MONISHA GUPTA
The research work I have been involved in since June 2021 focuses on inhibiting HIV-1 viral entry and spread of infection through developing novel macrocyclic peptide triazoles (cPT). I conduct research in the Chaiken Lab in the Department of Biochemistry and Molecular Biology in the College of Medicine. The research techniques I have gained experience in include solid phase peptide synthesis, organic chemistry reactions such as click-chemistry (triazole formation) and Suzuki Coupling, as well as purification techniques using HPLC/MS. I have also been able to assist in the analysis of the synthesized peptides through assisting with kinetic studies via Surface Plasmon Resonance. To further guide project developments, I have interpreted infection inhibition data and chemical modeling results from a lab we collaborate with at Drexel.
Prior to explaining the most recent findings in the cPT project, a bit of background is provided here: Regarding HIV-1, the gp120 trimer Env is part of the viral spike, and the target host cell receptor is CD4. One of the operational domains of interest to the Chaiken lab group is the Phe 43 pocket in CD4. The Chaiken lab group focuses on developing a cyclic peptide to inhibit the viral entry of HIV-1 and cause the irreversible inactivation of HIV-1 viruses. This cyclic peptide is known as a macrocyclic peptide triazole. It binds to the gp120 trimer of HIV-1 which then binds to CD4 Phe 43 pocket; however, the presence of the cPT in this interaction prevents viral entry. The cyclic peptide triazole complex N2 (AAR029N2) is the current parent molecule with a pharmacophore of the isoleucine, tryptophan, and triazole residues. The three other amino acids in the cPT are aspartic acid, lysine and asparagine. Current work has focused on exploring modifications at both the tryptophan and triazole moieties of the pharmacophore.
As stated earlier, the cPT work involves solid phase peptide synthesis, other critical organometallic chemistry reactions, and then purification and validation via HPLC/MS. Recent efforts have been focusing on exploring the Trp and triazole residues of the cPT. The lead compound N2 houses a thiophene group in the triazole component. We have been exploring a thiophene-methyl group and other pyrazole attachments as well (difluoro-methyl and a pyrazole by itself). The thiophene-methyl has shown similar affinities to N2 in initial testing. The reasoning to explore a pyrazole attachment was based on the previously proposed difluoro-methyl pyrazole replacement of the thiophene of N2. This was regarding hypothesized improvements in potency and effects on kinetics. From recent SPR binding studies, we can see that there is a slight loss of affinity from this adjustment. From using the pyrazole by itself as a replacement for the thiophene, the affinity is strengthened compared to the difluoro-methyl. This comparison signals to us that perhaps the pocket in which the triazole fits is quite small and the room for adjustment or optimization may not need a larger modification than something in the relative ballpark of the thiophene. A possible modification to test this further is to attach a bromine to the thiophene and test whether there is a loss of affinity, which could help confirm the tightness of the triazole moiety interaction in gp120.
Furthermore, a larger focus of modifications has been on the indole component of the Trp residue in the pharmacophore. Based on prior computer modeling conducting by a collaborating group at Drexel, there have been scenarios where the indole has been shown to not be interacting as intimately with its target site in gp120. Studying the indole has shown that it is in fact critical to the binding interactions with gp120. For example, one derivative made housed a bromine on the benzene component of the indole. Which has detrimental effects on binding kinetics, as the off rate and affinity of the compound are significantly worsened due to this addition. We hypothesize this is due to the larger electron cloud which may be impeding the necessary pi-pi electron stacking between the indole and the gp120 target pocket (which has been shown in a previous computer model of N2). A similar binding profile was seen by applying a methyl to the nitrogen on the indole. The same effect of a significantly faster off rate is present, along with a loss of affinity as well. However, we do plan to run ITC experiments with the indole modifications to verify the same kinetic patterns are visible when the target is not immobilized as it is in SPR.
In addition to exploring the intimate binding interactions of triazole and indole modifications, we also have another goal to obtain a strong candidate molecule for x-ray crystallography. To date, the cPT has not been crystalized, and adding the bromine to the Trp residue was also a strategy to see if this derivative could be a good crystal candidate. However, as explained above, the visible loss of binding seems to be a hinderance in the x-ray crystallography process. In the summer of 2022, we sent some material of the N2 with a bromine on the indole to a research group in NYC to run some crystallography studies and it seems as though this derivative may not be the best candidate. We then decided to create a pyrazole (no methyl-difluoro) and bromine-indole variant of N2 to see perhaps if the addition of an improvement in triazole affinity could combat the disruption of bromine addition to the indole for binding interactions. However, current results show that this avenue is not significantly better regarding binding kinetics. Based on current data, one possible avenue is to synthesize an N2 derivative which has a penta-fluorine phenyl on the aspartic acid residue, which is not on the pharmacophore. We hypothesize that electron clouds from the penta-fluorine phenyl may be sufficient to be visible under x-ray crystallography. And perhaps without disruption pharmacophore binding interactions, this variant could result in a crystal structure! I am excited to continue my research work at the Chaiken Lab and continue to learn and grow as a student!
