  		  		  	

 (a) Education Background 
  

							
	 From-To
	  	 Institution
	  	 Area of specialization
	  	Degree
	 1981-1986
	  	Weizmann Institute of Science	  	Chemistry	  	Ph. D.
	 1977-1979
	  	Tel-Aviv University	  	Chemistry	  	M. Sc.
	 1975-1977
	  	Tel-Aviv University	  	Chemistry	  	B. Sc.

 Major research interest: Carbohydrate chemistry, Bioorganic and medicinal chemistry, Drug design and development,
Rational design of substrate and inhibitors, Mechanistic enzymology. 
 (b) Employment 

 

							
	 From-To
	  	 Institution
	  	 Research area
	  	Title
	 2004-
	  	Technion	  	Bioorganic Chemistry	  	Professor
	 3/1998-8/1998
	  	The Scripps Research Institute, Cal	  	Bioorganic Chemistry	  	Visiting Prof.

  
 10 

 Timor Baasov 

 

							
	 1998-2003
	  	Technion	  	Bioorganic Chemistry	  	Assoc. Prof.
	 1990-1998
	  	Technion	  	Bioorganic Chemistry	  	Senior Lecturer
	 1988-1990
	  	Technion	  	Bioorganic Chemistry	  	Lecturer
	 1986-1988
	  	Harvard University	  	Bioorganic Chemistry	  	Post-Doct. Res.

 VI. T. Baasov - List of Publications last three years (2011-2014) 

 

	1.	C. Brendel, V. Belakhov, H. Werner, E. Wegener, J. Gaertner, I. Nudelman, T. Baasov, P. Huppke. Readthrough of Nonsense Mutations in Rett Syndrome: Evaluation of novel aminoglycosides and generation of a new
mouse model. Journal Molecular Medicine, 89, 389-398, (2011). 

  

	2.	J. Kandasamy, D. Atia-Glikin, V. Belakhov, T. Baasov. Repairing faulty genes by aminoglycosides: Identification of new pharmacophore with enhanced suppression of diseases-causing nonsense mutations. Medicinal
Chemistry Communications, 2, 165-171 (2011). 

  

	3.	S.M. Rowe, L.P. Tang, P. Sloane, K.Backer, M. Mazur, J. Buck;ey-Lauriel, I. Nudelman, V. Belakhov, Z. Belok, E. Schwiebert, T. Baasov, D.M. Bedwell. Suppression of CFTR
Premature Termination Codons and Rescue of CFTR Protein and Function by the Synthetic Aminoglycoside NB54. Journal Molecular Medicine (Berl), 89, 1149-1154 (2011). 

 

	4.	M. Vecsler, B. Ben Zeev, I. Nudelman, Y. Anikster, A. J. Simon, N. Amariglio, G. Rechavi, T. Baasov, E. Gak. Ex Vivo Treatment with a Novel Synthetic Aminoglycoside NB54 in Primary Fibroblasts from Rett Syndrome
Patients Suppresses MECP2 Nonsense Mutations. PLoS ONE, 6 (6), e20733 (2011). 

  

	5.	H-L. R. Lee, C-C. Chen, T. Baasov, Y. Ron, J. P. Dougherty. Post-transcriptionally Regulated Expression System in Human Xenogeneic
Transplantation Models. Molecular Therapy, 19(9), 1645-1655 (2011). 

  

	6.	D. Wang , V. Belakhov, J. Kandasamy, T. Baasov, S-C. Li, Y-T Li, D.M. Bedwell, K.M. Keeling. The designer aminoglycoside NB84
significantly reduces glycosaminoglycan accumulation associated with MPS I-H in the Idua-W392X mouse. Molecular Genetics and Metabolism 105, 116-125
(2012). 

  

	7.	T. Goldmann, N. Overlack, F. Möller, V. Belakhov, M. van Wyk, T. Baasov, U. Wolfrum, and K. Nagel-Wolfrum. A comparative evaluation of NB30, NB54 and PTC124 in translational read-through efficacy for
treatment of an USH1C nonsense mutation. EMBO Molecular Medicine, 4, 1-14, (2012). 

  

	8.	J. Kandasamy, D. Atia-Glikin, E. Shulman, K. Shapira, M. Shavit, V. Belakhov T. Baasov. Increased Selectivity toward Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic
Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations. J. Med. Chem. 55(23), 10630-10643 (2012). 

 

	9.	M. Schalev, J. Kandasamy, N. Skalka, V. Belakhov, R. Rosin-Arbesfeld, T. Baasov. Development of generic immunoassay for the detection of a series of aminoglycosides with
6’-OH group for the treatment of genetic diseases in biological samples. Journal of pharmaceutical and biomedical analysis. 75, 33-40 (2013).

  

	10.	K.M. Keeling, D. Wang, Y. Dai, S. Murugesan, B. Chenna, J. Clark; V. Belakhov, J. Kandasamy, S.E. Velu, T. Baasov, D.M. Bedwell. Attenuation of Nonsense-Mediated mRNA Decay Enhances In Vivo Nonsense Suppression.
PLoS ONE 8 (4), e60478 (2013). 

  

	11.	M. Schalev, J. Kondo, D. Kopelyanskiy, C.L. Jaffe, N. Adir, T. Baasov. Identification of the molecular attributes required for Aminoglycoside activity against Leishmania. PNAS 110 (33), 13333-13338
(2013). 

  

	12.	M. Kamei, K. Kasperski, M. Fuller, E. Parkinson-Lawrence, L. Karageorgos,, V. Belakhov, T. Baasov, J.J. Hopwood, D.J. Brooks. Am inoglycoside-Induced Premature Stop Codon Read-Through of Mucopolysaccharidosis
Type I Patients Q70X and W402X Mutations in Cultured Cells. Journal of Inherited Metabolic Disease Reports. 13, 139-147 (2014) 

 

	13.	X. Xue, V. Mutyam, L.P. Tang, S. Biswas, M. Du, L. A. Jackson, Y. Dai, V. Belakhov, M. Shalev, F. Chen, J. Schacht, R. Bridges, T. Baasov, J. Hong, D. M. Bedwell, S.M. Rowe. Synthetic Aminoglycosides Efficiently
Suppress CFTR Nonsense Mutations and Are Enhanced by Ivacaftor. Am. J. Respir. Cell Mol. Biol. 50 (4), 805- 816 (2014). 

 

	14.	E. Shulman, V. Belakhov, G. Wei, A. Kendall, E. G. Meyron-Holtz, D. Ben-Shachar, J. Schacht, T. Baasov. Designer aminoglycosides that selectively inhibit cytoplasmic rather
than mitochondrial ribosomes show decreased ototoxicity: a strategy for the treatment of genetic diseases. J. Biol. Chem. 289(4), 2318-2330 (2014). 

  

	15.	G. Gunn, Y. Dai, M. Du, V. Belakhov, J. Kandasamy, T.R. Schoeb, T. Baasov, D.M. Bedwell, K.M. Keeling. Long-term nonsense suppression therapy with NB84 moderates MPS IH disease progression. Molec. Genet.
Metabol. 111, 374-381 (2014). 

  
 11 

 Timor Baasov 

 

	16.	M. Shalev, T. Baasov. When Proteins Start to Make Sense: Fine-tuning of Aminoglycosides for PTC Suppression Therapy. Med. Chem. Commun. 5, 1092-1105 (2014). Invited Review Perspective
Article 

 Chapters in Books and other Publications 

 

	1.	V. Mutyam, X. Xue, X. Jackson, L. Hong, S. Biswas, D. Bridges, T. Baasov, V. Belakhov, D. Bedwell, S. Rowe. Use of transepithelial conductance as a screening technique for identification of drugs that promote
readthrough of premature stop codons. Pediatric Pulmonology 48, page 232 (supplement 36; meeting abstract). ISSN: 8755-6863 (2013). 

  

	2.	K. Nagel-Wolfrum, T. Baasov, U. Wolfrum. Therapy strategies for Usher syndrome Type 1C in the retina. Advances in experimental medicine and biology. Vol. 801, pp.
741-747 (2014). 

  

	3.	T. Baasov, M. Fridman. Foreword-The 17th European Carbohydrate Symposium-EuroCarb17. Carbohydrate Research 389, 1 (2014). (Guest Editor of the special
issue.) 

  

	4.	S. Garneau-Tsodikova, T. Baasov. Editorial – Carbohydrates Themed Issue. Med. Chem. Commun., 5, 1010-1013 (2014). (Guest Editor of the special issue.) 

  
 12

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