The research work I have been involved in since June 2021 focuses on inhibiting HIV-1 viral entry and spread of infection through developing novel macrocyclic peptide triazoles (cPT). I conduct research in the Chaiken Lab in the Department of Biochemistry and Molecular Biology in the College of Medicine. The research techniques I have gained experience in include solid phase peptide synthesis, organic chemistry reactions such as click-chemistry (triazole formation) and Suzuki Coupling, as well as purification techniques using HPLC/MS. I have also been able to assist in the analysis of the synthesized peptides through assisting with kinetic studies via Surface Plasmon Resonance. To further guide project developments, I have interpreted infection inhibition data and chemical modeling results from a lab we collaborate with at Drexel.
Prior to explaining the most recent findings in the cPT project, a bit of background is provided here: Regarding HIV-1, the gp120 trimer Env is part of the viral spike, and the target host cell receptor is CD4. One of the operational domains of interest to the Chaiken lab group is the Phe 43 pocket in CD4. The Chaiken lab group focuses on developing a cyclic peptide to inhibit the viral entry of HIV-1 and cause the irreversible inactivation of HIV-1 viruses. This cyclic peptide is known as a macrocyclic peptide triazole. It binds to the gp120 trimer of HIV-1 which then binds to CD4 Phe 43 pocket; however, the presence of the cPT in this interaction prevents viral entry. The cyclic peptide triazole complex N2 (AAR029N2) is the current parent molecule with a pharmacophore of the isoleucine, tryptophan, and triazole residues. The three other amino acids in the cPT are aspartic acid, lysine and asparagine. Current work has focused on exploring modifications at both the tryptophan and triazole moieties of the pharmacophore.
As stated earlier, the cPT work involves solid phase peptide synthesis, other critical organometallic chemistry reactions, and then purification and validation via HPLC/MS. Recent efforts have been focusing on exploring the Trp and triazole residues of the cPT. The lead compound N2 houses a thiophene group in the triazole component. We have been exploring a thiophene-methyl group and other pyrazole attachments as well (difluoro-methyl and a pyrazole by itself). The thiophene-methyl has shown similar affinities to N2 in initial testing. The reasoning to explore a pyrazole attachment was based on the previously proposed difluoro-methyl pyrazole replacement of the thiophene of N2. This was regarding hypothesized improvements in potency and effects on kinetics. From recent SPR binding studies, we can see that there is a slight loss of affinity from this adjustment. From using the pyrazole by itself as a replacement for the thiophene, the affinity is strengthened compared to the difluoro-methyl. This comparison signals to us that perhaps the pocket in which the triazole fits is quite small and the room for adjustment or optimization may not need a larger modification than something in the relative ballpark of the thiophene. A possible modification to test this further is to attach a bromine to the thiophene and test whether there is a loss of affinity, which could help confirm the tightness of the triazole moiety interaction in gp120.
Furthermore, a larger focus of modifications has been on the indole component of the Trp residue in the pharmacophore. Based on prior computer modeling conducting by a collaborating group at Drexel, there have been scenarios where the indole has been shown to not be interacting as intimately with its target site in gp120. Studying the indole has shown that it is in fact critical to the binding interactions with gp120. For example, one derivative made housed a bromine on the benzene component of the indole. Which has detrimental effects on binding kinetics, as the off rate and affinity of the compound are significantly worsened due to this addition. We hypothesize this is due to the larger electron cloud which may be impeding the necessary pi-pi electron stacking between the indole and the gp120 target pocket (which has been shown in a previous computer model of N2). A similar binding profile was seen by applying a methyl to the nitrogen on the indole. The same effect of a significantly faster off rate is present, along with a loss of affinity as well. However, we do plan to run ITC experiments with the indole modifications to verify the same kinetic patterns are visible when the target is not immobilized as it is in SPR.
In addition to exploring the intimate binding interactions of triazole and indole modifications, we also have another goal to obtain a strong candidate molecule for x-ray crystallography. To date, the cPT has not been crystalized, and adding the bromine to the Trp residue was also a strategy to see if this derivative could be a good crystal candidate. However, as explained above, the visible loss of binding seems to be a hinderance in the x-ray crystallography process. In the summer of 2022, we sent some material of the N2 with a bromine on the indole to a research group in NYC to run some crystallography studies and it seems as though this derivative may not be the best candidate. We then decided to create a pyrazole (no methyl-difluoro) and bromine-indole variant of N2 to see perhaps if the addition of an improvement in triazole affinity could combat the disruption of bromine addition to the indole for binding interactions. However, current results show that this avenue is not significantly better regarding binding kinetics. Based on current data, one possible avenue is to synthesize an N2 derivative which has a penta-fluorine phenyl on the aspartic acid residue, which is not on the pharmacophore. We hypothesize that electron clouds from the penta-fluorine phenyl may be sufficient to be visible under x-ray crystallography. And perhaps without disruption pharmacophore binding interactions, this variant could result in a crystal structure! I am excited to continue my research work at the Chaiken Lab and continue to learn and grow as a student!
